Intro + Task Intro

Question 1

What is clinical pathology?

Answer 1

A medical speciality that is concerned with the diagnosis of disease based on the laboratory analysis of bodily fluids e.g. blood, urine and tissue homogenates or extracts using the tools of chemistry, microbiology, haematology and molecular pathology.

Question 2

Why are diagnostic tests performed?

Answer 2

– Investigate disease process underlying clinical signs.
- Make a problem list based on history / PE findings.
- Make differential diagnosis lists for each (primary) problem.
- Test selection to exclude / confirm differential diagnoses.

– Investigate risk of disease / presence of positive traits in healthy animals.

– Minimise risk of adverse events during / following treatment or diagnostic procedure.

– To provide baseline parameters for future monitoring.

Question 3

Types of samples that can be analysed.

Answer 3

Fluids e.g. Blood, serum, urine, effusions, joint fluid, CSF.
Cells e.g. FNA, washes.
Tissues e.g. biopsies.

Question 4

Ways of sample processing.

Answer 4

Machine vs manual.
As submitted vs concentrated / enriched.
With or without processing / additives / fixatives / stains (huge range of options).

Question 5

Information gained from testing.

Answer 5

Organ / tissue damage.
Organ function.
Susceptibility to disease.
Cell pathology – metabolic derangements, inflammation, neoplasia.
Presence (or historic evidence) of infectious agents.
Host response to disease.

Question 6

Considerations for sample submission.

Answer 6

Appropriate sample collection used.
Correct tube e.g. EDTA for haem / cytology, serum for biochemistry, heparin for rapid biochemistry, oxalate fluoride for glucose, citrate for clotting function.
Accurately fill sample container.
Mix well (gently).
Store / transport correctly with consideration re temp (room, fridge, freezer), light etc.

Question 7

What is clinical biochemistry?

Answer 7

Measurement and interpretation of various analytes from blood plasma or serum.

Question 8

Clinically significant differences between serum and plasma.

Answer 8

More plasma proteins in plasma than in serum due to loss in clot.
More potassium in serum than plasma as it’s released from platelets during clot formation.

Question 9

1. What diagnostics is serum required for?
2. What diagnostics is plasma better for?

Answer 9

1. Bile acids and haptoglobin concentration measurements and serum protein electrophoresis.
2. Better in emergency setting for rapid processing.
   Required for some parameters (e.g. PTH and ammonia).

Question 10

Factors affecting result variability.

Answer 10

Biological – Inter-individual and intra-individual.
Analytical – Pre-analytical, analytical, post-analytical.

Question 11

Inter-individual variations.

Answer 11

PCV varies between cats (1/4-1/2) and dogs (1/3-2/3) and is also different in sighthounds (2/3).
PCV can also be affected by age (lower in younger).
Age can also affect indicators of bone growth (ALP, calcium (produced by bone so higher in younger)).
Cell morphology varies between mammals, birds and reptiles (nucleated RBCs) (some w/ green plasma) and can also be different in the camelid subset (ovular w/ no nucleus) (can’t fit through haem machine so must be run manually).

Question 12

Intra-individual variations.

Answer 12

Diet (and time from most recent meal).
Reproductive status.
Drugs / therapy.
Stress / excitement / fear.

Question 13

What is the impact of stressful sampling on the test results?

Answer 13

Neutrophilia and/or lymphocytosis
- From marginating to circulating pools – by combo of adrenaline and cortisol.
Increased red cell mass and increased reticulocytes.
- Splenic contraction.
Decreased red cell mass (if haemolysis) by damage of RBCs through needle by increased pull pressure by sampler.
Increased glucose
- Increased cortisol so insulin resistance (physiologically normal for sustained energy and survival).
- Cats more susceptible (predation).
Increased creatine kinase (CK) activity (muscular tension).
- Stressful/repeated restraint.
Activation of clotting cascade.

Question 14

Pre-analytical errors causing analytical variability.

Answer 14

Sample collection, handling, presence of interfering substances.
Incorrect blood tube (various anticoagulants – presence and type).
Contamination - EDTA increases potassium and decreases calcium.
Tube incorrectly filled.
Incorrect storage.
Haemolysis, lipaemia, icterus.
** RECOMMENDED TO FAST! **

Question 15

Haemolysis.

Answer 15

Pink/red sample due to lysed RBCs.
False increase in RBC constituents e.g. potassium.
In vivo, sampling, post sampling.
Mimicked by synthetic oxygen carrying solutions.

Question 16

Lipaemia

Answer 16

Visible turbidity (cloudiness).
Can be physiological or pathological.
Can cause a false decrease in other substances in the serum e.g. electrolytes.

Question 17

Icterus/jaundice.

Answer 17

Yellow – due to increased bilirubin.
Notable that plant-eating herbivores such as horses and cows have carotene-rich plasma so has a slightly yellow discolouration (so is NOT jaundice!)

Question 18

What can cause analytical errors?

Answer 18

Lab environment e.g. temperature / humidity outside range.
Personnel e.g. incorrect use of machine / inaccurate pipetting.
Equipment e.g. lack of appropriate maintenance (e.g. filter needs cleaning), expired reagents/blood tubes, lack of calibration.
Analytical procedure e.g. technique not validated for spp / sample used (e.g. serum vs plasma).

Question 19

What is spectrophotometry?

Answer 19

The measurement of colour/turbidity in a solution by determining amount of light absorbed in the ultraviolet, infrared or visible spectrum, widely used in clinical chemistry to calculate concentration of substances in solution.

Question 20

Enzymatic assays…
1. Liver enzymes that can be analysed.
2. Muscle enzymes that can be analysed.
3. End-point vs kinetic.
4. What issues can arise as many enzymatic assays are kinetic?

Answer 20

1. ALT (alanine aminotransferase), ALP (alkaline phosphatase).
2. CK (Creatinine kinase).
3. End-point = amount of substance at a pre-determined end point.
   Kinetic = Activity between 2 time points.
4. Can generate falsely low results if high amounts of enzyme present due to substrate depletion.

Question 21

1. What must be done for the sake of direct comparison between results?

Answer 21

1. Repeat samples should be run on the same machine at the same lab. Inter-machine variations such as environment of machine (temp, humidity etc.), reference ranges.

Question 22

1. What can cause post-analytical errors to occur?
2. How can these be reduced?

Answer 22

1. Inaccurate interpretation / recording of results.
2. Show working out and reasoning in clinical notes. Show you know why a certain level was lower/higher than expected. (e.g. increased urea due to lack of fasting).

Question 23

1. Considerations for patient and test selection.
2. Considerations for test result interpretation.

Answer 23

1. Diagnostic tests are not perfect.
   Expense / invasiveness / patient factors (e.g. aggression) may limit whether a diagnostic test is performed and whether surrogate markers are used.
2. True result vs error / artefact.
   Knowing normal vs abnormal.
   ‘Normal’ is NOT ALWAYS normal.
   Abnormal – diagnostic vs screen (i.e. indicates need for further diagnostics).

Question 24

What can be given in context as to why the diagnostic test was performed?

Answer 24

• To investigate disease process underlying clinical signs.
• To investigate risk of disease / presence of positive traits in healthy animals.
• To minimise risk of adverse events during / following treatment or diagnostic procedure.
• To provide ‘baseline’ parameters for future monitoring.

Question 25

1. By definition, what % ‘normal’ animals will fall outside the reference range?
2. Is this representative?

Answer 25

1. 5%.
2. No, this is based off the target population which may not match the patient population.

Question 26

Overcoming problems with results…
1. How can an approach be standardised with:
a. Haematology
b. Serum biochemistry
c. Fats
d. Others

Answer 26

1. a. RBCs, WBCs, platelets, other.
   b. Proteins – albumin, globulin.
   Kidney parameters – urea, creatinine, PO43-, Ca2+, [urinalysis].
   Carbohydrates – glucose.
   Electrolytes – Na+, K+, Cl-, Ca2+.
   Liver parameters.
   Enzymes – ALT, ALP, GGT, GLDH etc.
   Function – bilirubin, bile acids, ammonia, albumin, glucose, cholesterol, urea.
   c. cholesterol, triglycerides.
   d. Markers of systemic inflammation – APP response, specific APP proteins (CRP, a1-HAGP).
   Hormones – T4 (+/-TSH), cortisol, insulin.

Question 27

Describing the results…
1. What must first be considered?
2. What must be done for each abnormal parameter?

Answer 27

1. The individual patient (signalment / demographics) context.
   Why was the test performed?
   How do findings contribute to your original problem / differential diagnoses lists?
   - Can they confirm/exclude a differential diagnosis?
   - Are further diagnostic tests needed?
2. Describe the changes.
   - Increased vs decreased.
   - Magnitude.
   - Note inappropriate ‘normal’ results.
   - Interpretation (in context w/ other results).