MSAT Clin Path Flashcards
How can you do macroscopic evaluation of semen?
- Volume
- Colour
No discolouration or blood, pus, urine discolouration - Consistency
Affected by: Semen production, collection and method (electroejaculation = dilute semen)
Scale:
0 - clear
1- Cloudy < 200
2- Milky 200-500
3- Thick milk 500-1000
4- Creamy 1000-1500
5- Thick creamy 1500 + - Smell: e.g. Urine contamination, pus
- pH: Normal pH of bull is 6.2-7.4. Decreases overtime due to build of lactic acid
How can you do a microscopic evaluation of semen?
Mass Activity: Small drop of semen on pre-warned slide with no cover slip
–> Use: reserved for ruminant semen as highly concentrated
–> Calculate: Concentration & motility
Scale:
0 - No swirl
1 - Very slow swirl
2- Slow swirl
3- Moderate Swirl
4- Fast swirl
5- Very fast swirl
Individual motility
a) Total motility: % of sperm moving in any direction
b) Progressive motility: % of sperm moving progressively across the field of view (moving actively forward), assess different parts of the slide
Results: Tick -> 60%, Q -> 30-59%, X - <30%
c) Speed of movement
Method: Dilute sample (warm isotonic) and place on drop on a pre-warmed slide with a coverslip or dip wooden applicator in semen & dilutant.
Morphology
Determining the % of normal vs abnormal sperm whilst immotile
Methods:
a) Wet prep: Drop of semen on slide with coverslip –> Phase contrast or DIC microscope
b) Eosin-nigrosin stain: Mix small semen drop with large stain drop –> place on slide near frosted edge and use blood smear method to spread.
–> bright field microscope
Major defects: Proximal cytoplasmic droplets pyriform heads, folded/coiled tails, mid piece defects, maldeveloped, craters
Result: Tick > 70%, Q - 50-60%, X - <50%
Foreign cell smear: Smear made from raw semen directly or from a pellet of semen after centrifuging
Cells seen: Sperm, neutrophils, lymphocytes, epithelial cells, spermatogenic cells
Smegma contamination: WBC’s
High no. of neutrophils from sheath ejaculation may be normal, but high numbers if penis is exteriorised/washed is worrying.
Concentration
Results: Tick - > 200, Q - >200, X - < 200
Methods:
- Haemocytometer
Step 1: Dilute sperm in saline/phosphate buffered glutaraldehyde –> Preserved sperm
Step 2: mix well, load an aliquot on eac side of the haemocytometer
Step 3: Count no. of sperm heads within counting area (25 squares) boarded by 3x lines, if too many sperm, count sperm in 5 corner/middle squares and multiple by 5
ONLY count sperm heads in contact with upper/left border and NOT lower/right border
Step 4: Calculate concentration taking in dilution factor
= Count in 25 squares x 10,000 x Dilution Factor
(AKA 1:100 factor = x 100)
Other ways of measuring sperm concentration:
- Nucelocounter
- Photometer (Spermacute)
- CASA: Computer assisted semen analysis
What is the method to make a blood smear?
Method: Wedge slide by Maxwell Wintrobe
- Place drop of blood from EDTA from capillary tube or wooden stick ~3mm in size
- Place spread slide at 30–45-degree angle, drawn backwards first to distribute blood
- Spread forward at a 45-degree angle in a single motion
Preparing the stain:
Preparation – Romanowski Stain:
Dip 4-5x for 2s each → Fixative (Methanol) → Acidic dye (Eosin) → Basic dye (Methylene)
Basic care: Wipe slides between each jar, keep sealed & regularly replace, write date on jar
PARTS OF A BLOOD SMEAR:
- Application point
- Body
- Monolayer
- Feathered Edge
What is the Systematic Examination for a blood smear?
On 10x: Assess smear quality
Feathered edge look for platelet clumps, parasites, large or neoplastic cells, WBC clumping
On 100x: Estimate platelet count and observe platelet morphology
Feline platelets prone to clumping
Monolayer/Feathered Edge
On 10x: Estimate WBC count
Count number of WBC in 3 (10x) fields in the monolayer. Divide by 3 to get the average.
Divide by 4 to get white cell concentration.
and 100x perform WBC differential count:
Tallying, differential grid, manual cell counter
Count neutrophils, band neutrophils, lymphocytes, monocytes, eosinophils, basophils
Scan on 10x for erythrocyte distribution and 100x for morphology
- Assess density, shape, colour, size, regeneration/cell types
Rouleaux: Linear branching/non-branching aggregates resembling stacked coins
Common in horses, less in cats
Formed via interactions between RBC membranes and plasma macromolecules.
Increased rouleaux with hyperglobulinaemia and hyperfibrinogenaemia
Aggregates due to agglutination (RBC being held together via antibodies). Immune-mediated response
–> Saline Dispersion/Dilution Test: Disperse RBC aggregates into individual with TPP is diluted
Density/Anaemia:
Reduced RBC density or increased space between RBC
Morphology:
Size: Microcytes, macrocytes, anisocytosis (Different sizes)
Shape: Round, or poikilocytes
What are the different WBC types and how to differentiate?
Neutrophils: Sausage shaped nucleus, granules on the cytoplasm
Band neutrophil: Shaped like a horseshoe
Lymphocytes: Round nucleus.
Metarubicyte: Purple, poly cytoplasm
Natural killer cells: High amount of granules in the cytoplasm
Blue: producing protein
Monocytes
Basophils: Purple or lavender coloured granules
Eosinophils: Bubbly looking
Describe species variation in erythrocytes?
Canine: Largest in size, greatest in central pallor
Feline: Second largest, minimal central pallor, rouleaux/aniscocytosis mild
Equine: Mid range, central pallor. Prominent rouleaux, no anisocytosis
Bovine: Smaller in size, central pallor, no rouleaux, aniscocytosis
How to identify blood smear species?
Identify blood smear species
o Horse – raspberry eosinophils
o Cat – eosinophil rod-shaped granules
o Cat, dog, horse mainly neut
o Sheep, cow, goat mainly lymphocytes
o Cat and horse has normal rouleaux (horse more)
o Cat and cow have anisocytosis
How to do a WBC count?
o Clean slides
o Dilute –> fill up wills
o Count
o Do calculation
How to do a platelet count?
Platelet Count:
- Automated Analyser (EDTA)
- Blood smear (EDTA): Not accurate count as platelets are clumps. 100x objective and count 10 points. Normal 10 plt/hpf (100x). If there is less than 3 platelets: Spontaneous haemorrhage (IMT)
What are characteristic features of RBC in dogs?
- Greatest Central pallor
- Largest RBC of all species
- Some polychromatophils (immature RBCs)
What are characteristic features of RBC in cats?
- Mild rouleaux (stacking)
- Less central pallor compared to canines
- Anisocytosis (Variation in RBC size)
- Some polychromatophils (Immature RBCs)
- Platelet anisocytosis –> Giant platelets
- Frequent platelet clumping
What are characteristics of RBC in horses?
- Prominent rouleaux (stacking)
- Little to no central pallor
- RASPBERRY APPEARANCE OF EOSINOPHILS
What are characteristics of RBC in bovine?
- Little to no central pallor
- Anisocytosis (variation in RBC size)
- Small RBC compared to dogs + cats
- Lymphocytes predominante WBC type
- Lymphocytes vary in morphology
RBC characteristics in ovine?
- Very small
- Lymphocytes predominate WBC type
RBC characteristics in Goat?
- Smallest RBC of all
- Lymphocytes predominate
