(W1) Evaluation of Blood Smears

Question 1

Why do you need to examine blood smears (not just using haematology analysers)?

Answer 1

• Even the best haematology analysers can’t identify:
• Morphological changes of erythrocytes (e.g. Heinz bodies, schistocytes, etc.)
• Nucleated RBCs (some may flag possible presence but smear confirmation required)
• Band neutrophils (some may flag possible presence but smear confirmation required)
• Toxic changes of neutrophils
• Atypical/neoplastic cells (some may flag possible presence but smear confirmation required)
• Platelet clumps (some may flag possible presence but smear confirmation required)
• Infectious organisms

Question 2

What does examining a blood smear require and what should you do as a clinician if you don’t feel confident about your examination?

Answer 2

Properly examining a blood smear requires quiet time and knowledge, if you do not feel confident send the blood smear/s to a referral lab to be reviewed by a clinical pathologist

Question 3

When you send blood to the lab for haematology testing, what should you also provide and why?

Answer 3

include a fresh blood smear (make blood smear within 1 hour of blood collection)

• Over time during storage, cells accumulate morphological changes which distort their morphology and increase the degree of diagnostic inaccuracy

Question 4

What are the steps to prepare a blood smear?

Answer 4

1) Mix blood sample by inverting tube several times (invert 7-10 times so get good representation of cells in tube - when sample left standing, cells go to bottom and fluid remains at top)
2) Check for clots using applicator stick (move applicator stick inside the tube)
3) Place a small drop of blood near the frosted edge
4) At ~40⁰ angle pull spreader slide back to edge of drop
5) Wait for blood to spread across the edge of spreader
6) Push spreader slide smoothly, steadily and gently
7) Label your smear and let it air dry

Question 5

What kind of blood is best for morphological preservation of cells?

Answer 5

EDTA blood (EDTA prevent blood from clotting)

note blood smears are always made from anticoagulated blood (to prevent blood from clotting)

Question 6

What do you need to do to ensure that cells have good degree of cytological detail? Where is the best point on the slide to view blood cells?

Answer 6

spread the blood enough to form a monolayer

view cells near the feathered edge (but not at the feathered edge - spaces between the RBCs would be too large)

Question 7

How would the cell arrangement appear if the area on the slide had a layer that was too thin, and too thick?

Answer 7

Too thin - cells flattened and morphology is not well preserved
- this happens when you go to feathered tip (can get the impression that there are many spherocytes)

Too thick - cells on top of each other so don’t see cytological detail

Question 8

What is the difference between monocyte and basophil cytoplasm?

Answer 8

monocyte cytoplasm is blue but basophil cytoplasm is lavender coloured

Question 9

What is different about feline and equine eosinophilic granules?

Answer 9

feline - small granules

equine - large granules

Question 10

How does the appearance of activated platelets differ from normal platelets

Answer 10

activated platelets develop projections on their surface

Question 11

What is the normal appearance of a platelet?

Answer 11

• 1/3 size of RBC

- red/magenta granules in cytoplasm

Question 12

How does the visibility of platelets on a blood smear differ in cats?

Answer 12

platelets are less visible on blood smear (than in dogs)

Question 13

How can you differentiate between Rouleaux formations and auto-agglutination?

Answer 13

mix unstained EDTA blood and normal saline on a glass slide (to break up rouleaux formations)

Question 14

What are the common abnormalities in RBC shape (names only)

Answer 14

• echinocytes (aka burr cells)
• acanthocytes (aka spurr cells)
• schistocytes
• keratocytes
• target cells (aka codocytes)
• eccentrocytes
• heinz bodies
• howell-jolly bodies
• metarubricytes
• spherocytes

Question 15

What are echinocytes (aka burr cells) and what causes them?

Answer 15

- the projections of the cell
membrane may be sharp
or blunt
- usually numerous
- tend to be evenly spaced around the circumference 

Causes:
- prolonged storage of blood before film is produced

• excessive concentrations of EDTA
• pathological states

Question 16

What are acanthocytes (aka spurr cells) and what can cause them?

Answer 16

• Spherical cells with
  blunt-tipped or club-shaped spicules of different lengths projecting from their surface at irregular intervals
• i.e. projections unevenly spaced, sized and shaped

Causes:

• liver disease
• DIC
• haemangiosarcoma in dogs
• iron deficiency
• alterations of RBC membrane lipid composition
• others

Question 17

What are schistocytes and what can cause them?

Answer 17

• RBC fragments,
  generally taken to reflect
  mechanical injury to
  erythrocytes
• A wide variety of forms may be observed
• Usually seen in relatively
  low numbers
• associated with conditions causing roughening of endothelial lining e.g. DIC, haemangiosarcoma, PSS and vascular neoplasms
• also seen in iron deficiency due to increased fragility of RBCs

Question 18

What are keratocytes?

Answer 18

Erythrocytes with a blister-like vesicle, which may rupture, leaving a “bite-shaped” defect in the cell outline or one or two horn-like projections on the same
side of the cell

• low numbers found in health

Causes (DNTK):

• fragmentation (e.g. DIC)
• oxidative damage
• liver disease

Question 19

What are target cells (aka codocytes) and what are its causes? What species is it typically observed in and in what context are they typically seen in?

Answer 19

Target cells have a “lump” of haemoglobinised cytoplasm within the area of normal
central pallor, causing them to resemble a “bullseye” target

• reflect an increased SA:V ratio
• typically observed in dogs
• Young erythrocytes have excess membrane relative to mature cells; therefore,
  polychromatophilic target cells are often seen in the context of a regenerative
  anaemia

Causes:
- liver disease (associated with normocytic-normochromic target cells)

• hypothyroidism (related to o high systemic cholesterol concentrations that are seen in this disorder in dogs)

Question 20

What causes eccentrocytes?

Answer 20

• Form under conditions of
  excess oxidant stress to
  the erythrocytes (oxidative damage due to e.g. paracetamol)
• this induces cross-linking of membrane proteins
• distortion of Hb causes distortion of RBC shape

note often (but not always) seen in association with Heinz bodies

Question 21

What are Heinz bodies and what causes them?

Answer 21

seen as a (irregular round) projection from RBC
- Hb denatures and forms aggregates that appear as projections

Cause:
- Oxidation of Hb causes distortion of the Hb molecule

• The result is precipitation of haemoglobin, which may then coalesce to form intracellular inclusions called Heinz bodies (reduces
  RBC life span)
• Toxins that cause oxidative damage:
• copper (sheep)
• naphthalene (dogs)
• onions
• enzyme deficiencies
• drugs e.g. acetaminophen (aka paracetamol)

Question 22

In what species can non-pathologic ‘endogenous’ Heinz bodies be found?

Answer 22

found in healthy cats

Question 23

What is the diagnosis of an oxidant-induced haemolytic anaemia based on?

Answer 23

the finding of a regenerative anaemia with characteristic red blood cell morphologic abnormalities of Heinz bodies and eccentrocytes

Question 24

Are Heinz bodies easy to visualise on a blood smear, if not, what staining would be used

Answer 24

difficult to visualise

• could be visualised using routine staining but would be difficult
• use new-methylene blue (they will appear as dark blue dots near the surface of the cell)

Question 25

What are Howell-Jolly bodies and when are they seen?

Answer 25

Small fragments of non-functional nucleus which
was not extruded as the
erythrocyte left the bone
marrow

• may be seen in normal cats and horses s (they have non-sinusoidal spleens) as
  Howell-Jolly bodies are not
  removed as readily as in
  dogs or cattle
• seen in regenerative anaemia
• others

Question 26

What are metarubricytes and when are they seen?

Answer 26

nucleated erythrocytes (latest nucleated stage of RBC maturation process)

• most often noted peripheral blood in
  dogs, cats and camelids
  in the context of strongly regenerative anaemia
• seen less often in
  regenerative anaemia in
  cattle and rarely seen in
  horses
• lead poisoning, altered
  or abnormal splenic function, or bone marrow hypoxia or injury
• others

Question 27

What are spherocytes, how do they differ from normal RBCs and in what species are they difficult to identify in (why)?

Answer 27

• Erythrocytes which have assumed the form of a sphere rather than the normal discoid shape
• Smaller and more dense than normal RBCs of the species, and have a reduced area of central pallor
• Because cats, horses and cattle normally have RBCs with little central pallor, recognition of spherocytes is more difficult in these species than in dogs

Question 28

Apart form RBC abnormalities, what other blood smear findings are there?

Answer 28

Infectious organisms e.g.

• Babesia canis
• microfilaria
• mycoplasma haemofelis

Leukaemia:
- lymphoid leukaemia (would see neoplastic cells of lymphoid origin)

etc