Blood film analysis is an inexpensive but highly useful control for in-house haematology instrumentation and provides a quick evaluation of the peripheral blood. Components of complete blood count (CBC) include white blood cell count, packed cell volume (PCV), platelet count, total protein and review of the blood film.

Evaluation of Blood Film

 10x magnification (low power). Evaluate cellularity (>50 cells/low power field (LPF) is usually a high count, <20 cells/LPF is usually a low count); evaluate the feathered edge for platelet clumps, large cells, parasites.

 40x magnification. Requires good uniform film preparation, used to support manual or automated count:

 Raw leucocyte count/number of fields counted) x 2000 = estimated leucocyte count.

 Corrected count = (estimated count x (actual PCV/ normal PCV)):

 Example: 100 cells/10 fields:
10 x 2000 = 20.0 x 10⁹/l

 PCV 22%: corrected count
20.0 x 10⁹/l x 22/45 = 9.8 x 10⁹/l

 Anatomy of a blood film:

 Counting area--just in from the feathered edge, area to perform white blood cell differential and evaluate red cell (RBC) morphology.

 Body of film--RBCs touching or just overlapping. Area where platelet estimate is performed.

 Platelet estimate--average number/oil immersion field x 20,000 (>10 platelets/oil immersion field--normal platelet mass).

Evaluation of Erythrocytes

 RBC morphology: changes in shape (poikilocytosis), inclusions, polychromasia or no polychromasia, changes in size (anisocytosis), immature or nucleated red blood cells

 Poikilocytes: abnormal red blood cell shapes:

 Echinocytes (crenation)--most common artefact, can be seen with dehydration, toxins, RBC metabolic defects.

 Acanthocytes--altered lipid membrane. Seen with severe liver disease, haemangiosarcoma, renal disease.

 Eccentrocytes--oxidative injury, fused inner cell membrane, most commonly seen in dogs.

 Target cells (codocytes)--usually an artefact, usually seen in regenerative anaemia, may be associated with liver disease, endocrinopathies, iron deficiency.

 Schistocytes--hallmark of RBC fragmentation, shearing of RBC by fibrin (microangiopathy), turbulent blood flow, caval syndrome, valvular stenosis, intrinsic RBC abnormalities (severe iron deficiency, chronic doxorubicin toxicosis).

 Spherocytes--hallmark of immune-mediated anaemia (large numbers), also seen with RBC fragmentation (small numbers), hypophosphataemia, toxins (zinc).

 Howell-Jolly Bodies--micronuclei (nuclear remnants), seen with regenerative anaemia, post splenectomy, marrow injury.

 Heinz bodies--oxidative injury, most common in cats, associated with toxicity (acetaminophen, onions, propylene glycol), metabolic disease (diabetes mellitus).

 Basophilic stippling--punctate aggregates of RNA seen with Wright-Giemsa stain, regenerative anaemia, lead poisoning.

Blood Film Evaluation in the Anaemic Patient

Evidence of Regeneration From CBC

 Macrocytosis (increased mean cell volume (MCV)) is commonly seen with regeneration. Young 'stress' reticulocytes are often larger than normal RBCs.

 Presence of polychromasia. Polychromasia does not always correlate with reticulocyte count--not all reticulocytes appear polychromatophilic.

 Reticulocyte counts--necessary to evaluation of all anaemic patients:

 Dog--aggregate reticulocytes only

 Cat--both aggregate and punctate reticulocytes. Aggregate indicates recent regeneration. Punctate indicates regeneration sometime in the past.

 Metarubricytosis (normoblastosis) can be part of the regenerative process. Must also see polychromasia! Can also be seen with alterations in splenic function, sudden hypoxia, bone fractures. Inappropriate metarubricytosis (not associated with regeneration): lead toxicity, myeloproliferative disease (erythemic myelosis in the cat), extramedullary haemopoiesis, sepsis, neoplasia.

Evidence of Chronic Blood Loss (Iron Deficiency)

 Hypochromasia: visual assessment and measured (mean corpuscular haemoglobin concentration):

 Iron deficiency--true decrease in haemoglobin

 Marked regeneration--large polychromatophilic cells have less haemoglobin/volume

 Increased MCHC is always an artefact--Heinz bodies, lipaemia, paraproteinaemia.

Evidence of Intravascular Haemolysis

Intravascular haemolysis is relatively rare: haemoglobinaemia results in artefactual increase in MCHC, may see 'ghost cells', should see haemoglobinuria. It may be difficult to differentiate from increased fragility where cells lyse in vitro. No haemoglobinuria is seen with in vitro lysis.

Evidence of Extravascular Haemolysis

Most common form of immune injury, not associated with haemoglobinaemia or haemoglobinuria. See spherocytes in large numbers.

Evaluation of Leucocytes

 Significant variation in normal differentials: cow, sheep, goats, pigs--lymphocytes predominate; dog, cat, horse--neutrophils predominate:

 Band neutrophils are the hallmark of inflammation. Variation in band neutrophil morphology. Dog neutrophils are not as segmented as those of horse or human. Bands are often over-estimated on differential by human reference laboratories.

 Neutrophil toxicity is the hallmark of sepsis. Dog--basophilic foamy cytoplasm and Döhle bodies; cat--basophilic foamy cytoplasm. (Döhle bodies are commonly seen in cat neutrophils with no evidence of toxicity.)

 Regenerative versus degenerative left shift:

 Regenerative--leucocytosis with greater number of mature neutrophils compared to bands.

 Degenerative--greater number of bands than mature neutrophils regardless of total count or normal or low count with significant numbers of bands.

Significant Haemoparasites

 Mycoplasma haemocanis, Mycoplasma haemofelis

 Babesia canis

 Dirofilaria immitis

 Ehrlichia canis

Viral Disease

 Canine distemper virus: inclusions in RBCs and WBCs

References

1.  Allison RW, Meinkoth JH. Hematology without the numbers: in-clinic blood film evaluation. Veterinary Clinics of North America, Small Animal Practice 2007; 37(2): 245-266.

2.  Reagan WJ, Sanders TG, Denicola DB. Veterinary Hematology: Atlas of Common Domestic Species. Ames, IA: Blackwell Publishing Professional, 1998.

3.  Thrall MA, Rebar A, et al. Veterinary hematology and clinical chemistry: text and clinical case presentations set. Ames, IA: Blackwell Publishing, 2004.