PREPARATION OF BLOOD SMEAR

PREPARATION OF BLOOD SMEAR

Blood smear is a valuable diagnostic tool for differential count of white blood cells and evaluation of pathologic abnormalities in leukocytes (toxic neutrophils, left shift, blast cells), erythrocytes (polychromasia, anisocytosis, inclusions, irregular shape, parasites) and platelets (macroplatelets, platelet clumps). But to get the relevant information from it, the preparation of the smear requires a proper technique, which creates a monolayer of dispersed cells and a cell distribution that reflects the cell concentration in the blood. Inadequately prepared smear can present different artifacts and lead to errors in the differential count.

Blood films should be made immediately after collection of the blood, because cell morphology deteriorates rapidly after sample collection. EDTA sample or fresh blood immediately from the collection needle, before the contact with anticoagulant can be used. Samples with heparin are unusable for preparation of the smears.

The most common technique of blood smear preparation is called the “wedge or push” technique. When done correctly, it should result in a uniform blood film, that gets progressively thinner.

  • A small drop of blood is placed on the midline at the end of a glass slide.
  • Second slide (ideally narrower than smear slide, to avoid spreading the cells over the edge) is placed on the smear slide in front of the blood spot in a way that it forms a 30-45°angle on the side of the blood drop.
  • Second slide is pulled back into the blood drop, so that the blood spreads along its edge, and then pushed in other direction to the end of the smear slide. The last push should be done with rapid single movement and minimal downward pressure.
  • Ideally, the smear should be approximately 2/3 of the length of the slide.

Advices:

  • Pushing the slide too slow will generate too thin film, which results in poor distribution of leukocytes and artifacts in erythrocytes. This is the most common problem in technique among veterinarians.
  • In case of reduced viscosity of the blood (severe anaemia), increase the angle to avoid too thin slide.
  • Ensure that slides are clean and free of grease in order to avoid streaks and holes in the smear.

Once the blood smear is made and stained, we must know, how to properly examine it. First, scan the whole smear at low power. The largest part (body) of film is unusable for examination, because the cells lie one over another and their evaluation is therefore compromised. The end of smear is called a feathered edge and can be recognized by loss of erythrocyte central pallor and a reticular pattern of erythrocyte distribution. This is the part, where large particles are gathered, so it is useful to quickly check it, to detect some abnormalities like microfilaria, platelet clumps and unusual large cells. The counting area is actually only a small portion of the smear between body and feathered edge, that consists of a monolayer of evenly dispersed cells which is optimal for microscopy. Focus on this area and examine it under high power, using oil immersion. Examining the wrong part of the smear can lead to false interpretation.

It is important to know the artifacts and distinguish them from morphologic abnormalities. Inadequately prepared smears with streaks or lacking the feathered edge and monolayer of cells are not appropriate for evaluation of erythrocyte morphology and performing a differential count.

 

 

LITERATURE:                                                                                             

Thrall M. A., Weiser G., Allison R. W., Campbell T. W.: Veterinary Hematology and Clinical Chemistry. 2nd Ed. Wiley-Blackwell, 2012

Bexfield N., Lee K.: BSAVA Guide to Procedures in Small Animal Practice. 2nd Ed. BSAVA, 2014