6.4.1 Procedure

• gather all supplies needed (see Figure 6.1)
  
• label slides with patient name and date
  
• gently mix the blood sample by either inverting it several times (five to eight inversions) or placing it on a blood tube rocker for five to eight rotations as described in Chapter 3.
  
• using either a micropipette, blood‐filled hematocrit tube, or two wooden applicator sticks, place a small drop of blood (approximately 3 mm in diameter) on one end of a glass slide (see Figure 6.2a and b)
  
• hold the glass slide with the blood drop firmly in place, use a second glass slide (the “spreader” slide) to back up into the drop of blood, keeping the drop of blood behind the spreader slide in an acute angle of about 45° if the PCV is within reference interval (see Figure 6.3)

  

  

  

  

  
  
• if the PCV is below the reference interval, the angle between the two slides should be greater than 45° (see Figure 6.4)

  

  
  
• if the PCV is above the reference interval, the angle between the two slides should be less than 45° (see Figure 6.5)
  
• once the spreader slide has reached the drop of blood and the blood has spread out along the entire width of the spreader slide, quickly advance the spreader slide using light downward pressure and a quick fluid forward motion to the end of the horizontal slide (see Figure 6.6a and b)
  
• this procedure should produce a blood film that covers about 2/3 of the horizontal glass slide with a body, monolayer, and feathered edge (see Figure 6.7).

Staining the blood film: see Chapter 5, Buffy Coat.

6.4.2 Evaluating the Blood Film

Appropriate review of the blood film requires a well‐defined protocol to ensure that the maximal information is obtained. Establishing a standard operating procedure for blood film review will result in consistent results regardless of who is performing the evaluation. The systematic review of the blood film should include:

• a gross evaluation of the blood film to note the quality of the blood film and to identify any abnormal staining like a blue tinge suggestive of high total protein or a “reverse” feathered edge suggesting significant erythrocyte agglutination

  

  

  
  
• using the 10× objective:
  
  
  
  • review of the feathered edge for platelet clumping, hemoparasites, and large, atypical cells
    
  • evaluate the density and distribution of leukocytes (low, appropriate, high); noting if the leukocytes are inappropriately concentrated at the feathered edge
  
  
• using the 40× objective:
  
  
  
  • estimate the white blood cell count; perform differential on samples with leukopenia
  
  
• using the 100× oil immersion objective:
  
  
  
  • evaluate platelet morphology and estimate platelet numbers
    
  • evaluate erythrocyte morphology with an estimate of frequency of each abnormality
  
  
• evaluate leukocyte morphology and perform a leukocyte differential count

6.4.3 Gross Examination of the Blood Film

• A properly prepared blood film should have a body, monolayer counting area, and feathered edge (see Figure 6.8). There should be no streaks, breaks, or clear droplets in the film.
  
• Assess the staining quality and density of the blood film. The blood film should not be too orange or too blue. If the staining quality is poor and the staining protocol has been followed, the stains should be replaced and a new slide stained.
  
  
  
  • Occasionally, increased blue staining of the blood film is seen with significantly high total protein. This can also be seen with altered plasma protein composition with hypergammaglobulinemia, resulting in a prominent blue hue to the stained slide (see Figure 6.9).
  
  
• If there is significant erythrocyte agglutination in the sample, the large aggregates of erythrocytes are carried to the feathered edge rather than being evenly distributed throughout the blood film, resulting in a “reverse” feathered edge (see Figure 6.10).

  

  

  
  
  
  
  • Slides with a “reverse” feathered edge are difficult to evaluate. The differential cell count and any estimate of cell numbers should be interpreted with care or not done. The large erythrocyte aggregates will also result in significant artifactual changes in the red cell count and indices on the automated CBC.
  
  
• If too much pressure is applied to the push slide or the blood film is made too slowly, the leukocyte distribution will be altered. A large number of leukocytes will be carried to the feathered edge altering the leukocyte distribution and invalidating an estimate of the WBC count from the blood film (see Figure 6.11).

  

6.4.4 Microscopic Examination of the Blood Film

The blood film is initially reviewed using the 10x microscope objective . This degree of magnification allows an overall view of the density of leukocytes, a more precise assessment of the quality of the blood film, and evaluation of the feathered edge. After reviewing the slide with the 10x objective, most microscopists will move directly to the 100x oil emersion objective to evaluate platelet numbers and erythrocyte and leukocyte morphology, and perform a differential cell count. In doing so, the 40x objective is often overlooked as a tool in evaluation of the blood film, primarily because the use of this objective requires a coverslip to be placed on the microscope slide. Most practices do not coverslip their blood films, making it difficult to use the 40x objective, since it cannot be adequately focused without one. To utilize this objective, a temporary coverslip, using immersion oil as the mounting media, can be placed on the slide. The 40x objective allows the microscopist to estimate white blood cell count and perform a differential cell count quickly, especially in patients with leukopenia. Following a review of the blood film at 40x, a closer evaluation of erythrocyte and leukocyte morphology and platelet estimate can be done using the 100x oil emersion objective.

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