The Blood Film


6
The Blood Film


Sharon M. Dial


6.1 Procedural Definition: What Is This Test About?


The blood film is a hematological preparation in which whole blood is spread on a glass slide and stained with a Romanowsky stain to provide visual evaluation of peripheral blood cellular element morphology, number, and distribution. Historically, the blood film was an essential part of the complete blood count (CBC) and provided a differential count of the leukocytes from which absolute numbers were calculated as well as providing the opportunity for the evaluation of erythrocyte, leukocyte, and platelet morphology. With the increased availability of in‐house hematology instrumentation, blood film evaluation has, regrettably, been underutilized in veterinary clinics.


6.2 Procedural Purpose: Why Should I Perform This Test?


Blood film review is now recommended for all blood samples submitted for an automated CBC by the American Society of Veterinary Clinical Pathologists (ASVCP) [1]. The blood film is considered a nonstatistical quality assurance procedure and provides an inexpensive and readily available internal quality control for the values reported using in‐house hematology instruments. It also provides information not found in the automated CBC, which is essential for the evaluation of an ill patient. Identification of significant erythrocyte and leukocyte abnormal morphology or circulating hemoparasites and atypical cells cannot be done using in‐house automated instruments.


While instruments can suggest the presence of a shift to immaturity in neutrophils as a component of an inflammatory leukogram, evaluation of the blood film is necessary to confirm the shift and to evaluate for changes in the neutrophils that assist in determining the degree of inflammation. While it is tempting to only evaluate a blood film when there are significant changes in erythrocyte, leukocyte, and platelet numbers, significant changes in the character of these cell populations can be present when numbers are within the reference limits.


Preparation of a blood film from a freshly collected blood sample will allow confirmation of abnormalities, such as thrombocytopenia, in samples submitted to a reference laboratory. Transport from the clinic to the laboratory and the delay in performing the automated CBC can introduce artifactual changes in morphology and cell number that can be either confirmed or refuted by a review of the blood film in the clinic. In addition, a quick review of the blood film at the clinic can provide a preview of the results that will be reported by the laboratory and allow the development of an initial treatment plan to be refined by the fully automated CBC once received. The blood film, when used in conjunction with the history and physical exam findings, will provide excellent, in the moment, information for the development of a differential diagnosis and the expedient choice of additional tests necessary for the refinement of the differential diagnosis.


6.3 Equipment



  • whole blood (preferably in EDTA)
  • glass slides
  • microhematocrit tubes
  • wooden applicator sticks
  • quick Romanowsky stain
  • distilled water
  • gloves

6.4 Procedural Steps: Preparing the Blood Film How Do I Perform This Test?


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)
    Photo depicts equipment necessary for preparing a blood film include whole blood in anticoagulant, glass slides, microhematocrit tubes (or wooden applicator sticks), quick Romanowsky stain set, distilled water, and gloves.

    Figure 6.1 Equipment necessary for preparing a blood film include whole blood in anticoagulant, glass slides, microhematocrit tubes (or wooden applicator sticks), quick Romanowsky stain set, distilled water, and gloves.


    Source: Courtesy of Jeremy Bessett.

    Photo depicts (a) a microhematocrit tube is used to place a small drop in the glass slide; (b) two wooden applicator sticks are used to place the blood on the slide.

    Figure 6.2 (a) A microhematocrit tube is used to place a small drop in the glass slide; (b) two wooden applicator sticks are used to place the blood on the slide.


    Source: Courtesy of Jeremy Bessett.

    Photo depicts the slide used to spread the blood film should be held at a 45° angle and backed into the blood film.

    Figure 6.3 The slide used to spread the blood film should be held at a 45° angle and backed into the blood film.


    Source: Courtesy of Jeremy Bessett.

    Photo depicts the angle of the spreader slide is greater than 45° in this image to illustrate the angle used with blood that has a low PCV.

    Figure 6.4 The angle of the spreader slide is greater than 45° in this image to illustrate the angle used with blood that has a low PCV.


    Courtesy of Jeremy Bessett.


    Source: Courtesy of Jeremy Bessett.


  • if the PCV is below the reference interval, the angle between the two slides should be greater than 45° (see Figure 6.4)
    Photo depicts the angle of the spreader slide is smaller than 45° in this image to illustrate the angle used with blood that has a high PCV.

    Figure 6.5 The angle of the spreader slide is smaller than 45° in this image to illustrate the angle used with blood that has a high PCV.


    Source: Courtesy of Jeremy Bessett.


  • 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
    Photos depict (a) allow the blood to spread out to the edge of the spreader slide; (b) rapidly push the spreader slide in a continuous motion to the end of the slide.

    Figure 6.6 (a) Allow the blood to spread out to the edge of the spreader slide; (b) rapidly push the spreader slide in a continuous motion to the end of the slide.


    Source: Courtesy of Jeremy Bessett.

    Photo depicts an example of a well-made blood film with a good-feathered edge and monolayer area.

    Figure 6.7 This is an example of a well‐made blood film with a good‐feathered edge and monolayer area.


    Source: Courtesy of Jeremy Bessett.


  • 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).
    Photo depicts the blood film shows no streaks, breaks, or droplets and has an identifiable feathered edge and monolayer.

    Figure 6.8 The blood film shows no streaks, breaks, or droplets and has an identifiable feathered edge and monolayer.


    Source: Courtesy of Jeremy Bessett.

    Photo depicts the blood film in this image is from a sample with a plasma total protein of 10.5 g/dl.

    Figure 6.9 The blood film in this image is from a sample with a plasma total protein of 10.5 g/dl.


    Source: Courtesy of Jeremy Bessett.



    • 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).
    Photo depicts the side on the left is from a sample from a dog with a PCV in the reference interval.

    Figure 6.10 The side on the left is from a sample from a dog with a PCV in the reference interval. The slide on the right is from a dog with immune mediate hemolytic anemia and marked erythrocyte agglutination. Note the red blood cells are concentrated at the feathered edge.


    Source: Courtesy of Jeremy Bessett.


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.

Photo depicts the blood film in this case was made with too much downward pressure and too slow, resulting in concentration of the leukocytes at the feathered edge.

Figure 6.11 The blood film in this case was made with too much downward pressure and too slow, resulting in concentration of the leukocytes at the feathered edge.


Source: Courtesy of Jeremy Bessett.

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May 3, 2023 | Posted by in SMALL ANIMAL | Comments Off on The Blood Film

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