Evaluation of the Blood Smear
World Small Animal Veterinary Association World Congress Proceedings, 2004
Mary M. Christopher, DVM, PhD, DACVP, DECVCP
University of California Davis, Department of Pathology, Microbiology, and Immunology School of Veterinary Medicine
Davis, CA, USA

Despite the advanced technologies now available for the evaluation of peripheral blood, the blood smear remains an important part of the hematologic evaluation of dogs and cats. Some hematologic abnormalities are found only on a blood smear. These include left shifts, red cell morphology, and the characterization of atypical cells. Thus, to be complete, a CBC must include evaluation of a blood smear. In this presentation, I will review the preparation and staining of blood smears, the components involved in the microscopic evaluation of blood, and provide examples of important abnormal findings.

Blood Smear Preparation

Blood smear preparation is a technical skill that requires practice. Smears may be made using coverslip or push-smear slide techniques. Coverslip smears result in more equitable distribution of leukocytes. Push-smear techniques result in a more consistent "feather-edge" and a better monolayer (red cell area) for examination. With either technique, always use clean new slides and coverslips. Smears should be dried quickly to prevent RBC artifacts. Smears should be identified by writing with pencil on the frosted or label end of the microscope slide. Label the smear with the date and the animal's identification name or number. Allow smears to "set" for a few minutes after preparation; then stain within 2 hours.

If you are mailing smears to a laboratory, check with the lab for instructions on fixing slides. Usually, air-drying alone is sufficient fixation. A lag of more than 48 hours before staining can result in poor staining and bluish RBCs. Any Romanowsky-type stain is acceptable. Romanowsky stains are composed of methylene blue, polychromes (azures) and eosin. Staining requires fixation, buffering, and staining. Acidic components of cells stain deep blue to purple (DNA, RNA, some granules). Basic components of cells stain pink (hemoglobin, eosinophil granules). "Azurophilic" components stain pink-purple (monocyte, platelet, and myeloid precursor granules). And "neutrophilic" components stain with both acidic and basic portions of the dye (neutrophil granules). Rapid stains, such as Diff-Quikμ provide reasonably good staining in a short time.

Diff-QuikTM Staining Technique

Fixative

About 5 minutes

Solution I

8-10 dips (1 second each)

Solution II

6-8 dips (1 second each)

Distilled water

Rinse till clean

Touch off excess liquid at the container edge to reduce carryover from one solution to another. Dry the smear by standing the slide at a steep angle; do not blot! Cover solutions to prevent evaporation. Smears that are inadequately stained can be restained

Artifacts may result from improper handling and staining of smears. Avoid contact of smears and stains with water! Exposure of smears to formalin vapors causes a bluish background, making it more difficult to differentiate cells. Don't store formalin in the same area where staining is done. Also, don't package surgical pathology specimens with blood smears. Never store smears in a refrigerator or freezer, since water condensation, RBC lysis, and hemoglobin crystallization may occur. Always be sure a slide is completely dried before placing it in a sealed container.

New methylene blue is another stain useful for hematologic examination. It is an aqueous rather than alcohol-based stain (it does not fix cells and therefore it is not a permanent stain). New methylene blue can be used as a supravital stain to examine reticulocytes, or may be applied to dried smears. New methylene blue also is a handy stain for urine sediment and cytologic preparations.

Microscopic Evaluation of the Blood Smear

A systematic procedure should be used to examine a blood smear so that all elements are examined and can be recorded. When quantitative CBC information is available, the qualitative findings on the blood smear should correlate with the quantitative values obtained by automated analysis. Because errors can occur in automated analysis, examination of the smear is an important quality control tool for confirming quantitative values.

1.  Observe the smear grossly to check smear orientation and the location of the feather edge.

2.  4X objective

a.  Scan the smear for microfilaria.

b.  Locate the feature edge and counting area or red cell area of the smear, where red cells are adjacent but not overlapping. All subsequent observations should be made in and around this area.

3.  10X objective

a.  Evaluate RBC density (normal, low, high) and check that it matches with the patient's PCV.

b.  Evaluate RBC distribution. Move from the red cell area towards the thicker area of the smear. Rouleaux refers to RBCs that line up singly, one on top of the other, like a stack of coins. Rouleaux usually is the result of increased plasma protein concentration, specifically fibrinogen and immunoglobulins. Rouleaux also may form as an artifact caused by slow drying of blood smears. Agglutination refers to irregular, variably-sized clumps of RBCs caused by "stickiness" and crosslinking of attached antibodies. Agglutination is indicative of bound RBC antibodies, as occur in immune-mediated hemolytic anemia. Agglutination may be distinguished from rouleaux by diluting the blood 1:10 with saline; rouleaux is dispersed by the dilution but agglutination is not. Agglutination can falsely decrease the PCV and increase the MCV.

c.  Evaluate whether platelet clumps are present at the feature edge. Platelet clumping is especially common in cats and can result in falsely low platelet counts.

d.  Estimate the leukocyte count by determining an average number per field and multiplying by 100 (note: if the WBC count is high, the estimate is more accurate at 40X objective--see below). For accurate estimates WBCs must be evenly distributed over the smear; check the feather edge to be sure WBCs are not disproportionately located at the edge (a common occurrence).

e.  Scan for atypically large or deep blue cells. Examine these more closely at higher magnification.

4.  40X objective

a.  Place a drop of oil and then a coverslip over the smear to improve resolution at 40X (another drop of oil can then be placed on top of the coverslip to use the 100X objective).

b.  Estimate the leukocyte count: average number of WBCs per field X 1600. Check to see that the estimate matches with the quantitative WBC count.

c.  Perform a differential leukocyte count by counting 200 cells. A 200-cell differential count is more accurate than a 100-cell differential. Check to see that the manual differential count matches with the automated differential count. Note any nucleated RBCs separately and correct the total WBC count using the following formula: corrected WBC = (100+NRBC+100) X total WBC. Multiple the percentages obtained in the differential count by the total WBC count to obtain absolute counts. Absolute counts are necessary for interpretation.

5.  100X objective

a. Evaluate WBC morphology--Toxicity, inclusions, organisms

Quantitation of Neutrophil Toxicity

"Slight" or 1+

Döhle bodies (may be normal in cats).

"Moderate" or 2+

Döhle bodies, cytoplasmic basophilia or foaminess.

"Marked" or 3+

Severe Döhle bodies, cytoplasmic basophilia and foaminess;

or 4+

may be difficult to distinguish from monocytes

b. Evaluate RBC morphology--Anisocytosis, polychromasia/hypochromasia, poikilocytosis, inclusions, organisms. The types of poikilocytes should be noted.

Quantitation of RBC Morphologic Abnormalities

"Slight" or 1+

An abnormal feature that is slightly but significantly
increased in number. It is present in small amounts in each field.

"Moderate" or 2+

An abnormal feature that is moderately increased.
It is consistently present in noticeable amounts in each field.

"Marked" or 3+

or 4+

An abnormal feature that is markedly increased.
It is
unmistakable and outstandingly present in each field

c. Estimate platelet number--The average number of platelets per field X 15,000 = estimated platelets/μl. Check to see that the platelet estimate matches the quantitative platelet count. In cats, platelet clumping may interfere with quantitation by many hematology analyzers. Platelet morphology also should be evaluated, including the presence of macroplatelets or atypical shapes.

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Mary M. Christopher, DVM, PhD, DACVP, DECVCP
University of California-Davis, School of Veterinary Medicine
Davis, CA


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