9 LABORATORY EVALUATION OF LEUKOCYTES
1 What laboratory test results are included in the leukogram?
The leukogram consists of the laboratory tests used in the routine evaluation of leukocytes on the complete blood count (CBC). The total leukocyte concentration is measured by a cell-counting instrument. The next step is to determine the relative percentage of each leukocyte type. The stained blood smear is examined with a microscope using high-power (50× or 100×) oil-objective lenses. A tally is recorded of the leukocyte types observed in the thin, monolayer portion of smear. The percentage of each leukocyte type is multiplied by the total leukocyte count to obtain their absolute concentration. The absolute concentration of each leukocyte type (not their percentages) is compared to appropriate reference intervals for that animal to detect any increases or decreases. In addition, microscopic changes in the morphology of leukocytes are noted on the stained blood smear, typically using the 100× oil-objective lens.
2 How is the total leukocyte concentration measured?
In the manual procedure, leukocytes in an aliquot prepared with a dilution kit (e.g., Unopette) are counted on a hemacytometer using a microscope. This method is very time consuming and is relatively inaccurate, with up to a 20% margin of error. Automated cell counters, which make precise dilutions and count the leukocytes in a larger volume of blood, provide more accurate results (<5% error). Impedance cell counters detect the voltage changes caused by the nonconducting leukocytes as they pass through an electrical field. Newer cell counters use flow cytometric methods to channel leukocytes through the path of a laser light beam. Laser optical detectors obtain both size and light scatter information (“interrogation”) about each leukocyte to differentiate it from other types of leukocytes and blood cells.
3 How are leukocytes counted in avian and nonmammalian blood?
Automated cell counters are unable to differentiate the nucleated erythrocytes and thrombocytes from leukocytes in avian and nonmammalian species. The leukocyte count can be calculated using the leukocyte differential from the stained blood smear and a hemacytometer count of the granulocytic leukocytes. The stain most often used to obtain the granulocyte count is phloxine B, which is marketed as a blood dilution kit for counting human eosinophils. In birds and reptiles, phloxine B stains the heterophils and eosinophils, not the lymphocytes and monocytes.
The formula for calculating the total leukocyte count (cells/μl) follows:
With number of (#) stained granulocytes obtained from hemacytometer count (sum of both chambers) and percentages (%) obtained from stained smear.
4 How many leukocytes should be counted in the manual differential count?
When the total leukocyte count is within the reference interval, a percentage distribution based on the differential counting of 100 leukocytes is typically performed. Although counting more cells could produce greater accuracy, other sources of error are present (e.g., uneven distribution of different leukocytes across the blood smear). When significant leukocytosis is present, more leukocytes must be counted to accurately reflect leukocytes present in low numbers. One rule of thumb is to count 100 leukocytes for each 20,000 cells/microliter (μl) increment of the total leukocyte count. For example, when the total leukocyte count is 40,000/μl, a 200-cell differential would be performed. If there is significant leukopenia, however, it can be difficult to find 100 leukocytes on the smear. Therefore a percentage distribution of leukocytes may be obtained from a differential count of 50 or even 25 leukocytes.
5 Can automated hematology analyzers provide differential leukocyte concentrations?
Some hematology analyzers (e.g., Vet ABC, IDEXX QBC) produce a three-part leukocyte differential (neutrophils, eosinophils, and combined lymphocyte/monocytes). The newer flow cytometric analyzers (e.g., Bayer Advia, Abbott Cell-Dyne, IDEXX LaserCyte) produce a five-part differential leukocyte counts. These instruments perform well with leukocytes from healthy animals. However, differential counts during abnormal conditions such as left shifts and leukemias are inaccurate. Therefore, despite its own limitations, microscopic examination of the stained blood smear currently remains the “gold standard” for obtaining the differential leukocyte concentrations in animals.
6 Which hematologic stains are used to evaluate leukocyte morphology?
The classic hematologic stains are Romanowsky stains, such as Wright’s and Giemsa, which have eosinophilic and basophilic dyes dissolved in alcohol. Although they produce optimal staining of blood cells, these dye solutions are unstable, and the staining procedure requires several minutes. Modified Romanowsky stains (e.g., Diff-Quik) that use water-soluble dyes were developed. These dyes are more stable and rapidly stain the cells on blood smear (<1 minute). The only shortfall of the quick stains is failure to consistently stain the metachromatic granules in basophils (and mast cells) and the azurophilic granules in granular lymphocytes.
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