Blood Laboratory

Blood cell indices > Differential white cell count > Practice When the blood smear has dried
and been stained with Diffquick, the slide is placed on a microscope and scanned at low power to find a good distribution of cells. A drop of oil is placed on the slide and the cells are examined with the oil immersion objective. The percentage of each type of white blood cell is determined. Preparation of the slide: (procedure not done during the laboratory session; however, pre-stained slides will be available for leukocytes differential count)

1) A fresh (non-heparinized) sample of blood is added to one side of the slide

2) The edge of another slide is pushed against the drop of blood and smeared onto the rest of the slide (see 3 and 4 below).

3)

4)

The smeared slide is allowed to dry. The staining procedure:

Diff-Quik Stain set is a modification of the Wright Stain technique: Blood smears are fixed using the methanolic fixative solution in order to stabilize cellular components. Solutions I and II are then applied individually to the fixed smear to differentially stain specific cellular components.

The dried slide is dipped several times in the Fixative solution. The excess is allowed to drain.

Then this slide is dipped several times in Solution I, which is a buffered solution of Xanthene dye (an anionic dye). The dye stains the granules in the cytoplasm, a bright orange colour. The same slide is dipped several times in Solution II, which is a buffered solution of thiazine dyes (cationic dyes) consisting of methylene blue and Azure A. The resultant basophilic staining of nucleoli and cytoplasm is due to the methylene blue component of the mixture. The anionic component of the nucleoli and cytoplasm is stained with the cationic methylene blue.

The slide is rinsed with distilled water and allowed to dry. Leukocytes:

Granular Polymorphonuclear neutrophils nucleus: dark blue cytoplasm: pale pink granules: reddish lilac Eosinophils nucleus: blue cytoplasm: blue granules: red-orange Basophils nucleus: purple or dark blue granules: dark purple, almost black Non-granular monocytes nucleus (lobated): violet cytoplasm: sky blue Lymphocytes nucleus: violet cytoplasm: dark blue

The slide is examined under oil immersion.

Expected Ranges Neutrophil (%) Eosinophil (%) Basophil (%) Monocyte (%) 50-70 1-4 0.1 2-8

Lymphocyte (%) 20-40 Click here to open a window which mimics what you might see looking through the 100x objective of the microscope using oil immersion. Try to determine the differential white cell count.