tap water is not neutral, wash the slide with distilled water. The stain and the metallic film must be floated off to prevent streaking, so keep the slide flat and horizontal in the stream of water. If the slide is tilted, the metallic film will settle to the surface of the smear and remain there.
4. Wipe the stain from the bottom of the slide. Let it air-dry. A good smear should be thin and evenly distributed, and it must be dry before staining. Always make at least two smears for each patient, as the additional smear should be examined to verify any abnormal findings.
Staining times will vary with different batches of Wrights stain. One batch may give best results with 3 minutes of staining and 2 minutes of buffering, another may give better results with 2 minutes of staining and 3 minutes of buffering. The only way to determine the best time interval for a particular stain is by experimentation.
Next to incorrect time intervals, the most common cause of poor results with Wrights stain is incorrect pH of the staining fluid. If the stain is too acid, the red cells in the smear will stain a bright pink or may even be colorless, while an alkaline stain will cause the red cells to appear blue-gray, with poor color definition. In either case, the pH of the buffering solution should be checked.
1. Place the stained slide on the microscope with a drop of immersion oil and adjust the ocular lenses.
2. Using the oil immersion lens (red) (highest power), scan the fields for areas where the red cells just touch. In this area the blood smear is thinner, and consequently, the white cells are easier to identify.
3. Count 100 consecutive white cells (see figure 6-10 for identification), pressing the correct key on the cell counter for each type of white cell identified.
4. If you count 20 lymphocytes among the 100 cells, then the patient has 20 percent lymphocytes. If an absolute count has been requested, the total leukocyte count is multiplied by individual percentages.
Example: Patient has a white count of 8,000.
20 percent lymphocytes.
20 percent x 8,000 = 1,600
The patient has 1,600 lymphocytes per cubic millimeter of blood.
The ability to identify the different types of white cells is not difficult to develop, but it does require a thorough knowledge of staining characteristics and morphology that can be gained only by extensive, supervised practice. The beginner is likely to encounter some difficulty in learning to differentiate between monocytes and large lymphocytes, or between monocytes and atypical (not typical) lymphocytes. It is possible also to confuse eosinophils with basophils, since an alkaline stain may cause the orange granules in an eosinophil to appear deep blue or purple. If this happens, the reddish cast in the granules can often be detected by judicious use of the minor focusing knob. Also, the granulations of an eosinophil are generally much finer than those of a basophil, and they tend to concentrate in the cytoplasm.
The neutrophils are subclassified according to age, and the age is indicated by the nucleus. If there is any doubt as to the identity of the neutrophil, always classify it with the older stage. For instance, if a single irregularity in a horseshoeshaped nucleus appears as a break or a concentration of color, classify it as a segmented neutrophil, not as a neutrophilic band. Practice is the key. And remembereven experienced technicians often disagree as to the identity of a particular cell.
If it is desirable to save a smear for reexamination, remove the immersion oil by placing a piece of lens tissue over the slide and moistening the tissue with xylol. Draw the damp tissue across the slide and dry the smear with another piece of lens paper.
A good smear (thin and evenly distributed) is essential for accurate identification and counting.
The cytoplasm of an eosinophil contains numerous coarse, reddish-orange granules, which are lighter colored than the nucleus.
Scattered large, dark-blue granules, which are darker than the nucleus, characterize the cell as a basophil. Granules may overlay the nucleus as well as the cytoplasm.