Unopette Procedure The Unopette disposable diluting pipette system used to count WBCs is almost identical in shape and application to the Unopette system for RBC counts. The only major difference is that the reservoir contains a different diluent and the capillary pipette capacity differs (RBC 10 Fl and WBC 20 Fl). To assist you in performing the Unopette procedure for WBCs, we will refer to illustrations for the Unopette procedure for RBCs in this section. The Unopette procedure for counting white blood cells is as follows: 1. Puncture the diaphragm in the neck of the reservoir with the tip of the capillary pipette shield. See figure 7-9. 2. After you obtain free-flowing blood from a lancet puncture of the finger, remove the protective plastic shield from the capillary pipette. Hold the capillary pipette slightly above the horizontal and touch the tip to the blood source (fig. 7-10, view A). The pipette will fill by capillary action. When blood reaches the end of the capillary bore in the neck of the pipette, filling is complete and will stop automatically. The amount of blood collected by the capillary tube is 20 Fl. Wipe any blood off the outside of the capillary tube, making sure no blood is removed from inside the capillary pipette. (An alternative source of blood is a thoroughly mixed fresh venous blood sample obtained by venipuncture. See figure 7-10, view B.) 3. With one hand, gently squeeze the reservoir to force some air out, but do not expel any diluent (fig. 7-11). Maintain pressure on the reservoir. With the other hand, cover the upper opening of the capillary overflow chamber with your index finger and seat the capillary pipette holder in the reservoir neck (fig. 7-11). 4. Release pressure on the reservoir and remove your finger from the overflow chamber opening. Suction will draw the blood into the diluent in the reservoir. 5. Squeeze the reservoir gently two or three times to rinse the capillary tube, forcing diluent into but not out of the overflow chamber, releasing pressure each time to return diluent to the reservoir. Close the upper opening with your index finger and invert the unit several times to mix the blood sample and diluent. See figure 7-12. 6. For specimen storage, cover the overflow chamber of the capillary tube with the capillary shield. 7. Immediately prior to cell counting, mix again by gentle inversion, taking care to cover the hole with your index finger. 8. Place the coverglass on the hemacytometer counting chamber, making sure the coverglass is clean and grease-free. (Fingerprints must be completely removed.) 9. Remove the pipette from the reservoir. Squeeze the reservoir and reseat the pipette in the reverse position. Release pressure to draw any fluid in the capillary tube into the reservoir. Invert and fill the capillary pipette by gentle pressure on the reservoir. After discarding the first 3 drops, load (charge) the counting chamber of the hemacytometer by gently squeezing the reservoir while touching the tip of the pipette against the edge of the coverglass and the surface of the counting chamber (fig. 7-13). A properly loaded counting chamber should have a thin, even film of fluid under the coverglass (fig. 7-14, view A). Allow 3 minutes for the cells to settle. If fluid flows into the grooves (moats) at the edges of the chamber or if you see air bubbles in the field, the chamber is flooded and must be cleaned with distilled water, dried with lens tissue, and reloaded (fig. 7-14, view B). If the chamber is underloaded, carefully add additional fluid until properly loaded. 10. Place the loaded hemacytometer into a petri dish with a piece of dampened tissue to keep the hemacytometer from drying out (fig. 7-15). Allow 5 to 10 minutes for the cells to settle. 11. Once the cells have settled, place the hemacytometer on the microscope. Using the high-power objective, count the WBCs in the four corner fields of the hemacytometer chamber (fields A, B, C, and D of figure 7-16). Each field is composed of 16 small squares. To count the cells in each field, start in the upper left small square and follow the pattern indicated by the arrow in field B of figure 7-16. Count all of the cells within each square, including cells touching the lines at the top and on the left. Do not count any cells that touch the lines on the right or at the bottom. 7-18