A disposable Unopette (see fig. 7-8) for RBC counts. The Unopette consists of

-a shielded capillary pipette (10 microliter (Fl) capacity), and
-a plastic reservoir containing a premeasured volume of diluent (1:200 dilution).
Hemacytometer and coverglass
Microscope with light source

Hand-held counter
Laboratory chit

Unopette Procedure
The Unopette procedure for counting red blood cells is as follows:

1. Puncture the diaphragm in the neck of the diluent reservoir with the tip of the capillary shield on the capillary pipette. See figure 7-9.

2. After obtaining free-flowing blood from a lancet puncture of the finger, remove the protective plastic shield from the capillary pipette. Holding the capillary pipette slightly above the horizontal, touch the tip to the blood source (see fig. 7-10, view A). The pipette will

Figure 7-7.-Improved Neubauer Ruling.

Figure 7-8.-Unopette(r) for RBC count.

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 10 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 (see 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 the diluent. See figure 7-12.

6. For specimen storage, cover the overflow chamber of the capillary tube with the capillary shield.

Figure 7-9.-Puncturing the diaphragm of diluent with the capillary pipette shield.

Figure 7-10.-Drawing blood into the Unopette capillary tube: A. From a finger puncture; B. From a venous blood sample.

Figure 7-11.-Preparing reservoir to receive blood from the capillary tube.

7. Immediately prior to cell counting, mix again by gentle inversion, taking care to cover the upper opening of the overflow chamber with your index finger.

8. Place the coverglass on the hemacytometer counting chamber, making sure coverglass is clean and free of grease. (Fingerprints must be completely removed.)

9. Remove the pipette from the reservoir. Squeeze the reservoir and reseat the pipette in the reverse position, releasing 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 cells to settle. If fluid flows into the grooves (moats) at the edges of the chamber or if air bubbles are seen in the field, the chamber is flooded and yes"> 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 hemocytometer on the microscope. Use the low-power lens to locate the five small fields (1, 2, 3, 4, and 5) in the large center square bounded by the double or triple lines. See figure 7-16. Each field measures 1/25 mm 2 , 1/10 mm in depth, and is divided into 16 smaller squares. These smaller squares form a grid that makes accurate counting possible.

12. Switch to the high-power lens and count the number of cells in field 1. Move the hemacytometer until field 2 is in focus and repeat the counting procedure. Continue until the cells in all five fields have been counted. Note the fields are numbered clockwise around the chamber, with field 5 being in the center.

Figure 7-12.-Mixing blood sample and diluent.

Figure 7-13.-Loading the counting chamber.

Count the fields in this order. To count the cells in each field, start in the upper left small square and follow the pattern indicated by the arrow in field 1 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.

13. Total the number of cells counted in all five fields and multiply by 10,000 to arrive at the number of red cells per cubic millimeter of blood.

NOTE: The number of cells counted in each field should not vary by more than 20. A greater variation may indicate poor distribution of the cells in the fluid, resulting in an inaccurate count. If this happens, the test must be repeated.