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
