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