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The Unopette System (Becton-Dickinson and Co.) consists of a disposable uniform-bore glass capillary pipet with an attached plastic tab for handling. The pipet (see figure 2-1) is attached to a plastic reservoir in which predetermined amounts of diluting fluids, depending on their purpose, can be placed. The blood aspirated into the diluting fluid can be mixed and dispensed with great ease and speed.
Figure 2-1. Unopette System.
The Unopette System can be used for a variety of hematological procedures. In general, the Unopette System is used as follows.
(1) Puncture diaphragm. Using the protective shield on the capillary pipette, puncture the diaphragm of the reservoir as follows.
(a) Place reservoir on a flat surface. Grasping reservoir in one hand, take pipette assembly in other hand and push tip of pipette shield firmly through diaphragm in neck of reservoir, then removed (figure 2-2).
Figure 2-2. Unopetted filling procedure (puncturing diaphragm of reservoir).
(b) Remove shield from pipette assembly with a twist (figure 2-3).
Figure 2-3. Unopetted filling procedure (removing shield).
(2) Add sample. Fill capillary with whole blood and transfer to reservoir as follows:
(a) Holding pipette almost horizontally, touch tip of pipette to blood. Pipette will fill by capillary action. Filling is complete and will stop automatically when blood reaches end of capillary bore in neck of pipette.
Figure 2-4. Unopetted filling procedure (filling pipette).
(b) Wipe excess blood from outside of capillary pipette making certain that no sample is removed from capillary bore.
(c) Squeeze reservoir slightly to force out some air. Do not expel any liquid. Maintain pressure on reservoir (figure 2-5).
Figure 2-5. Unopetted filling procedure (forcing out air)
(d) Cover opening of overflow chamber of pipette with index finger and seat pipette securely in reservoir neck (figure 2-6).
Figure 2-6. Unopetted filling procedure (seating pipette).
(e) Release pressure on reservoir. Then remove finger from pipette opening. Negative pressure will draw blood into diluent.
(f) Squeeze reservoir gently two or three times to raise capillary bore, forcing diluent up into, but not out of, overflow chamber, releasing pressure each time to return mixture of reservoir (figure 2-7).
Figure 2-7. Unopetted filling procedure (squeezing reservoir).
(g) Place index finger over upper opening and gently invert several times to thoroughly mix blood with diluent (figure 2-8).
Figure 2-8. Unopetted filling procedure (mixing).
(h) Let stand for ten (10) minutes to allow red cells to hemolyze.
BLOOD CELL COUNTING CHAMBERS
The most common type of hemacytometer consists of two counting chambers separated by grooves or canals. On the smooth glass surface of the counting chambers are straight lines etched into glass in a gridwork pattern. The Neubauer ruling, preferred for hematological work, consists of a gridwork with dimensions of 3 mm by 3 mm. It is further divided into 9 smaller squares with dimensions of 1 mm by 1 mm; 4 of these squares are used for the white count. The 8 outer squares are further subdivided into 16 squares 0.25 mm on a side. The central square is divided into 25 squares, 0.20 mm on a side, which are used for the platelet count. Thus the large squares are 1 square mm, the 16 small squares in the outer large squares are 1/16 square mm and the 25 central squares are 1/25 square mm (see figure 2-9).
Figure 2-9. Rulings on a hemacytometer.
The cover glass must be free of visible defects and must be optically plane on both sides within + 0.002 mm according to the United States (US) Bureau of Standards. When the cover glass is placed on the platform, the space between it and the ruled platform should be 0.1 mm.
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