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TITLE: DETERMINATION TOTAL RED BLOOD CELL COUNT

(MANUAL METHOD)

PRINCIPLE

To facilitate counting and prevent lyses of the red blood cells, whole blood is diluted with an isotonic diluting fluid.

CLINICAL APPLICATION

Diagnosis and management of patients with anemia or Polycythemia depends on knowledge of the total mass of red cells and the rate of their production and destruction. Increased values are found in Polycythemia Vera and Secondary Polycythemias whilst decreased values are found in Anaemia.

SAMPLE REQUIREMENTS: Whole blood using EDTA or heparin as the anticoagulant. Capillary blood may also be used.

PROCEDURE REQUIREMENT:

Automated Pipette

Neubauer counting chamber.

Microscope

Cover glass

Clean gauze

Red cell diluting fluid

Test tubes

REAGENT PREPARATION

Refer to preparation of reagent on page 62 (sodium citrate).

PROCEDURE (Manual)

1. Prepare all dilutions in duplicate

20ul pipette (1:201 dilution)

(a) Place exactly 4.0 ml of diluting fluid into 10 x 75 mm test tube.

(b) Add 20ul of well-mixed whole blood to the test tube and mix.

(c) For a 1:2000 dilution, remove 20ul of diluting fluid from the 4.0ml in step I above prior to adding the blood specimen.

2. Clean the counting chamber. Mix the diluted RBC for 3 minutes.

3. Fill the counting chamber (one red count dilution filling each side of the haemocytometer). Once the counting chamber is filled, allow approximately 3 minutes for the red cells to settle prior to counting.

4. Count the red blood cells (Figure 2.)

(a) Carefully place the filled counting chamber on the microscope stage.

(b) Using low power (10 x objective), place the large center square in the middle of the field vision. Examine the entire large square for even distribution for red blood cells.

(c) Carefully change to the high dry objective (40 x)

(d) Move the counting chamber so that the small...