DETERMINATION OF RBC BY UNOPETTE SYSTEM

HARRY PERSAUD, MS, RPA-C, PBT (ASCP), MT

RBC DETERMINATION• Red blood cells are calculated for a number of

reasons including the diagnosis of disease states and the management of the patient.

• Whole blood is added to isotonic saline which preserves the red cells and prevents it from lysis or crenation.

• It also preserves leukocytes. • This method (Unopette method) also uses Sodium

Azide as an antibacterial agent to inhibit bacterial growth

RBC DETERMINATIONREAGENTS

• 1. Unopette reservoirs containing Sodium azide, sodium chloride, and purified water as the diluent. Check expiration dates and do not use expired Unopettes.

• 2. Unopette capillary pipette, 10 μL

RBC DETERMINATIONDILUTION RATIO

• Sample to total volume...........................1:200

• That is 1.99 ml of diluent to 10μl of sample

PROCEDURE FOR MANUAL RBC• 1. Specimen should be well mixed and left on a

rocker for at least 5 minutes before using.

• 2. Check Unopettes for clarity and contents. If the Unopette chambers appear cloudy or the amount of reagent looks questionable, do not use.

PROCEDURE FOR MANUAL RBC• 3. With the reservoir on a flat surface, puncture the

diaphragm of the reservoir using the protective shield of the capillary pipette

• A. Using a twist action, remove protective shield from the pipette assembly

• B. Holding the pipette and the tube of blood almost horizontally, touch the tip of the pipette to the blood

The pipette will fill by capillary action and will stop automatically when the blood reaches the end of the capillary bore in the neck of the pipette

PROCEDURE FOR MANUAL RBC• C. Wipe the excess blood from the outside of the capillary

pipette. Be careful not to touch the tip of the capillary when wiping off excess blood

• D. Before entering the reservoir, it is necessary to force some air out of the reservoir. Do not expel any liquid and maintain pressure on reservoir

• E. Place an index finger over opening of overflow chamber and position pipette into reservoir neck

• F. Release pressure on reservoir and then remove finger. The negative pressure will draw blood into pipette

PROCEDURE FOR MANUAL RBC• G. Rinse the capillary pipette with the diluents by

squeezing the reservoir gently two or three times. This forces diluent up into, but not out of, the overflow chamber and releases pressure each time to ensure the mixture returns to the reservoir

• H. Return protective shield over upper opening and gently invert several times to mix blood adequately

• I. Allow the Unopette to stand for 10 minutes to allow RBCs to settle

• RBC counts should be performed within 3 hours.

PROCEDURE FOR MANUAL RBC• 4. Charge the hematocytometer

• A. Mix the dilution by inversion and convert the Unopette to the dropper assembly.

• B. Gently squeeze Unopette and discard first 3 or 4 drops. This allows proper mixing, with no excess diluent in the tip of the capillary.

• C. Carefully charge hematocytometer with the diluted blood, gently squeezing the reservoir to release contents until chamber is properly filled. Be sure to charge both sides and not to overfill chambers

PROCEDURE FOR MANUAL RBC• 5. Place the hematocytometer in the pre-

moistened Petri dish and leave for 15 minutes. This allows the sample to settle evenly

• 6. Cell count can now be performed

• 7. RBC count should be performed within 6 hours of making dilution

RBC CELL COUNT AND CALCULATION• 1. Place the hematocytometer on the microscope.

Use low power to locate the five small fields in the large center square. Switch to high power lens and count the number of cells in each of the 5 fields

RBC CELL COUNT AND CALCULATION• Note: To count the cells, start in the upper left small square. • Count all of the cells within each square, including the cells

touching the lines at the left and on the bottom• Count both sides of the chamber and average the count

RBC CELL COUNT AND CALCULATION• 2. The number of red cells per cubic millimeter of blood =

Total the numbers of cells counted in all 5 fields multiply by 10,000

OR

• Use the following formulas to calculate the RBC.

• Cells/mm3 = Average No. of cells X dilution factor (200) divide by the Area counted (0.2) X Depth (0.1)

• Cells/mm3 = (# cells X 200) / (0.2 X 0.1)• Cells/mm3 = (# cells X 200) / 0.02• Cells/mm3 = # cells X 10,000

RBC CELL COUNT AND CALCULATION

Example:

• If have 360 cells on one side and 440 cells on the other sideAverage = 800/2 = 400 cells

• Cells/mm3 = 400 x 10,000 = 4,000, 000

• Total RBC = 4,000, 000 mm3

RBC DETERMINATIONNormal Value:

• Adult Male: 4.7 – 6.1 million/mm3

• Adult Female: 4.2 – 5.4 million/mm3

• Newborn: 4.0 – 6.0 million/mm3

SOURCES OF ERRORSErrors can be caused by the Apparatus or during the Technique

Errors caused by apparatus include:

• Pipette tips that may be chipped• Using the wrong cover glass• Dirty hematocytometer or cover slip• Inaccurate chamber rulings (lines)• Markings on the pipette or the hematocytometer

RBC DETERMINATIONErrors caused by technique include:

• Blood not mixed properly • Failure to discard the first 3-4 drops• Chamber not loaded properly (trapper air bubbles,

overfilling)• Inadequate cell count (such as skipping cells, not

counting the correct borders, or counting cells twice)

• Errors in calculation• Clerical errors

ELEVATED RBC• RBC elevation may be seen in:

• Cardiovascular disease• Polycythemia Vera• Tobacco abuse• Renal cell carcinoma• High altitude• Dehydration

DECREASED RBC• A decrease in RBC may be seen in:

• Anemia• Hemorrhage• Bone marrow disease• Chronic renal failure

RBC DETERMINATION

THE END