Laboratory Techniques: RBC, WBC and Platelet Counting (pp 764-768)

Normal RBC Values

Newborn: 4.4 - 5.8 x 106/µL 4.4 - 5.8 x 1012/L Adult Male: 4.7- 6.1 x 106/µL 4.7- 6.1 x 1012/L Adult Female: 4.2 - 5.4 x 106/µL 4.2 - 5.4 x 1012/L

RBC Determination

(1) Dilute the blood sample using the RBC Unopette diluting system. The micropipette is calibrated to draw 0.01 ml (10 μl) of blood. The diluent reservoir contains 1.99 ml of isotonic saline. Therefore, the dilution of blood to diluent is 200. (2) Repeat the dilution process using a new Unopette diluting system. (3) Let the diluted samples stand for 10 minutes. (4) Charge one side of the cleaned hemacytometer with one sample and repeat using the other diluted sample on the other side of the hemacytometer. (5) Place the charged hemacytometer in a moist Petri dish for an additional 10 minutes. (6) After 10 minutes, the specimen is now ready for counting using a microscope

WBC Determination

(1) Dilute the blood sample using the WBC Unopette diluting system. The micropipette is calibrated to draw 0.02 ml (20 μl) of blood. The reservoir contains 1.98 mL diluent consisting of ammonium oxalate and phosphate buffer. Therefore, the dilution of blood to diluent is 100. (2) Repeat the dilution process using a new Unopette diluting system.43 (3) Let the diluted samples stand for 10 minutes. (4) Charge one side of the cleaned hemacytometer with one sample and repeat using the other diluted sample on the other side of the hemacytometer. (5) Place the charged hemacytometer in a moist Petri dish for an additional 10 min. (6) After 10 minutes, the specimen is now ready for counting using a microscope.

Hemacytometer

A calibrated counting chamber used for counting of microscopic elements

Blood sample dilution

Automation Thoma diluting pipette Glass pipette with expanded mixing region and calibrated stem Requires careful cleaning and drying No longer used Unopette system

Normal WBC Values

Newborn 9.0 - 30.0 x 103/μL 9.0 - 30.0 x 109/L Child/Adult 4.8 - 10.8 x 103/μL 4.8 - 10.8 x 109/L

RBC Counting Procedure

(1) Lower microscope stage and condenser to their lowest positions. (2) Place the charged hemacytometer on microscope stage. (3) Scan chamber under 10X objective until able to find grid and bring it into focus. (4) Using the 10X and then 40X objectives find the RBC counting area on the grid. The 4 corner secondary squares and the center middle secondary square of the center primary square are the areas where the RBCs will be counted. (5) Count all cells in the 5 designated RBC squares. Each of the 5 RBC contains 16 tertiary squares. (6) Count all cells that touch any of the upper or left lines but do not count any cells that touch the lower or right lines. This prevents duplicate counting of cells. (7) Keep a record of the number of RBCs in each of the 5 RBC squares. (8) After all 5 squares are counted on one side of the hemacytometer, total the number of cells on that side. (9) Repeat the same counting procedure on the other side of the hemocytometer. (10) Compare the total RBCs counted after both sides of the hemacytometer have been counted. (11) The total numbers from both sides should agree within 10%. If they do not, dilutions and counts should be repeated. (12) Using the following calculation, determine the # of RBCs for each side counted. (13) Average the two results to determine the final result. (14) 1 mm3 equals 1.00003 µL (15) Report the result as: #RBC x 106/µL or #RBC x 1012/L (format for S.I. units)

WBC Counting Procedure

(1) Lower microscope stage and condenser to their lowest positions. (2) Place the charged hemacytometer on microscope stage. (3) Scan chamber under 10X objective until able to find grid and bring it into focus. (4) Using the 10X and then 40X objectives find the WBC counting area on the grid. The 4 corner primary squares are the areas where the WBCs will be counted. (5) Each corner primary square contains 16 smaller secondary squares. (6) Starting at the upper left corner primary square, count all cells in the 16 secondary squares in each of the 4 corner primary squares. (7) Count all cells that touch any of the upper or left lines but do not count any cells that touch the lower or right lines. This prevents duplicate counting of cells. (8) After all 4 corner squares are counted on one side of the hemacytometer, total the number of cells on that side. (9) Repeat the same counting procedure on the other side of the hemacytometer. (10) Compare the total WBC counts after both sides of the hemacytometer have been counted. (11) The total numbers from both sides should agree within 10%. If they do not, dilutions and counts should be repeated. (12) Using the following calculation, determine the # of WBCs for each side counted. (13) Average the two results to determine the final result. (14) 1 mm3 equals 1.00003 µL (15) Report the result as: #WBC x 103/µL or #WBC x 109/L (format for S.I. units)

Platelet Counting Procedure

(1) Lower microscope stage and condenser to their lowest positions. (2) Place the charged hemacytometer on microscope stage. (3) Scan chamber under 10X objective until able to find grid and bring it into focus. (4) Using the 10X and then 40X objectives find the platelet counting area on the grid. The entire center primary square is the area where the platelets will be counted. (5) The center primary square contains 25 smaller secondary squares. (6) Starting at the upper left corner secondary square, count all cells in the 25 secondary squares. (7) Count all cells that touch any of the upper or left lines but do not count any cells that touch the lower or right lines. This prevents duplicate counting of cells. (8) After all 25 secondary squares in the center primary square are counted on one side of the hemacytometer, total the number of platelets on that side. (9) Repeat the same counting procedure on the other side of the hemocytometer. (10) Compare the total platelet counts after both sides of the hemacytometer have been counted. (11) The total numbers from both sides should agree within 10%. If they do not, dilutions and counts should be repeated. (12) Using the following calculation, determine the # of platelets for each side counted. (13) Average the two results to determine the final result. (14) 1 mm3 equals 1.00003 µL (15) Report the result as: #Platelets x 103/µL or #Platelets x 109/L (format for S.I. units)

Preparing the Hemacytometer for Charging with Diluted Sample

(1) Prepare a moist petri dish to house the hemacytometer after charging. (2) Clean hemacytometer surface and coverslip with alcohol and bleach. (3) Rinse with distilled water. (4) Place dry coverslip on dry hemacytometer. (5) Charge the hemacytometer

Charging the Hemacytometer

(1) Remove capillary tube from reservoir and reseat it in the reverse position. (2) Wipe the exterior of the tube towards the tip to remove excess fluid. (3) Squeeze the reservoir gently and discard 3 or 4 drops of diluted blood. (4) Place the capillary tube opening against the edge of the coverslip (5) Gently squeeze reservoir until diluted cells fill the chamber (6) Place charged hemacytometer in prepared Petri dish and allow to sit for 10 minutes

Unopette Diluting Procedure

(1) Separate shield from capillary pipette. (2) Break diaphragm with pipette shield tip. (3) Fill capillary tube to plastic intersection. (Finger stick or venous blood specimen) (4) Wipe outside of capillary with tissue to remove excess blood. (5) Squeeze diluent reservoir slightly. (6) Place finger over plastic end of capillary tube and introduce capillary tube into the diluent. (7) Seat pipette firmly. (8) Release pressure on diluent reservoir. (9) Remove finger from plastic tip of capillary tube. (10) Squeeze reservoir gently rinsing capillary tube 2 or 3 times. Mix. (11) Let stand for 10 minutes

Platelet Determination

(1) The same Unopette diluting system that is used for counting WBCs is used for counting platelets. (2) If the same diluted samples are to be counted on the charged hemacytometer, make sure that the fluid has not dried out on the grid under the microscope. The platelets should be counted immediately after the WBCs are counted.

What are the five sources of error?

1) Blood sample 2) Dilution 3) Charging 4) Counting 5)Calculations

Normal Platelet Value

130,000 - 400,000 platelets/μl 130 - 400 x 103/μL 130 - 400 x 109/L (S.I. units)