Module 11 Cell Counting Manual Acknowledgments Ministry of Health Guyana Centers for Disease Control and Prevention CDC Global AIDS Program GAP Guyana Centers for Disease Control and Prevention CDC Atlanta American Society for Clinical Pathology ASCP Objectives for Manual WBC Describe the manual procedure for counting white cells red cells and platelets Describe the hemocytometer and how it is used Discuss the general formula for calculating cells Define technical factors affecting the manual count General Methods Used in Cell Counting Diluting the blood sample quantitatively with appropriate diluent Determining the number of cells in a diluted specimen Converting the number of cells in the diluted specimen to the the number of cells in 1 L of whole blood Units Reported International Committee for Standards Units per liter of blood L Also reported out as per cubic millimeters of blood cumm or mm3 Also reported out as per cubic liter 1 cumm 1 ul 10 6 liter Manual Cell Counts The Standard Unopette system consists of a self filling pipette available in various diluents depending which cell is to be counted WBC and PLT ammonium oxalate destroys RBC but keeps WBC and PLT RBC Isotonic Saline maintains the RBC do not use water or won t have any RBC Each pipette is color coded and marked with a specific volume WBC and PLT 20 ul RBC 10 ul Manual Counts The reservoir contains a pre measured volume of diluent WBC and Plt 1 98 ml of ammonium oxalate 20 ul pipet Final dilution 1 100 RBC 0 85 saline 10 ul pipet Final dilution 1 200 Manual Counts Are performed with the use of a Hemacytometer or counting chamber For leukocytes erythrocytes platelets and body fluids different dilutions and diluting fluids are used with a varied area of the chamber to be counted A specially designed coverglass is placed over the ruled area of the counting chamber Hemocytometer Neubauer chamber Consists of 9 large squares that are 1 X 1 mm The center square is divided into 25 smaller squares 2 mm The distance between the counting area and the coverslip is 0 1 mm Total area of chamber 9 mm2 Total Volume is 9mm3 Hemacytometer WBC Use the 4 large corner boxes Platelet Center square all 25 sm sqs RBC Use center square small sqs count the top and bottom corner squares and the center square Body Fluid Count all nine squares Count under 40X objective Unopette System Commercially available from Becton Dickinson Consists of a disposable reservoir containing a premeasured diluting fluid and a capillary pipette WBC and Platelet ammonium oxalate is used for the diluting fluid to hemolyze the RBC s and leaves the WBC s and platelets intact Procedure for WBC and PLT Count Pierce the thin plastic cover Hold the pipet horizontally Fill the 20 ul pipet from end to end with no bubbles Wipe the pipet but be careful not to touch the end Procedure Squeeze the reservoir to let out all the air Hold this position Place the pipet on top and release the reservoir The sample will be drawn into the diluting fluid Rinse the pipet several times until the pipet is free of blood Procedure Mix well Allow the specimen to stand for 10 minutes to allow the red blood cells to lyse Mix well invert and expel a few drops of fluid Procedure Turn pipet upside down and use as a dropper Squeeze the reservoir slightly maintaining a steady flow charge fill the hemacytometer Fill both sides and do not overflow the grid Allow to stand for 2 minutes Cell Counting Pattern The counting pattern should be followed to ensure that cells are counted once The count begins in the upper left corner of the first square and continue in a serpentine manner Single double or triple lines divide the squares Cells that touch the left boundary and top are counted The cells that touch the right a bottom are not counted Calculations Total cells ul of cells counted X dilution of sqs counted X volume of each sq WBC volume 0 1 ul Platelet Volume 0 1 ul RBC Volume 0 004 ul Technical Factors Make sure there is no lint or dirt on the coverslip or counting chamber Always charge both sides of the chamber When charging the chamber do over or under fill Counts are done under the high dry objective Use a low light bright light will fade out the elements Count quickly the fluid starts to dry under the microscope