Automated Cell Counter Practical Session

Automated Cell Counter

Sample Requirements

• Venous blood: Anticoagulant of choice is dried dipotassium EDTA (violet or lavender top tube)
• Capillary blood: Special dipotassium EDTA-coated tubes available
• Instrument aspirates 30 μL of patient sample

Principles of Automated Cell Counting

• Electrical Conductivity (Coulter Principle)
  • Cells are sized and counted by detecting changes in electrical resistance when a particle passes through a small aperture
  • Blood sample is diluted in saline, and cells are pulled through an aperture by creating a vacuum
  • Electrical resistance or impedance occurs as cells pass through the aperture, causing a change in voltage
  • Pulse height is directly proportional to cell volume or size
• Optical Scatter
  • Sample is diluted with isotonic diluent and hydrodynamically focused through a quartz flow cell
  • Cells pass through a flow cell on which a beam of laser light is focused
  • Scattered light is detected by a photomultiplier or photodiode and converted to an electric pulse
• Flow Cytometry
  • Passing of single cell through a laser source
  • Measurement of scattered and emitted fluorescence using detectors
  • Data presentation: Graphic displays include histograms and scatter-plots, and numerical values for reporting to clinicians
  • Flags are signals that occur when an abnormal or out-of-range result is detected, mandating a review of blood smear examination

RBC Parameters

• Red Blood Cell Count
  • Typically one channel is used to detect RBCs and platelets
  • Red cell count and cell volume are directly measured by aperture impedance or light scatter
  • Detector is set to count cells between 36-360 fL as RBCs
• Hemoglobin Measurement
  • Measured directly by a modification of cyanmethemoglobin method
  • Spectrophotometric method is used after red cells are lysed in WBC/HB chamber
• RED CELL INDICES
  • MCH measures both red cell size and hemoglobin concentration
  • MCHC is calculated from MCH and is used to alert user about possibility of spurious results
  • MCV is a measure of red cell size
  • RDW is a measure of the degree of variation of size of red cells
  • RDW-CV and RDW-SD are two types of RDW measurements

WBC Parameters

• White Blood Cell Count
  • Determined in whole blood in which red cells have been lysed
  • Particles larger than 35 fL are counted as leukocytes by impedance method
  • Factitiously low automated WBCs may occur due to leucocyte agglutination, prolonged sample storage, or abnormally fragile cells
• WBC Differential
  • Most automated differential counters use flow cytometry incorporated into a full blood counter
  • Cells are classified into different categories based on volume and granularity
  • Five- to seven-part differential counts classify cells as neutrophils, eosinophils, basophils, and monocytes

Platelet Parameters

• Platelet Count
  • Counted by electrical impedance method in the RBC aperture
  • Platelets can be counted between 2 and 30 fL
  • Factitiously low impedance platelet counts may occur due to giant platelets, EDTA-induced platelet clumping, or EDTA-dependent platelet satellitism
• Mean Platelet Volume (MPV)
  • Derived from the impedance platelet size distribution curve
  • Normal MPV is 7-11 fL
  • Increased MPV results from presence of immature platelets in circulation, peripheral destruction of platelets stimulates megakaryocytes to produce such platelets

Automated Cell Counter

Sample Requirements

• Venous blood: Anticoagulant of choice is dried dipotassium EDTA (violet or lavender top tube)
• Capillary blood: Special dipotassium EDTA-coated tubes available
• Instrument aspirates 30 μL of patient sample

Principles of Automated Cell Counting

• Electrical Conductivity (Coulter Principle)
  • Cells are sized and counted by detecting changes in electrical resistance when a particle passes through a small aperture
  • Blood sample is diluted in saline, and cells are pulled through an aperture by creating a vacuum
  • Electrical resistance or impedance occurs as cells pass through the aperture, causing a change in voltage
  • Pulse height is directly proportional to cell volume or size
• Optical Scatter
  • Sample is diluted with isotonic diluent and hydrodynamically focused through a quartz flow cell
  • Cells pass through a flow cell on which a beam of laser light is focused
  • Scattered light is detected by a photomultiplier or photodiode and converted to an electric pulse
• Flow Cytometry
  • Passing of single cell through a laser source
  • Measurement of scattered and emitted fluorescence using detectors
  • Data presentation: Graphic displays include histograms and scatter-plots, and numerical values for reporting to clinicians
  • Flags are signals that occur when an abnormal or out-of-range result is detected, mandating a review of blood smear examination

RBC Parameters

• Red Blood Cell Count
  • Typically one channel is used to detect RBCs and platelets
  • Red cell count and cell volume are directly measured by aperture impedance or light scatter
  • Detector is set to count cells between 36-360 fL as RBCs
• Hemoglobin Measurement
  • Measured directly by a modification of cyanmethemoglobin method
  • Spectrophotometric method is used after red cells are lysed in WBC/HB chamber
• RED CELL INDICES
  • MCH measures both red cell size and hemoglobin concentration
  • MCHC is calculated from MCH and is used to alert user about possibility of spurious results
  • MCV is a measure of red cell size
  • RDW is a measure of the degree of variation of size of red cells
  • RDW-CV and RDW-SD are two types of RDW measurements

WBC Parameters

• White Blood Cell Count
  • Determined in whole blood in which red cells have been lysed
  • Particles larger than 35 fL are counted as leukocytes by impedance method
  • Factitiously low automated WBCs may occur due to leucocyte agglutination, prolonged sample storage, or abnormally fragile cells
• WBC Differential
  • Most automated differential counters use flow cytometry incorporated into a full blood counter
  • Cells are classified into different categories based on volume and granularity
  • Five- to seven-part differential counts classify cells as neutrophils, eosinophils, basophils, and monocytes

Platelet Parameters

• Platelet Count
  • Counted by electrical impedance method in the RBC aperture
  • Platelets can be counted between 2 and 30 fL
  • Factitiously low impedance platelet counts may occur due to giant platelets, EDTA-induced platelet clumping, or EDTA-dependent platelet satellitism
• Mean Platelet Volume (MPV)
  • Derived from the impedance platelet size distribution curve
  • Normal MPV is 7-11 fL
  • Increased MPV results from presence of immature platelets in circulation, peripheral destruction of platelets stimulates megakaryocytes to produce such platelets

A practical session on automated cell counters, focusing on venous blood samples and the importance of correct anticoagulant usage. Learn about the effects of excess or insufficient EDTA on platelet counts.