Questions and Answers
What is the anticoagulant of choice for venous blood collection?
dried dipotassium EDTA
What can happen if there is excess EDTA in a blood sample?
Platelets swell and disintegrate
Capillary blood collection is usually done from the ring finger in older children.
False
The Coulter Principle involves sizing and counting cells by detecting changes in __________ when a particle passes through a small aperture.
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Match the cell counting principle with its description:
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What is the anticoagulant of choice for venous blood collection?
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Platelet counts on capillary blood are usually higher than those on venous blood.
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How should the anticoagulant be mixed with blood for CBC?
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During hydrodynamic focusing, the technique narrows the stream of cells to _____.
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Study Notes
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
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Description
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.
