Electrical Impedance
and
Colorimetric Measurements
Joanna Ellis, MLS(ASCP)
Updated: Keri Brophy-Martinez
Objectives
• Cite the electrical impedance principle of cell
counting.
• Identify and interpret microcytic and
macrocytic RBC histograms
• Define coincidence
• Identify the cell populations represented on a
3-part differential WBC histogram
History
You CAN patent a hole
• Prior to the 1950s blood cell counts were performed by
manual methods:
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Hemacytometer blood counts
Spun hematocrits
Spectrophotometrically determined hemoglobins
Peripheral blood cell evaluation for all differentials
• In 1953, Wallace Coulter patented the Coulter Principle
in which particles are counted in fluid passed through a
hole. The incredulous attorneys who had told him
“You can’t patent a hole” were proven wrong.
• Hematology automation has since grown to include
additional principles such as optical light scatter and
flow cytometry.
The Coulter Principle
• The poorly conductive blood cells are suspended in a
conductive diluent (liquid).
• The diluent is passed through an electric field created
between two electrodes.
• The liquid passes through a small aperture (hole).
• The passage of each particle through the aperture
momentarily increases the impedance (resistance) of
the electrical path between the electrodes.
• The increase in impedance creates a pulse that can be
measured.
• The number of pulses = blood cell count
• The amplitude (height) of the pulse = Volume of cell
Electrical Impedance (The Coulter Principle)
Sweep Flow: Steady stream of diluent that flows behind each aperture to
prevent cells from re-entering the aperture
Counting Chambers
RBC/Platelet Chamber
WBC Chamber
Differential Chamber
Reticulocyte Channel
• Most common chambers using impedance:
RBC/Platelet chamber
WBC chamber
RBC/Platelet Chamber
Particle Size
Cell the instrument Counts
2-20 femtoliters (fL)
Platelet
36-360 fL
Red Blood Cell
RBC and Platelet Histograms
The black line represents normal cell distribution. The red line on
the RBC histogram graphically represents a microcytic red cell
population.
Bimodal Histogram
Bimodal peak can be seen in situations such
•Cold agglutinin disease
•Post-transfusion
•Post-treatment of IDA
Coincidence
Coincidence: Multiple cells passed through the
aperture at once.
WBC Chamber
WBCs
Lysing agent
Aperture in
electric field
Hgb released
In some analyzers the WBC count is directly measured by
electric impedance after the red cells have been destroyed by a
lysing agent. The lysing agent also shrinks the leukocyte cell
membrane and cytoplasm; therefore, the WBC count
represents the measure of the cell volume not native cell size.
Colorimetric measurements are used to determine hemoglobin.
Three-part Differential
WBC Histogram
Lymphocytes: 35-92L
Mononuclear Cells: 92-152 fL
Granulocytes: 152-450 fL
Colorimetric Measurements
• Hemoglobin is often determined by a colorimetric method.
– Imidazole
• Non-cyanide reagent with color change and read at 540nm
• Instruments
– Abbott CELL-DYN Sapphire
– Sodium Lauryl Sulfate
• Non-cyanide reagent with color change and read
spectrophotometrically
• Instruments
– Sysmex XT and XE
– Lysing agent converts free hemoglobin to cyanmethemoglobin
and read spectrophotometrically at 540nm.
• Instruments
– Advia 120
– Some Beckman Coulters
Differential Chamber
–VCS Technology
–V- Volume analysis
–C- Conductivity
–S- Scatter
Reticulocyte Chamber
–Used to measure reticulocytes
References
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"Abaxis Veterinary Diagnostics - VetScan HM2 Technology." Abaxis Medical
Diagnostics - Redirect. Abaxis Medical Diagnostics, 2006. Web. 12 Sept. 2010.
<http://www.abaxis.com/veterinary/vetscan_hm2_technology.html>.
Graham, Marshall Don. "The Coulter Principle: Foundation of an Industry." The
Association for Laboratory Automation :: Home. JALA Volume 8, Issue 6, Dec. 2003.
Web. 12 Sept. 2010. <http://labautomation.org>.
Kelly, Kathleen. "Modules :: CLIA :: CME." Hematology in the Physician Office
Laboratory Section I. University of Iowa Carver College of Medicine, 2008. Web. 12
Sept. 2010.
<http://www.medicine.uiowa.edu/cme/clia/modules.asp?testID=4#02>.
Krantz, Alexander. "Residency & Fellowship Programs | Education." Department of
Pathology & Cell Biology | CUMC. Columbia University of Physicians and Surgeons.
Web. 12 Sept. 2010. <http://pathology.columbia.edu/education/residency>.
Harmening., Denise, Clinical Hematology and Fundamentals of Hemostasis, 3rd
edition, pp. 593-599.
Turgeon, Mary Louise, Clinical Hematology - Theories and Procedures, 3rd edition,
pp373, 376-382.
Rodak, Bernadette, Diagnostic Hematology, 1st edition, p.605-606.
Coulter STKS Operating Manual
McKenzie, Shirlyn, Clinical Laboratory Hematology, 2nd edition,pp 813-829.