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Electrical Impedanceand
Colorimetric Measurements
Joanna Ellis, MLS(ASCP)
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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
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HistoryYou CAN patent a hole
• Prior to the 1950s blood cell counts were performed by manual methods:– 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.
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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
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Electrical Impedance (The Coulter Principle)
Sweep Flow: Steady stream of diluent that flows behind each aperture to prevent cells from re-entering the aperture
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Counting Chambers
• Most common chambers using impedance:RBC/Platelet chamber WBC chamber
RBC/Platelet Chamber
WBC Chamber
Differential ChamberReticulocyte Channel
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RBC/Platelet Chamber
Particle Size Cell the instrument Counts
2-20 femtoliters (fL) Platelet
36-360 fL Red Blood Cell
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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.
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Bimodal Histogram
Bimodal peak can be seen in situations such •Cold agglutinin disease•Post-transfusion•Post-treatment of IDA
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Coincidence
Coincidence: Multiple cells passed through the aperture at once.
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WBC Chamber
Lysing agent
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.
Aperture in electric field
WBCs
Hgb released
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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
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Three-part Differential WBC Histogram
Lymphocytes: 35-92LMononuclear Cells: 92-152 fLGranulocytes: 152-450 fL
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References• "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.