electrical impedance and colorimetric measurements
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Electrical Impedance and Colorimetric Measurements. Joanna Ellis, MLS(ASCP). Objectives. Cite the electrical impedance principle of cell counting. Identify and interpret microcytic and macrocytic RBC histograms Define coincidence

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objectives
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
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.
the coulter principle
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
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
Counting Chambers
  • Most common chambers using impedance:
    • RBC/Platelet chamber
    • WBC chamber

RBC/Platelet Chamber

WBC Chamber

Differential Chamber

Reticulocyte Channel

rbc and platelet histograms
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 Histogram
  • Bimodal peak can be seen in situations such
  • Cold agglutinin disease
  • Post-transfusion
  • Post-treatment of IDA
coincidence
Coincidence

Coincidence: Multiple cells passed through the aperture at once.

wbc chamber
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.

colorimetric measurements
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
three part differential wbc histogram
Three-part Differential WBC Histogram

Lymphocytes: 35-92L

Mononuclear Cells: 92-152 fL

Granulocytes: 152-450 fL

references
References
  • "Abaxis Veterinary Diagnostics - VetScan HM2 Technology." Abaxis Medical Diagnostics - Redirect. Abaxis Medical Diagnostics, 2006. Web. 12 Sept. 2010. .
  • 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. .
  • 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. .
  • Krantz, Alexander. "Residency & Fellowship Programs | Education." Department of Pathology & Cell Biology | CUMC. Columbia University of Physicians and Surgeons. Web. 12 Sept. 2010. .
  • 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, 2ndedition,pp 813-829.
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