1. Electrical Impedance and Colorimetric Measurements
Joanna Ellis, MLS(ASCP)
Updated: Keri Brophy-Martinez

2. 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

3. History You 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.

4. 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

5. Electrical Impedance (The Coulter Principle)
Sweep Flow: Steady stream of diluent that flows behind each aperture to prevent cells from re-entering the aperture

6. From Pulses to Histograms
Series of thresholds are used to sort particles by size and produce a distribution curve
Information is used to determine the MCV, RDW, and types of WBCs
Histogram is a frequency curve for ONE measured property of a cell.

7. Counting Chambers Most common chambers using impedance:
RBC/Platelet chamber
WBC chamber
RBC/Platelet Chamber
WBC Chamber
Differential Chamber
Reticulocyte Channel

8. Cell the instrument Counts
RBC/Platelet Chamber
Particle Size
Cell the instrument Counts
2-20 femtoliters (fL)
Platelet fL
Red Blood Cell

9. 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.

10. Bimodal Histogram Bimodal peak can be seen in situations such
Cold agglutinin disease
Post-transfusion
Post-treatment of IDA

11. Coincidence
Coincidence: Multiple cells passed through the aperture at once.

12. 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.

13. Three-part Differential WBC Histogram
Lymphocytes: 35-92L
Mononuclear Cells: fL
Granulocytes: fL

14. 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
Sysmex XT and XE
Lysing agent converts free hemoglobin to cyanmethemoglobin and read spectrophotometrically at 540nm.
Advia 120
Some Beckman Coulters

15. Differential Chamber VCS Technology V- Volume analysis C- Conductivity
S- Scatter

16. Reticulocyte Chamber
Used to measure reticulocytes

17. References
"Abaxis Veterinary Diagnostics - VetScan HM2 Technology." Abaxis Medical Diagnostics - Redirect. Abaxis Medical Diagnostics, Web. 12 Sept <
Graham, Marshall Don. "The Coulter Principle: Foundation of an Industry." The Association for Laboratory Automation :: Home. JALA Volume 8, Issue 6, Dec Web. 12 Sept <
Kelly, Kathleen. "Modules :: CLIA :: CME." Hematology in the Physician Office Laboratory Section I. University of Iowa Carver College of Medicine, Web. 12 Sept <
Krantz, Alexander. "Residency & Fellowship Programs | Education." Department of Pathology & Cell Biology | CUMC. Columbia University of Physicians and Surgeons. Web. 12 Sept <
Harmening., Denise, Clinical Hematology and Fundamentals of Hemostasis, 3rd edition, pp
Turgeon, Mary Louise, Clinical Hematology - Theories and Procedures, 3rd edition, pp373,
Rodak, Bernadette, Diagnostic Hematology, 1st edition, p
Coulter STKS Operating Manual
McKenzie, Shirlyn, Clinical Laboratory Hematology, 2nd edition,pp