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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 a3-part differential WBC histogram

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HistoryYou CAN patent a hole

Prior to the 1950s blood cell counts were performed bymanual methods: Hemacytometer blood counts

Spun hematocrits

Spectrophotometrically determined hemoglobins Peripheral blood cell evaluation for all differentials

In 1953, Wallace Coulter patented the Coulter Principlein which particles are counted in fluid passed through ahole. The incredulous attorneys who had told himYou cant patent a hole were proven wrong.

Hematology automation has since grown to includeadditional principles such as optical light scatter andflow cytometry.

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The Coulter Principle

The poorly conductive blood cells are suspended in aconductive diluent (liquid).

The diluent is passed through an electric field createdbetween two electrodes.

The liquid passes through a small aperture (hole).

The passage of each particle through the aperturemomentarily 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 theaperture at once.

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WBC Chamber

Lysing agent

In some analyzers the WBC count is directly measured byelectric 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 readspectrophotometrically

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: 35-92L

Mononuclear Cells: 92-152 fL

Granulocytes: 152-450 fL

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References

"Abaxis Veterinary Diagnostics - VetScan HM2 Technology."Abaxis MedicalDiagnostics - Redirect. Abaxis Medical Diagnostics, 2006. Web. 12 Sept. 2010..

Graham, Marshall Don. "The Coulter Principle: Foundation of an Industry." TheAssociation for Laboratory Automation :: Home. JALA Volume 8, Issue 6, Dec. 2003.Web. 12 Sept. 2010. .

Kelly, Kathleen. "Modules :: CLIA :: CME." Hematology in the Physician OfficeLaboratory Section I. University of Iowa Carver College of Medicine, 2008. Web. 12Sept. 2010..

Krantz, Alexander. "Residency & Fellowship Programs | Education." Department ofPathology & Cell Biology | CUMC. Columbia University of Physicians and Surgeons.Web. 12 Sept. 2010. .

Harmening., Denise, Clinical Hematology and Fundamentals of Hemostasis, 3rdedition, 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, 2

nd

edition,pp 813-829.