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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
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 cant patent a hole were proven wrong. Hematology automation has since grown to include additional principles such as optical light scatter and flow cytometry.
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
RBC/Platelet Chamber
Particle Size
2-20 femtoliters (fL)
36-360 fL
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
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
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
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.