Module 14 Cell Counting Automation Acknowledgments Ministry of Health Guyana Centers for Disease Control and Prevention CDC Global AIDS Program GAP Guyana Centers for Disease Control and Prevention CDC Atlanta American Society for Clinical Pathology ASCP Objectives for Automation Define the principle of electrical impedance in cell counting Describe the use of radio frequency in cell counting State the principles of light scatter in cell counting Identify sources of error in automated cell counting Discuss methods of quality control in automation Instrumentation Analyzers provide an electronic measurement of red cell count RBC white cell count WBC platelet count Plt mean platelet volume MPV hemoglobin concentration Hb mean red cell volume MCV Instrumentation Calculated parameters include Hematocrit Hct Mean cell hemoglobin MCH Mean cell hemoglobin concentration MCHC Red cell distribution width RDW Instrumentation Analyzers include white cell differential counts Relative or percent Absolute number Reticulocyte analysis Five part differential which includes neutrophils lymphocytes monocytes eosinophils and basophils Principles of Automation Electrical Resistance Impedance Method of counting and volumetric sizing based on the detection and measurement of changes in electrical resistance produced by a particle suspended in a conductive liquid as it is drawn through a small aperture A blood sample is diluted in saline saline because water destroys red cells via osmosis which is a good conductor of electrical current Impedance DC DC current is applied between the two electrodes Electrical resistance or impedance occurs as the cells pass through the aperture causing a change in voltage Each cell momentarily increases the electrical resistance between two electrodes The amplitude and size of the pulse depends on the cell volume Electrical Resistance The number of pulses is proportional to the number of cells counted The size of the voltage pulse is also directly proportional to the volume or size of the cell Threshold limits are established for the enumeration of each cell population based on the cell volume Pulses are channelized by their height or amplitude Histogram Histograms are created for the red blood cell white blood cell and platelet populations based on cell volume measured in femtoliters fl and relative number Histogram A display of the distribution of cell volume and frequency Each channel on the X axis represents size in fl The Y axis represents the relative number Hydrodynamic Focusing A process to generate a narrow channel through which cells separate and align in a single file in front of a detection system one at a time This process eliminates the recirculation of cells and the counting of cells twice Radiofrequency RF Conductivity or radio frequency measurements provide information about the internal characteristics of the cell RF resistance is a high voltage electromagnetic current flowing between the electrodes to detect the size of cells based on the cellular density The nuclear to cytoplasmic ratio nuclear density and cytoplasmic granulation are determined Principle of Light Scatter Flow Cytometry A single cell passes across a laser light beam the light will be reflected and scattered The patterns of scatter are measured at various angles forward scatter 180 degrees and right angle 90 degrees Principles of Light Scatter Flow Cytometry Forwarded Light Scatter Side Light Scatter Provides information about cell structure shape and reflectivity These characteristics can be used to differentiate the various types of white blood cells Scatter plots with a 5 part differential Scatterplots Graphic representations of two or more measurable characteristics of cells These three dimensional plots visualize and analyze data of cells size shape depth Provides information about abnormalities of populations and sub populations of cells The plots use conductivity and light scatter Lymphocytes granulocytes and monocytes are the prominent populations viewed Cytochemistry Using light scatter stain is added Myeloperoxidase Stain Then light scatter is used for cell size Light absorption or stain intensity for cell identification of cell type Forward light scatter direction 1 3 degree angle Example of Automated CBC Report Flagging Condition Flags WBC Suspect Flags Normal Abnormal Immature Granulocytes Variant Lymphs Blasts RBC Suspect Flags Dimorphic RBC Micro Fragments Anis Flagging Platelet Suspect Flags Giant Platelets Platelet Clumping Small platelets Definitive Flags Predetermined Neutrophillia Monocytosis Anemia lab limits Slide Review Scan WBC RBC Neut 80 W suspect flag Band Mono 15 Eos 15 MCV 70 110 RDW 20 All R Flags Plt 50 000 600 000 Manual Diff WBC 4 00 15 00 Baso 3 Lymphs 60 6 yrs age Variant Lymphs Suspect Blast Automation Considerations RBC agglutinin dec RBC HCT MCH inc MCV Lipemia inc HGB MCV MCH MCHC Platelet Clumps inc WBC dec plt count High WBC inc RBC HGB HCT MCH NRBC inc WBC Automation Considerations Cryoproteins inc WBC RBC HGB HCT MCH MCHC dec MCV Platelet Satellitosis inc WBC dec plt count Extremely small microcytes inc plt count Dec RBC Old Specimen Inc MCV MPV Dec WBC plt Quality Control The process of monitoring the testing system for accuracy and precision Instrumentation function adequately Accuracy of unknowns Methods Calibration of the instrument Controls or assayed material Previous patients Delta checks and XB analysis Quality Control Three levels of control are run on each shift Control material have known values Normal Low High Delta Check Compare patient parameters with previous run parameters If a difference considered an analytical error precision and accuracy Example Hemoglobin and MCV Failed Delta within 3 days Summary We Defined the principle of electrical impedance in cell counting Described the use of radio frequency in cell counting Stated the principles of light scatter in cell counting Identified sources of error in automated cell counting Discussed methods of quality control in automation XB Use stabilized parameters MCV MCH MCHC 20 sample batch then compared to target value Should not vary by 3 Monitors function of analyzer Critical Values Each laboratory will establish their own Normals and Critical Values or panic values Repeat for verification Confirm with differential or alternative method Notify appropriate parties Document Be aware of all lab policies for notification of these results