EXERCISE 7: Automated Differential MLAB 1315 HematologyAUTOMATED DIFFERENTIALLAB OBJECTIVE Dry lab1. The student will interpret 5 differentials from the Coulter STKS, the Bayer Advia and the Abbott Cell-Dyne by examining the instrument printout of the CBC as well as the histograms and scattergrams.2. The student will state the theory of the technology used to perform the automated differential from 4 different instruments.Wet labThe student will examine the scatterplots of CBCs performed at CPL and interpret the results stating whether the automated differentials are normal or abnormal.PRINCIPLECoulter STKS (currently in use as a primary instrument at CPL and as a backup instrument at Seton Medical Center.)The Coulter STKS performs a five-part differential consisting of monocyte, lymphocyte, granulocyte, eosinophil and basophil. The Coulter VCS established WBC differential technologyuses three measurements: individual cell volume, high-frequency conductivity and laser-light scatter as explained below:Volume Analysis - Electronic leukocyte volume analysis uses a low-frequency current measure volume of the WBCs as they impede the current when they pass through the aperture.Conductivity - Cell walls act as conductors to high-frequency current. As the current passes through he walls and through each cell interior, it detects differences in the insulating properties of cell components. It characterizes the nuclear and granular constituents and the chemical composition of the cell interior.Scatter - Leukocytes are hydrodynamically focused and passed in a steady stream through a sensing zone on which a laser light is focused. As each cell passes through the sensing zone of the flow cell, it scatters and reflects the focused light which is detected by a photodetector. The patterns of scatter are measure at various angles (forward scatter at 180 and right angle scatter at 90). Scattered light provides information about cell structure, shape and reflectivity. The characteristics are used to differentiate the various types of WBCs and to produce scatterplots with a LAB EXERCISE #7 MLAB 1315Page 1EXERCISE 7: Automated Differential MLAB 1315 Hematologyfive-part differential.Bayer Advia (currently at Seton Medical Center and Brackenridge)The Bayer Advia utilizes 3 technologies to enumerate and identify cells: optical flow cytometry, cytochemistry and light scattering. WBCs are fixed with formaldehyde and stained with peroxidase in the peroxidase reaction chamber. The high heat in the chamber lyses platelets and RBCs and causes the WBCs to be fixed and dehydrated. Forward-angle light scatter and tungsten light optics are used to measure WBC size and peroxidase activity. Myeloperoxidase is a granulocyte enzyme marker that is present in varying degrees in neutrophils, eosinophils and monocytes but is absent from basophils, lymphocytes and blasts. A specific basophil count is determined separately in the basophil/lobularity chamber. Whole blood is exposed to an acid buffer that selectively lyses all cells except basophils. The resulting particles are sorted and quantified by measuring the forward angle and light scattering properties. An additional category of cells is reported by the Advia as LUC (large unstained cells). This category reflects atypical lymphocytes or blasts.Sysmex TOAThe Sysmex TOA uses radio frequency (RF) and direct current (DC) to enumerate and identify WBC populations. The RF method detects and sizes lyse-treated cells based on density and nuclear size, whereas the DC method sizes the entire cell, nucleus, and cytoplasm. Cells pulses detected by RF and DC methods are then displayed as a 3-dimensional WBC scattergram which contains information about the distribution of lymphs, monos and granulocytes. Separate histograms are generated for basophil and eosinophil populations. LAB EXERCISE #7 MLAB 1315Page 2EXERCISE 7: Automated Differential MLAB 1315 HematologyAbbott Cell-Dyne (currently at Seton Northwest)This system uses multiangle polarized scatter separation (MAPSS) flow cytometry with hydrodynamic focusing of the cell stream. The leukocyte differential is accomplished by light scatter. It features three independent measurements and focused flow impedance. Multidimensional light scatter and fluorescent detection are used as well.SPECIMENEDTA-anticoagulated bloodQUALITY CONTROLCommercial low, normal and high controlsMonitor the CBC and differential parameters. Latron controls monitor the performance of the volume, conductivity and light scatter for the automated differential. Control values are stored inthe instrument computer and can be monitored with the generation of a Levey-Jennings graph foreach parameter.REAGENTS, SUPPLIES AND EQUIPMENTAs indicated for specific instrumentINTERPRETATION OF RESULTSLEUKOCYTE HISTOGRAM ANALYSISSize-referenced leukocyte histograms display the classification of leukocytes according to size following lysis. It does not display the native cell size. The lytic agent lyses the cell and the cytoplasm collapses around the nucleus, producing differential shrinkage. The histogram subpopulations reflect the sorting of the cells by their relative size, which is primarily their nuclear size. As each leukocyte passes through the aperture, it causes a change in electrical resistance which is proportional to its volume. The histogram is a representation of the sizing of the leukocytes. The differentiation is as follows:Cell type Size range Cells that fall within this size rangeLymphocytes 35-90 fL lymphs and atypical lymphsMononuclear 90-160 fL Monos, promyelocytes, myelocytes, plasma cells and blastsGranulocyte 160-450 fL segs, bands, metas, eos and basosLAB EXERCISE #7 MLAB 1315Page 3EXERCISE 7: Automated Differential MLAB 1315 HematologyA valley ordepression should be seen between each population. Abnormal sized cells or abnormal particles can cause abnormal patterns and the instrument will print an alert specific for the region where the abnormal pattern exists. The following table lists the region (R) flags