1. By Dr. Richa Sharma

2. TYPES OF CELL COUNTING MANUAL SEMI AUTOMATED AUTOMATED 3. TYPES OF AUTOMATED FULLY AUTOMATED MORPHOLOGY BASED CoulterSTKS SysmexSE series,XE2100 Bayer H Series Adiva Abbot Cella Vision DM 96 DiffMaster SE-223 70 4. DISADVANTAGES OF MANUAL CELL COUNTING: Cell identification errors in manual counting: mostly associated with distinguishing lymphocytes from monocytes, bands from segmented forms and abnormal cells (variant lymphocytes from blasts) Slide cell distribution error: increased cell concentration along edges also bigger cells found there i.e. monocytes, eosinophils, and neutrophils Statistical sampling error 5. ADVANTAGES OF AUTOMATED: Objective (no inter-observer variability) No slide distribution error Eliminate statistical variations associated with manual count based on high number of cells counted Many parameters not available from a manual count, e.g. MCV, RDW More efficient and cost effective than manual method: Some cell counters can process 120-150 samples per hour 6. PRINCIPLES OF AUTOMATED CELL COUNTERS Impedance (conductivity) system (Coulter) Optical system (Light Scattering) Flow cytometry Selective lysis Special Stains 7. ELECTRICAL IMPEDANCE METHOD The Coulter Principle 8. Cell counting and sizing is based on the detection and measurement of changes in electrical impedance (resistance) produced by a particle as it passes through a small aperture Particles such as blood cells are nonconductive but are suspended in an electrically conductive diluent As a dilute suspension of cells is drawn through the aperture, the passage of each individual cell momentarily increases the impedance (resistance) of the electrical path between two electrodes that are located on each side of the aperture 9. ELECTRICAL IMPEDANCE METHOD The Coulter Principle Direct current - measurement of cell volume Radio frequency - measurement of internal cell structure. 10. OPTICAL METHOD Laser light is used A diluted blood specimen passes in a steady stream through which a beam of laser light is focused As each cell passes through the sensing zone of the flow,scatters the focused light Scattered light is detected by a photodetector and converted to an electric impulse The number of impulses generated is directly proportional to the number of cells passing through the sensing zone ina specific period of time 11. The application of light scatter means that as 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. Scattered light provides information about cell structure, shape, and reflectivity. These characteristics can be used to differentiate the various types of blood cells and to produce scatter plots with a three or five-part differential 12. Flow Cytometry PRINCIPLE:A beam of light ( laser light) of a single wavelength is directed onto a hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter ) and several perpendicular to it (Side Scatter ) and one or more fluorescent detectors. Each suspended particle from 0.2 to 150 m passing through the beam scatters the ray, and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a longer wavelength than the light source. 13. Optical System 14. This combination of scattered and fluorescent light is picked up by the detectors, and, by analysing fluctuations in brightness at each detector (one for each fluorescent emission peak),it is then possible to derive various types of information about the physical and chemical structure of each individual particle. Forward light scatter - cell size & volume Side light scatter- inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness) Side fluorescence - RNA/DNA information(nucleus) 15. Specific measurements 16. Analysis Parameter Whole WBC Count LYM% MXD% NEUT% LYM# MXD# NEUT# HGB RBC Count HCT MCV MCH MCHC RDW-CV RDW-SD PLT PDW MPV P-LCR Some extended parameters 17. WHOLE WBC DC detection method Wbc count in 1l of whole blood LYM% WBC Small cell ratio Ratio(%) lymphocyte to whole wbc MXD% WBC- Middle cell ratio Ratio(%) of basophil,eosinophil&monocyte to whole wbc 18. NEUT% WBC Large cell ratio Ratio(%) of neutrophils to whole wbc LYM# Absolute count of lymphocte in 1l of whole blood MXD# Absolute count of middle cell in 1l of whole blood 19. NEUT# Absolute count of neutrophil in 1l of whole blood HGB Non cyanide Hb analysis method Volume(gm) of Hb in1dl of whole blood RBC DC detection method Rbc count in 1l of whole blood 20. HCT RBC pulse height detection method Ratio(%) of whole rbc volume in whole blood MCV Mean rbc volume (fl) in whole blood HCT/ RBC MCH Mean Hb volume(pg) per rbc HGB/ RBC 21. MCHC Mean Hb concentration (g/dl) HGB/HCT RDW-CV Rbc distribution width-Coefficient of Variation Calculated from pts. defining68.26%of entire area spreading from peak of rbc particle distribution curve RDW-SD Rbc distribution width- Standard Deviation The distribution width(fl) at ht20% from bottom when peak rbc particle distribution curve is taken 100% 22. PLT DC detection method Platelet count in1l of blood PDW Platelet distribution width Distribution width at ht 20%from bottom with peakof platelet distribution curve taken 100% MPV Mean volume of platelet (fl) P-LCR Ratio of large platelet volume exceeding 12fl to the platelet volume 23. REAGENT DILUENT Cell Pack(approx. 30 ml) WBC/HGB LYSE REAGENT StromatolyserWH(approx. 1 ml) 24. Overview of analysis modes Whole Blood Mode o Analyse collected sample in whole blood status Pre diluted mode o Used in analyzing a minute amount of childs blood e.g collected from earlobe o Blood sample diluted into 1:26 25. Fully automated instruments perform TLC by impedance or light scattering or both. Counts performed on diluted WBC in which red cells are either lysed or rendered transparent. FLAGS Although threshold set to exclude platelet from being counted, giant platelet might be included. NRBC are usually included, hence. Count approx more to TNCC than to WBC. 26. FACTIOUS COUNTS LOW COUNT Leucocyte agglutination HIGH COUNT Failure to lyse RBC Microclots Platelet clumping Abnormal Hb 27. DLC Automated cell counters have a differential capacity of doing three-part, five-part or seven-part differential count Categorization based on different volume of various cells& cytoplasmic shrinkage Three-part differential Can be produced by single channel by both impedance or light scatter instruments. It designates cell as: a. Granulocyte/Large cell b. Lymphocyte/Small cell c. Monocyte/Middle cell 28. Five or Seven part differential Produced by two or more channels. Analysis dependent on volume and other physical characteristic of cell or binding of certain dyes to granules or activity of cellular enzyme. Technology used are Light scattering, Absorbance and Impedence with low or high frequency electromagnetic or radiofrquency current. Cell classification:- a) neutrophil b)eosinophil c)basophil d)monocyte e)lymphocyte extended classification:- *large immature cells *atypical lymphocytes 29. New White Cell Parameter Many instruments can flag presence of atypicalor variant lymphocyte by alteration in size,impedence and light scattering eg: identify eosinophils by ability of their granules to polarize light detect a left shift or presence of blasts by reduced light scattering of nuclei of more immature granulocytes. The ADVIA uses TWO separate channels to determine the white cell count: 1) Peroxidase channel 2) Basophil/lobularity channel 30. PEROXIDASE CHANNEL Specimen is diluted with peroxidase reagents, red cells and platelets are lysed Dark precipitate forms in cells containing peroxidase: Neutrophils, eosinophils, monocytes Lymphocytes, basophils, large unstained cells contain no peroxidase and therefore do not stain A tungsten-based optical system is used to measure: Individual impulses are counted to determine WBC Stain intensity (absorbance) for each cell Cell size (by forward light scatter) 31. Cells are plotted on a scattergram: scatter (size) on yaxis and peroxidase activity (staining intensity) on the x-axis Each cell is classified based on this information In this channel, basophils are classified with lymphocytes Lymphocytes: Small, unstained cells Larger atypical lymphocytes, plasma cells, and some blasts: large unstained cells Eosinophils: Strongest peroxidase activity and appear smaller than neutrophils. Neutrophils are large and have moderate peroxidase activity 32. BASOPHIL/LOBULARITY CHANNEL Surfacant and phthalic acid are added to lyse the RBCs and strip away the cytoplasmic membrane from all leucocytes except the basophils Laser light signal from a low-angle forward scatter plot identifies basophils, which, due to their retained cytoplasm, are larger than other cells A high-angle detector is sensitive to the shape and structure (lobularity and density) of remaining nuclei 33. Flags DLC ordered Automated Differential Performed flags no flags slide made automated DLC reported Review with Review with Microscope Cellavision 34. FLAGS GENERATED BY COMBINATIONS OF THE CHANNELS In addition to numerical data and various flags generated by the peroxidase and the basophil channels individually, other flags are generated by comparison of the channels: Discrepancy of the two WBC counts: Can be due to nonlysis of RBC in peroxidase channel Flag for immature granulocytes: Presence of more peroxidase-positive cells than expected from the number of PMN in basophil channel NRBC flag: Excess of cells in PMN in comparison with sum of eosinophils and neutrophils in peroxidase channel 35. HAEMOGLOBIN Measure Hb by modification of HiCN method with Sodium lauryl sulphate(non-toxic) Modification include : a) alteration in conc. of reagent b) temp & pH of reaction A non ionic detergent used for rapid cell lysis & to reduce turbidity by cell membrane and plasma lipid. 36. Counted on either aperture impedance or light scattering technology Renders red cell indices derived from RBC count ERRORS Inaccuracy of coincidence a. Two cells passing from aperture being counted as one or pulse being genrated during electronic dead time b. Red cell agglutination c. Recirculation of already counted cell d. Counting of bubbles or lipid drops or micro organisums Faulty maintainance Use of diluent other than the one recommended by manufacturer 37. hct & MCV Passage of cell through apperture of an impedence counter or through beam of light of light scattering instrument generates electrical pulse of ht proportional to cell volume No. of pulses generated= RBC count Average pulse ht = MCV MCV RBC Count= HCT OR Summation of pulse ht= HCT MCV= HCT/RBC Count 38. Its seen that apperture impedance system measures apparent volume which is greater than true volume of RBC and is influenced by shape factor < 1.1- young and flexible RBC 1.1-1.2 fixed RBC 1.5 Spheres 39. Reticulocyte Count o An automated retic count can be performed using the fact that various fluoro-chromes combine with the RNA of the reticulocytes. Fluorescent cells can then be enumerated using a flowcytometer. o An automated retic counter also permits the assessment of retic maturity since the more immature reticulocytes have more RNAfluoresce more strongly than the mature retics found normally in PB. o Size and Hb content of reticulocytes are measured by light scatter 40. Same principal of electrical or electro optical detection Upper Threshold needed to separate platelet from RBC Lower Threshold needed to separate platelet from debris and noise 3 technique for setting threshold:- a. Platelet can be counted between two fixed threshold 2 and 20 fl b. Platelet b/w fixed threshold can be counted with subsequent fitting of curve, so that, platelet falling outside threshold hold can be conted in computed count c. Threshold may vary automatically depending on characteristic of individual blood sample to make allowance for microcytic or fragmented RBC 41. NEW METHOD- BY FLOW CYTOMETRY Platelet in a blood sample labelled flourscently with specific monoclonal or combination of antibody and by measuring RBC : Platelet ratio the platelet count can be measured. Antibody suitable for labeling of platelet Antigen are CD41,CD42 and CD61. 42. ERRORS Factiously low count giant platelet identified as RBC EDTA induced platelet clumping Satelitism Factiously high count Markedly microcytic or fragmented RBC Cell fragments in leukemia Bacteria or fungi 43. Mean platelet volume Measured by same technology as for MCV. Dependent on technique of measurement and on length &condition of storage before testing blood. Other parameters are: PDW Plateletcrit= MPV x platelet count. 44. graphics Display of automated cell counters include coloured or black and white graphics Histogram of RBC, WBC & Platelet size Histogram of Red cell Hb concentration Scatter plots of size versus Hb conc 45. TRENDS Current trends include attempts to incorporate as many analysis parameters as possible into one instrument platform, in order to minimize the need to run a single sample on multiple instruments (e.g CD4 counts, smear preparation) Such instruments are being incorporated into highly automated combined chemistry/hematology laboratories, where samples are automatically sorted, aliquoted, and brought to the appropriate technique by a robotic track system 46. Tha nk you