AUTOMATED CELL COUNTER.pdf

AUTOMATED CELL COUNTER Practical Session By Asmaa Ahmed Hewila Resident Of Clinical &Chemical Pathology Sample Required 1- Venous blood: Anticoagulant of choice is dried dipotassium EDTA. ✓ Plastic tubes with spray-coated EDTA are available (violet or lavender top).. ✓ With excess...

AUTOMATED CELL COUNTER Practical Session By Asmaa Ahmed Hewila Resident Of Clinical &Chemical Pathology Sample Required 1- Venous blood: Anticoagulant of choice is dried dipotassium EDTA. ✓ Plastic tubes with spray-coated EDTA are available (violet or lavender top).. ✓ With excess EDTA, platelets swell and then disintegrate, causing an artefactually high platelet count. ✓ Less EDTA causes formation of microclots that leads to artificially low platelet count. ✓ The anticoagulant is mixed with blood by 8–10 inversions of tube. ✓ CBC should be done within 2 hours of blood collection. 2- Capillary blood: ✓ In infants and very young children, it is collected from heel, while in older children and adults it is collected from middle or ring finger of non-dominant hand. Special dipotassium EDA-coated tubes are available. ✓ Platelet counts on capillary blood are lower (by 9– 32%) than those on venous blood because some platelets immediately adhere to endothelium of damaged vessels and aggregate ✓ The instrument aspirates 30 μL of patient sample. Principles  Electrical Conductivity (The Coulter Principle) Optical Scatter Flow cytometry is an excellent method to determine the five- part WBC differential. Fluorescence flow cytometry: It is useful for analysis of platelets, nucleated RBCs, and reticulocytes. Peroxidase-based cell counter. Immunological-based cell counter Hydrodynamic Focusing  This is a technique that narrows the stream of cells to single file, eliminating data above and below the focus points. Hydrodynamic focusing allows greater accuracy and resolution of blood cells. Diluted cells are surrounded by a sheath fluid, which lines up the cells in a single file while passing through the detection aperture. After passing through the aperture, the cells are then directed away from the back of the aperture This process eliminates the recirculation of cells and the counting of cells twice. Electrical impedance (Coulter principle) Cells are sized and counted by detecting and measuring changes in electrical resistance when a particle passes through a small aperture. A blood sample is diluted in saline, a good conductor of electrical current, and the cells are pulled through an aperture by creating a vacuum. Two electrodes establish an electrical current. The external electrode is located in the blood cell suspension. The second electrode is the internal electrode and is located in the glass hollow tube, which contains the aperture. Low-frequency electrical current is applied to the external electrode and the internal electrode. 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. This change in voltage generates a pulse. The number of pulses is proportional to the number of cells counted. The height of the voltage pulse is also directly proportional to the volume or size of the cell. ⚫ Threshold limits are established for enumerating cells based on cell volume. ⚫ Particles (cells) of more than 36 fL are counted as erythrocytes (RBCs). ⚫ This was the principal parameter used in earlier analyzers for characterizing all cell types, but it is now used primarily for counting and sizing red blood cells and platelets. Optical Scatter A sample of blood is diluted with an isotonic diluent and then hydrodynamically focused through a quartz flow cell. Cells pass through a flow cell on which a beam of light is focused The light source is a laser light that is light amplification by stimulated emission of radiation. Laser light or monochromatic light is emitted as a single wavelength. As the cell passes through the sensing zone, light is scattered in all directions. The scattered light is detected by a photomultiplier or photodiode sense and collect the scattered rays at different angles. These data are then converted to an electric pulse. Flow cytometry Passing of single cell through a laser source. Measurement of scattered and emitted fluorescence using detectors When the laser strikes the cell, light is diffracted around the edges of the cell,producing a diffraction pattern along the path of the laser beam. This scattered light is approximately equivalent to the cell circumference and is the same wave- length as the exciting laser light. ) Termed forward scatter. ( it is collected along the same axis as the laser beam. Because this is a very strong signal, a simple photodiode can convert the forward- scattered light into an electrical pulse.  Fluorescence Flow Cytometry Data presentation ⚫ Most analyzers display two types of data: Graphic displays usually for internal laboratory review) and a series of Numerical values (for reporting to the clinician). ⚫ Graphic displays include: histograms (red cell, platelet, and WBC) in which relative numbers of red cells, white cells, and platelets are plotted against cell size, and scatter-plots (display of 5-part white cell population). ⚫ Flags” are signals that occur when an abnormal or out of range result is detected by the analyzer. Flags are displayed to reduce false positive and false negative results by mandating a review of blood smear examination RBC Parameters : 1.Red blood cell count Typically one channel is used to detect RBCs and platelets. Red cell count and cell volume are directly measured by aperture impedance or light scatter. The detector is set such that any cell between 2 and 30 fL will be counted as a platelet and any cell between 36 -360 fl will be counted as a red cell.  If there are large platelets, these will be counted as red cells and will also result in a falsely low platelet count. Similarly, if there are fragmented red cells, these smaller red cells will be counted as platelets. falsely elevated RBC count result from Very high WBC, numerous large platelets. falsely low RBC results from cold agglutinins and extreme microcytosis. 2. Hemoglobin measurement Hemoglobin is measured directly by a modification of cyanmethemoglobin method (all hemoglobins are converted to cyanmethemoglobin by potassium ferricyanide; cyanmethemoglobin has a broad absorbance peak at 540 nm By Spectrophotometry  falsely elevated hemoglobin level can result from Poorly mixed sample, high WBC count, hyperlipidemia, hypergammaglobulinemia. Spectrophotometric method is used after the red cells are lysed in WBC /HB chamber 4.RED CELL INDICES Both MCH and MCHC are both calculated values ✓ MCH measures both the red cell size and hemoglobin concentration. Low MCH indicates hypochromia or microcytosis or both A high MCH often coexists with high MCV, and thus, indicates macrocytosis.  ✓ MCHC is the most important parameter which will alert the user about the possibility of a spurious result since it is derived from multiple parameters. ✓ A high MCHC result can be caused by following factors: 1-Plasma : Cold agglutinin. 2-Red cells : Spherocytosis, sickle cell disease. 3-Technical: Improper mixing of sample ✓ MCV is a measure of red cell size; ✓ A low MCV indicates microcytosis and a high MCV indicates macrocytosis. ✓ Therefore, MCV is used for morphological classification of anemia into microcytic, macrocytic, and normocytic types. Falsely high MCV can result from storage of blood at room temperature, cold agglutinins, and very high WBC 5.RED CELL DISTRIBUTION WIDTH ⚫ RDW is a measure of the degree of variation of size of red cells, i.e., it reflects extent of anisocytosis. ⚫ Two RDW measurements are currently in use, RDW-CV (coeffificient of variation) and RDW- SD (standard deviation). ❖ RDW-CV = One SD of mean cell size/MCV, multiplied by 100. Normal range is 11%-15%. ❖ RDW-SD: It is an actual measurement of the width of the red cell distribution in femtoliter at the point, 20% above baseline. Normal range: 40-55 fL ⚫ The RDW expressed as the CV has been found of some value in distinguishing between : ✓ iron deficiency (RDW usually increased) and thalassaemia trait (RDW usually normal) and normal in anemia of chronic disease. ✓ megaloblastic anaemia (RDW often increased) and other causes of macrocytosis (RDW more often normal) WBC Parameters : 1-WHITE BLOOD CELL COUNT ⚫ The WBC is determined in whole blood in which red cells have been lysed. ⚫ Particles larger than 35 fL are counted as leukocyte by impedance method. ⚫ Factitiously low automated WBCs a consequence of 1. leucocyte agglutination, 2. prolonged sample storage or 3. abnormally fragile cells (e.g. in leukaemia). ⚫ Factitiously high counts are more common and usually result from failure of lysis of red cells. platelet clumping With certain instruments this may occur with the cells of neonates presence of an abnormal haemoglobin such as haemoglobin S. Microclots. 2-WBC Differential ⚫ Most automated differential counters that are now available use flow cytometry incorporated into a full blood counter rather than being stand-alone differential counters. ✓ cells with volume 35–90 fl are designated as lymphocytes, ✓ cells with volume 90–160 fl as mononuclear cells (monocytes, blasts, immature granulocytes, and reactive or atypical lymphocytes), ✓ cells with volume 160–450 fl as neutrophils A three-part differential count assigns cells to ⚫ categories usually designated ‘granulocytes’ or ‘large cells’ ‘lymphocytes’ or ‘small cells’ ‘monocytes’, ‘mononuclear cells’, or ‘middle cells’. ⚫ Five- to seven-part differential counts ⚫ classify cells as neutrophils, eosinophils, basophils,monocytes according to granularity and nuclear complexity. Platelet Parameters : PLATELET COUNT ⚫ Platelets are counted by electrical impedance method in the RBC aperture,.. If platelet count is low on analyzer, then its accuracy must always be confirmed by blood smear examination. ✓ platelets can be counted between 2 and 30fl) ❖ Factitiously low impedance platelet counts (Pseudothrombocytopenia) may be the result of giant platelets being identified as red cells EDTA-induced platelet clumping EDTA-dependent platelet satellitism ✓ The last two conditions are typically diagnosed when the slide is reviewed, blood should be recollected in citrate or heparin. ❖ Factitiously high platelet counts may be due to Markedly fragmented red cells, white cell fragments in leukaemia bacteria or fungi Mean platelet volume ⚫ The same techniques that are used to size red cells can be applied to platelets. ⚫ The mean platelet volume (MPV)s derived from the impedance platelet size distribution curve by drawing a vertical line from the peak to the baseline. ✓ Normal MPV is 7–11 fl. ❖ Increased MPV >11 fl results from presence of immature platelets in circulation; peripheral destruction of platelets stimulates megakaryocytes to produce such platelets (e.g. in idiopathic thrombocytopenic purpura). Decreased MPV (