Hematology 3: Leukocyte Count, Peripheral Blood Smear

(HEMATOLOGY-3) (Leukocyte Count, Peripheral Blood Smear) 1. COUNT TYPES OF LEUKOCYTES (Differential Count) Principle HJ or DC is identifying and counting the types of leukocytes in at least 100 cells, and expressed in %. Tools & Reagents Peripheral blood smear Work Procedures 1. Identification is carried out in the counting area. 2. Identify cells starting from one side moving to the other side, then returning to the original side in a zigzag direction-3 fields of view. 3. To make calculations easier, make boxes as follows 4. The type of leukocyte first seen is entered from the columns1, if the number of cells is 10, move to column 2. 5. Each column contains 10 cells that have been identified, and if all 10 columns are filled, it means that 100 leukocytes have been identified and counted. 2. EDGE BLOOD WIPE (HDT) Principle A drop of blood is exposed on a glass object, then stained, and then evaluated. Tools & Reagents 1. Glas Objects The object glass must be clean, dry and not greasy. The surface must be flat and smooth. If it is dirty, it must be washed first with soap or alcohol, then dried with a dry and clean cloth or cotton. 2. Glass Eraser Can be made from object glass by removing the corners. The edges should be even and clean. Work Procedures 1) Place one drop of blood sample on one end of the glass object. 2) Hold the wiper glass in such a way that the blood sample is at an angle between the object glass and the wiper glass (angle of 30O-45O) 3) Wipe the wiper glass towards the blood drop so that it touches it and the blood drop will be evenly distributed between the tip of the wiper glass and the object. 4) Slide the wiper glass in the opposite direction to the first direction. In this way, the blood droplets will spread evenly over the object glass as a thin layer. 5) Dry the wipe immediately by moving it in the air or using a fan, but don't blow it with your breath. 6) The thickness of the blood layer depends on -Slow movements or angles smaller than 30Owill produce a thin layer of - blood and otherwise a fast wiper or an angle greater than 30O - will produce a thick layer of blood. 7) Leukocytes should not be clustered at the end (feather edge) from deletion. If this happens then the distribution of the various leukocytes is not representative. Movement that is too slow or a dirty wiper glass can cause this error. 8) Drying the wipe immediately is very important. 9) If not, the erythrocytes will be damaged (crenation) and makes it easier to formrouleaux(rulo).The size of the blood dropQuickly, we move the eraser glass to the angle between the eraser glass and the object glass HDT staining There are many kinds of dyes that can be used. Usually one of the Romanosky dyes (Giemsa, Wright) is used. - Dye Reagent - Wright Stain This dye contains eosin and methylene blue with methanol as a solvent. - Phosphate Buffer pH 6.4 This phosphate buffer consists of KH2PO4 and Na2HPO4 -KH2PO4 ……………….... 6.63 g -Na2HPO4 ……………….. 3.20 g -Aquades ad ……………… 1 L Staining Work Procedure 1) The dried smear is fixed by dripping Wright's dye onto the blood smear so that the smear is completely covered.Fixation time is approximately 2 minutes. HDT Quality Assessment 2) In a good smear the erythrocytes are red orange and the leukocytes are blue and have purple nuclei. 3) If the erythrocytes are blue, this is because the buffer is too alkaline or the washing is not clean enough. 4) If the cell nucleus is not purple but blue, this is due to insufficient coloring. 5) If the staining is carried out for too long, then washed excessively, the cell nuclei are still visible but the cytoplasmic granules are no longer visible. 6) To avoid precipitationmetallic scumWhen smearing, do not tilt the slide to remove the dye. 7) The dye must be washed away by pouring enough distilled water, while the smear remains in a horizontal position on the staining rack. 8) Fixation and coloring times can be changed depending on the quality of the dye used. HDT Quality Assessment HDT quality assessment is carried out using small magnification (objective 10X), including: -The blood layer must be thin enough so that the erythrocytes and leukocytes are clearly separated from each other. -The smear should not contain color deposits. -Erythrocytes and leukocytes should be well stained. -Leukocytes should not cluster at the end of HDT. -If the HDT does not meet the requirements mentioned above, a new HDT should be made, so that it does not make it difficult to evaluate. HDT examination The HDT examination consists of, 1) Examination with small magnification (Objective 10X) -HDT quality assessment - Interpretation of the number of leukocytes and erythrocytes -Interpretation of the leukocyte type nose count -Check for young and abnormal cells 2) Inspection with immersion oil (objective 100X) -Erythrocytes: are there any abnormalities or morphological variations -Leukocytes : Count types Look for morphological abnormalities -Platelets: interpretation of their number and morphology - Abnormal cells: morphological examination.