Hemocytometer PDF

HEMATOLOGY 1 HEMACYTOMETER WEEK 2 LABORATORY WHAT IS HEMOCYTOMETRY? It is a technique used to enumerate the total cell count in the BLOOD or other Biological body fluids. This can be done either by using Hemocytometer manually or by Electronic cell counter automatically. HEMO = BLOOD CYTO = CEL...

HEMATOLOGY 1 HEMACYTOMETER WEEK 2 LABORATORY WHAT IS HEMOCYTOMETRY? It is a technique used to enumerate the total cell count in the BLOOD or other Biological body fluids. This can be done either by using Hemocytometer manually or by Electronic cell counter automatically. HEMO = BLOOD CYTO = CELL METRY = MEASUREMENT/COUNT PRINCIPLE OF CELL COUNTING Since the No. of blood cells is very high, it is difficult to count them even under microscope. This difficulty is partly overcome by diluting the blood to a known degree, with a suitable diluting fluids and then counting them. The sample of blood is diluted in a special pipette and the diluted blood drop is then placed under cover slip placed over special thick glass slide i.e. counting chamber. The drop of diluted blood spread under cover slip in single layer and make the counting of cells easy. Knowing the dilution applied, the no. of cells in a undiluted blood can be calculated and the result so obtained is expressed as cells per cubic mm. PURPOSE In certain pathological conditions the value of different type of cells may have the variation. Thus by counting the cells in the blood or body fluids it can be find out if an individual is normal or not . Broadly, The cell count is done To find out normal and abnormal count of the cells . To support and confirm clinical diagnosis of the patient . To find out the response of the patient to the treatment. HEMOCYTOMETER The hemocytometer was invented by Louis-Charles Malassez, It was modified by Neubauer and now a days improved Neubauer counting chamber is used . It is a special type of microscope slide consisting of two chambers. This is an instrument used for counting the cells in blood or fluid. COUNTING CHAMBER The center portion of chamber has double ruling area separated by troughs (these four troughs are extending across the slide and set parallel to each other. The fifth one is separating the two ruling areas from each other). The Central platform is slightly lower then the side ridges so when the glass cover slip is placed over ridges on either side of the vertical grooves of the H shape it held's at 0.1 mm above the surface of the counting grid. This gives the depth of chanter i.e 1/10 mm COUNTING CHAMBER COUNTING CHAMBER The center portion of chamber has double ruling area separated by troughs (these four troughs are extending across the slide and set parallel to each other. The fifth one is separating the two ruling areas from each other). The Central platform is slightly lower then the side ridges so when the glass cover slip is placed over ridges on either side of the vertical grooves of the H shape it held's at 0.1 mm above the surface of the counting grid. This gives the depth of chanter i.e 1/10 mm COUNTING GRID Each scale is 3mm wide and 3mm long. (9mm2 total area) The whole scale is divided into 9 big squares. Each square is 1 mm long and 1 mm wide. (1mm2 area) COUNTING GRID Red square = 1 mm^2 Green square = 0.0625 mm^2 Yellow square = 0.04 mm^2 Blue square = 0.0025 mm^2 CORNER SQUARES The four corner squares are further divided into sixteen smaller squares These squares are used for WBC counting. Total = 64 small squares CALCULATION OF THE VOLUME OF WBC SQUARES Length of one Corner square 1 mm Width of one Corner square 1 mm Area of one Corner square 1 mm2 Area of 4 Corner square 4 x 1 mm2 = 4mm2 As the Depth of Chamber is 1/10mm Therefore the Volume of diluted blood in 4 small square will be Area X Depth 4mm2 x 0.1mm 0.4mm3 CENTER SQUARE The central square is subdivided into twenty five (25) smaller squares and each of these smaller squares is further subdivided into sixteen smallest squares. This means that each smaller square is of (1/25mm2), 1/5mm wide and 1/5mm length. These squares are used for platelet and RBC counting. The platelets are counted in all the small squares of the central square, while the RBCs are counted in five small squares, four of corners and one of center (total 80 smallest squares). CALCULATION OF THE VOLUME OF RBC SQUARES Length of one Corner small square 0.2 mm Width of one Corner small square 0.2 mm Area of one Small Corner square 0.04 mm2 Area of 5 small square 5 x 0.04 mm2 = 0.2 mm2 As the Depth of Chamber is 1/10mm Therefore the Volume of diluted blood in 5 small square will be Area X Depth 0.2 x 0.1mm 0.02 mm3 RBC AND WBC PIPETTES PARTS OF DILUTING PIPETTES THE STEM the long narrow stem has capillary bore and well grounded conical tip. It is divided into 10 equal parts (graduations) but has only two marking of 0.5 in middle of stem & 1 at the junction of stem and bulb. BULB the stem at its upper portion widen into bulb which contains rolling bead that helps in mixing of blood with diluting fluids and helps in quick identification of pipette as the color of bead is red in RBC pipette while white in WBC pipette. THE RUBBER TUBE & MOUTH PIECE the bulb narrows again into short stem to which long narrow rubber tube (25 to 30 cm) with red mouth piece in RBC pipette & white mouth piece in WBC is attached. These are used to suck blood and diluents to the pipette. PARTS OF DILUTING PIPETTES SMALLER STEM marking of 11 in WBC and 101 in RBC pipette is etched. DIFFERENCES BETWEEN RBC AND WBC PIPETTE Differences RBC PIPETTE WBC PIPETTE Color of bead red white calibrations 0.5, 1.0 & 101 0.5, 1.0 & 11 size of bulb bigger smaller size of lumen narrow wider color of mouthpiece red white dilutions up to 100 or 200x up to 10 or 20x RBC PIPETTE WBC PIPETTE CHARGING OF COUNTING CHAMBER A measured unit of blood is diluted quantitatively with diluents (Hyaem's fluid for RBC and Turk's fluid for WBC counting)by using measuring devices ( i.e pipettes) Then mixed the content in the bulb by rolling the pipette between your both hands palms. Discard first 2 to 3 drops (fluid in stem because dilution takes place in bulb). Then this diluted blood is released smoothly after forming a drop onto the central platform of the chamber beneath the cover slip by placing the tip of pipette on the edge cover slip at the angle of 45 degree. Allow the fluid to flow under the cover slip through the capillary action and forming a uniform film. Wait for 2-3 minutes after charging to settle down cells on counting grid of chamber. CHARGING OF COUNTING CHAMBER For WBC counting 0.5 part of blood is mixed in 10 parts of Turks fluid Initial volume blood 0.5 and final volume 10 (11-1= 10) Thus, dilution factor for WBC counting is FV/IV = 10/0.5 = 20. For RBC counting 0.5 part of blood is mixed in 10 parts of Turks fluid Initial volume blood 0.5 and final volume 100 (101-1= 100) Thus, dilution factor for WBC counting is FV/IV = 100/0.5 = 200. IDEALLY CHARGED CHAMBER An ideally charged chamber is completely filled with diluted blood over counting grid under cover slip. If during charging diluted blood flows into the side grooves then it is known as over charging of chamber. If the diluted blood drop is insufficient to cover the counting grid or any air bubble then it is called as under charging . FOCUSING 4X to see the general formation of slide. 10X for WBC counting 40X for RBC counting COUNTING RULE Count the cells touching the triple lines of the left side and on the top of the square in the same square. Count the cells touching Bottom line, Right line in adjacent square.