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1 Prepared by Yasser El-dahdouh RBC Manual CountPrepared by Yasser El-dahdouh
2 HEMOCYTOMETER Hemo: blood Cyto: cell Meter: measurement/counter Thus, it is an instrument used to count the blood cells.
3 *** Hemacytometers a precision-made slide for performing manual cell counts with the aid of amicroscope.*** Hemacytometers are used when……1- automated cell counters and hematology analyzers are unavailable2- blood cell counts are extremely low3- to get a cell count for body fluids (spinal fluid, jointfluid, semen counts, and other bodily fluids)The most commonly used hemacytometer is the Neubauer chamber.
4 It includes:Neubauer’s slideCover slipRBC pipetteWBC pipette
5 NEUBAUER’S SLIDE** It is the name given to a thick glass slide. In the centre of the slide, there is anH- shaped groove. On the two sides of the central horizontal bar, there are scales for counting the blood cells.** The depth of the scales is 1/10mm or 0.1mm.
6 ** Each scale is 3mm wide and 3mm long. ** The whole scale is divided into 9 big squares.** Each square is 1mm long and 1mm wide.
7 Differences Between RBC AND WBC Pipette. RBC pipette WBC pipette 1)It has a red beadIt has a white bead2)It has graduations upto mark 101It has graduations upto mark 113)Size of bulb is largerSize of bulb is smaller
11 ** Clean the Neubauer chamber and the cover slip with 70% ethanol. The numberswritten on the chamber mean that the space between the chamber and the cover slip is mm andthat the smallest square on the grid has an area of mm2.Clean**The numberswritten on the chamber mean that the space between the chamber and the cover slip is mm andthat the smallest square on the grid has an area of mm2.** Clean the Neubauer chamber and the cover slip with 70% ethanol.
12 With the microscope, using a 4x objective, identify the nine main squares of the counting chamber delimited by three lines each as shown in the following image:
13 **The counting is performed in the ** Now change to 10x objective and focus one of the 9 main squares.**The counting is performed in thearea delimited by three lines.** Cells that touch the upper and left border are counted (black color) whilstcells that touch the right and lower border are not counted (red color).
14 The smallest square has an area of 0 The smallest square has an area of mm2 therefore each main square has an area of 0.04 mm2( x 16 = 0.04).
15 Erythrocyte Count Normal ranges: Is the number of erythrocytes per micro litter of blood.Normal ranges:Male – ^6/µLFemale – ^6/µLNew born ^6/µLErythrocyte count increased in case of polycythemia and decreased in anemia.
16 Erythrocyte Count Principle: In order to facilitate RBCs count a specified volume of blood is diluted with a specified volume of isotonic fluid.Red cell diluting fluid must be:anti-coagulant anti-hemolysis.anti-aggregation.anti-Rouleaux.preserve RBC shape.
17 Erythrocyte Count Diluting fluid: ** Dilution with normal Saline: One of the following solutions may be used:Isotonic saline:0.85% sodium chloride (NaCl) in distilled water.** Dilution with normal Saline:Maintain the normal disk shape of the RBCPrevents autoagglutinationHayam’s solution:Sodium Sulphate g.Sodium Chloride 2g.Mercuric Chloride g.Distilled Water mlNormal saline causes slight creation red blood cells and allows rouleaux formation.
18 4- Citrate-formalin solution: 3- Gower’s solution:Sodium Sulphate g.Glacial acetic acid mlDistilled water ml.4- Citrate-formalin solution:Tri-sodium CitrateFormalin.Formalin acts as preservative and checks bacterial and fungal growth.Sodium citrate prevents coagulation and provider correct osmotic pressure.
19 Note: Sample: Equipments: In certain conditions, Hayam’s solution may cause clumping of RBCs and Rouleaux formations, while Gower's solution prevents these problemsSample:Whole blood using EDTA or heparin as anticoagulant. Capillary blood may also be used.Equipments:(Pipettes) used one of the following:Thoma pipette (RBCs)Micropipette –20l is the desired volume.Improved Neubauer chamber with the cover slips.Conventional light microscope.Clean gauze.
20 Procedure: 1. Dilution of the blood: Micropipette (20) 1:201 dilution.Pipette 4.0ml of diluting fluid into a tubePipette 20l of will mixed anticoagulated whole blood to the tube.Thoma red count pipette.Draw the blood up to exactly the 0.5 mark and dilute to the 101 mark.Mix continuously for 2-3 minutes.Load the cleaned hematocytometer.Place the hematocytometer on the microscope stage, lower the condenser.
21 Procedure:Focus with x10 objective lens on the large central square. This square is ruled into 25 small squares, each of which is further divided into 16 smaller squares, of the 25 squares, only the four corner squares, and one middle square are used to count RBCs.Switch to 40 objective lens, and start counting in the five designated squares.
22 The large center square is used for RBC and PLT counts. The center square is divided into 25 smaller squares, which are each subdivided into 16 squares.Only 5 of the 25 squares are used to count red blood cells.These 5 are usually the 4 outer squares and the inner most center square.The entire large center square is used to count platelets.
25 Preparation of the slide Step 2Dilute the sample 1:200Step 1Place the cover slipStep 4Count in the small 5 center squares for RBCStep 3Load Step
26 Calculations: Total RBC Count = Where: Counted Volume: So, N x Dilution Factor x Volume Correction FactorWhere:N = the total number of red cells counted in the counting chamber.Dilution 1: 200Dilution Factor = 200Counted Volume:Each counted square has a volume of 0.2 X 0.2 X 0.1 = 0.0045 squares volume = 5 X = 0.02 cummVolume correction factor = 1/0.02 = 50So,Total RBC count =N X 200 X 50 = N Xcalculation.Count the number of cells in the small 5 squares in the center of hemocytometerThese 5 squares equals to 1/5 of the wholeCenter square= number of cells counted * dilution factor(200)/ volume(0.1)*1/5OR: number of cell counted x dilution factor x 5/volume (0.1)= cell/μL
27 Sources of errorsFalsely high counts:-* collection of blood from the area where there is hemoconcentration.* improper pipetting of blood as well as the fluid.* improper mixing.*Errors in calculation.Falsely low counts:-* Blood dilution with tissue fluid due to edem.* improper pipetting and dilution.* errors in calculation.
28 DiscussionIn certain conditions, such as polycythemia, the red blood cell count may be extremely high, which makes it difficult to obtain an accurate count. In this instance, make a larger dilution of blood. For a 1:301 dilution, add 20L of whole blood to 6.0mL of diluting fluid.For a patient who has severe anemia and in whom the RBC is low, make a 1:101 dilution by adding 20L of whole blood to 2.0mL of diluting fluid.
29 DiscussionMake certain the pipettes, hemacytometer, and cover glass are free from dirt, lint, and dried blood. Ensure that the diluting fluid is free from blood and other contamination.RBC takes longer to perform than a WBC because of the larger number of cells. Therefore, proceed as quickly as possible once the cells have settled. Drying of the dilution in the counting chamber causes inaccuracies in the final cell count.The range of error for a manual RBC is generally about 10 to 20%