Manual WBC Count PDF: Cell Counting with Neubauer Counting Chamber

CELL COUNTING WITH NEUBAUER COUNTING CHAMBER  INTRODUCTION ▪ The most frequently used haemocytometer is the Neubauer (or ‘Improved Neubauer’) chamber; other haemocytometers include the Burker, Thoma and Fuchs-Rosenthal. ▪ Using these, particles (e.g., WBCs, RBCs, platelets, b...

CELL COUNTING WITH NEUBAUER COUNTING CHAMBER  INTRODUCTION ▪ The most frequently used haemocytometer is the Neubauer (or ‘Improved Neubauer’) chamber; other haemocytometers include the Burker, Thoma and Fuchs-Rosenthal. ▪ Using these, particles (e.g., WBCs, RBCs, platelets, bacteria, fungus spores, pollen) are visually counted under a microscope.  GENERAL FEATURES OF THE NEUBAUER COUNTING CHAMBER ▪ A thick crystal slide with the size of a glass slide (30 x 70 mm and 4 mm thickness). ▪ The surface of the chamber contains two square ruled areas separated by an H-shaped moat. ▪ Each square has a total area of 9 mm2 (1 mm on each side). These squares are divided into nine primary squares with an area of 1mm2. ▪ The four corner primary squares are used when counting WBCs. ▪ The ruled area is 3mm2 divided into 9 large squares each with a 1 mm2 area. ▪ The large central square is divided into 25 medium squares with double or triple lines. ▪ Each of these 25 squares are is again divided into 16 small squares with single lines, so that each of the smallest squares has an area of 1/400 mm2. GLASS COVER ▪ A squared glass of width 22 mm and is placed on the top of the Neubauer chamber, covering the central area. ▪ The chamber is designed so that the distance between the bottom of the chamber and the cover is 0,1 mm. Pile of glass covers, and box Neubauer Counting Chamber Side view of Hemacytometer Neubauer-Improved chamber counting grid detail CELL COUNTING AREAS IN NEUBAUER CHAMBER ▪ WBC COUNTING AREA The four large squares placed at the corners are used for WBC count. ▪ PLATELET COUNTING AREA The large center square is used to count platelets. Platelets in all 25 squares within the large center square are counted. ▪ RBC COUNTING AREA The large center square is used for RBC counts. Of the 25 medium squares, only the four corner squares and the center square within the large center square are used to perform RBC counts.  USING THE NEUBAUER CHAMBER SAMPLE PREPARATION ▪ Depending on the type of sample, a preparation of a dilution with a suitable concentration should be prepared for cell counting. ▪ An appropriate dilution of the mixture with regard to the number of cells to be counted should be used. PREPARING THE NEUBAUER CHAMBER ▪ Clean the Neubauer chamber and the cover slip with 70% EtOH. ▪ Put the glass cover on the Neubauer chamber central area.  INTRODUCING THE SAMPLE INTO THE NEUBAUER CHAMBER A ▪ With a pipette, carefully draw up around 20 µl of the cell mixture. ▪ Place the pipette tip against the edge of the coverglass and slowly expel the liquid until the counting chamber is full. B ▪ Capillary action will help to ensure that the counting chamber is full. ▪ A volume of 10 µl is sufficient to fill one (A) Properly and (B) improperly counting chamber. filled counting chamber  MICROSCOPE FOCUSING AND CELL COUNTING ▪ Using the 10x objective, focus both the grid pattern and cell particles. ▪ The count should start at the uppermost left square. Direction for counting ▪ All cells that lie within the square and those touching the left or upper boundary are to be counted. ▪ As you move from square to square, continuously, but gently, focus up and down using the fine adjustment knob to Examples of cells that are counted in a representative area. see the details of each cell. Hand tally counter White Blood Cells have a rough or grainy surface, not very refractile. May have grayish or bluish tinge. Shape is generally round, but may be rougher or more irregular outer edges.  CALCULATING THE CELL COUNTS ▪ The total number of cells per microliter of sample can be calculated from the number of cells counted and area counted. # Cells/μL = average # cells x correction for dilution # squares counted x volume of one of those squares ▪ The dilution factor used in the formula is determined by the blood dilution used in the cell count. ▪ The depth used in the formula is always 0.1.  MANUAL WHITE BLOOD CELL COUNT  PRINCIPLE ▪ Free-flowing capillary or well-mixed anticoagulated venous blood is diluted with a 3% acetic acid solution, which lyses the mature RBCs but preserves WBCs and platelets. ▪ The standard dilution for WBC counts is 1:20; the dilution is mixed well and incubated to permit lysis of the RBCs. ▪ Following the incubation period, the dilution is mounted on a haemocytometer. ▪ The cells are allowed to settle and counted in the four large squares of the haemocytometer chamber. ▪ The count is multiplied by a dilution factor and reported as number of WBC s per L (x109/L) of blood. SPECIMEN ▪ EDTA-anticoagulated blood or capillary blood. SUPPLIES AND EQUIPMENT ▪ Haemocytometer with cover glass ▪ Petri dish with filter paper ▪ Hand counter ▪ Microscope REAGENTS ▪ Glacial acetic acid 2 mL ▪ Distilled water to 1L ▪ Gentian violet 1% w/v 2 drops PROCEDURE ▪ Put the cover glass on top of the grid area in the chamber ▪ Dilute you sample: 1:20 for WBC count ▪ Load your sample into the loading area in the chamber ▪ Count the cells in the 4 large squares for WBC ▪ Calculate the number of cells counted per L. CALCULATION Cells/L = average number of cells counted x dilution factor number of squares x depth (0.1mm) EXAMPLE: If 150 cells were counted in the four corner squares, the WBC count will be: = 150 x 20 x 1 4x 0.1 = 7,500 cells/mm3 = 7.5 x109/L Counts higher than 50 x 109/L ▪ When a count is higher than 50 x 109/L , repeat the count using 0.76 ml of diluting fluid and 20 L of blood; multiply the result by 2. Counts lower than 2.0 x 109/L ▪ When a count is lower than 2 x 109/L , repeat the count using 0.38 ml of diluting fluid and 40 L of blood; divide the result by 2. Correcting a WBC count when there are many nucleated RBCs ▪ When more than 10 nucleated RBCs per 100 WBC are present in the blood film, correct the WBC count as follows: Corrected WBC count = Uncorrected WBC count x 100 Nucleated RBCs* + 100 REFERENCE ADULT 2.5 – 8.5 x 109/L NEWBORN 9.0 – 30.0 x 109/L (18.1 x x109/L mean)  SOURCES OF ERROR ▪ Incorrect measurement of blood due to poor technique or using a wet or chipped pipette. ▪ When using anticoagulated blood, not mixing the blood sufficiently or not checking the sample for clots. ▪ Inadequate mixing of blood with diluting fluid. ▪ Not checking whether the chamber and cover glass are completely clean. ▪ Not using a haemocytometer cover glass. ▪ Over-filling a counting chamber or counting cells when the sample contains air-bubbles. ▪ Not allowing sufficient time (2 minutes) for the cells to settle in the chamber. ▪ Using too intense a light source or not reducing the iris diaphragm sufficiently to give good contrast (poor focusing and difficulty in seeing clearly the cells and rulings are common when using non-metallized haemocytometers). ▪ Not completing counting of the cells before the sample begins to dry in the chamber. ▪ Counting too few cells. ▪ Precision increases with the number of cells counted. ▪ Not correcting a count when the sample contains many nucleated RBCs.  COMMENTS ▪ WBC counts should be performed within three hours following dilution. ▪ The moistened filter paper retards evaporation of diluted specimen while cells settle on the haemocytometer. ▪ Technical sources of error in haemocytometer cell counts are: a. Errors resulting in false low count: I. Difficulty in obtaining capillary blood – cells become diluted with plasma. II. Excessive tissue trauma during collection of blood resulting in cell clumping. ▪ The presence of nucleated RBCs results in a falsely elevated WBC count, since they are indistinguishable from WBCs at 100x magnification and are preserved in 3% glacial acetic acid. ▪ A WBC differential must be performed to distinguish between nucleated erythrocytes and leukocytes.  EXERCISE EXAMPLE 1 WBCs counted in 4 corner large squares. Blood sample diluted 1/100. Side 1 = 27 Side 2 = 29 Average = 28 END