Hematology Lab - 02 Hemocytometry

HEMATOLOGY LABORATORY HEMACYTOMETRY OUTLINE • • • • • Hemacytometry Materials WBC Secondary Squares RBC Secondary Squares Procedure HEMACYTOMETRY • • Manual quantitative evaluation of formed elements of the blood (RBC, WBC, Platelets). Principle: o A small measured quality of blood is diluted with fluid (diluting fluid), which prevent coagulation, preserves the cell type to be counted, and eliminates the other cell types. The diluted blood is placed in a counting chamber and the number of cells in a known volume is counted with microscope. • • • Shaft - elongated part before bulb Bulb - bulged part in the middle Volume RBC pipette Shaft volume 1 Bulb volume 100 Total Volume 101 WBC pipette 1 10 11 MATERIALS • • • • • Compound microscope Thoma pipettes/diluting pipettes o RBC pipette - manual RBC counting o WBC pipette - manual WBC counting Diluting fluid Counting chamber/Hemacytometer Coverslip THOMA PIPETTE • • aka Diluting pipette Used to contain the blood sample to be diluted with diluting fluid RBC pipette WBC pipette Use For manual RBC For manual counting WBC counting Markings 0.5, 1, 101 0.5, 1, 11 Color of bead Red White Volume in the bulb 100 10 • DILUTING FLUID • • Bulb Shaft Syringe barrel - aspirator for pipette This lessen the concentration of blood sample in order to disperse the blood cells and facilitate its counting under the microscope Criteria: o RBC diluting fluid - isotonic ▪ Preserve morphology and structure of RBCs ▪ Preservation allow proper and accurate counting o WBC diluting fluid - hypotonic ▪ Hypotonic solution lyses RBC to leave WBC o Platelet diluting fluid – must preserve platelet integrity and inhibits aggregation ▪ Platelet has tendency to become dysmorphic & clumped ▪ Stains platelet for easy visualization o Has good preservative property ▪ Should not initiate growth of mold & yeast o Does not initiate growth of molds/yeasts o High specific gravity o With buffering action o Cheap and easy to prepare COUNTING CHAMBER • • • aka hemacytometer/hemocytometer “heart” of manual blood cell counting Improved Neubauer Counting Chamber o The most commonly used counting chamber IMPROVED NEUBAUER COUNTING CHAMBER • Characteristic: o Depth: 0.1 mm (1/10 mm) ▪ Distance between counting chamber & coverslip o Has two counting area (primary square) ▪ Area: • Primary Squares: 9.0 mm each • Secondary Squares: 1.0 mm each o 4 corner secondary squares – used for manual WBC counting ▪ Subdivided into 16 tertiary square o 1 central secondary squares – used for manual RBC counting ▪ Subdivided into 25 tertiary square ▪ Each RBC tertiary squares are further divided into 16 quaternary squares AREA OF WBC SQUARES • • • 1° square: 9.0 mm2 2° square: 1.0 mm2 WBC 3° square: o 16 per 1 secondary square o Area: 0.0625 mm2 RBC SECONDARY SQUARES WBC SECONDARY SQUARES • 4 corner secondary (2°) squares o o Get sum/total cells in squares in 1 counting area Get average of the total cells in both counting area RBC WBC AREA OF RBC SQUARES • • • • 1° square: 9.0 mm2 2° square: 1.0 mm2 RBC 3° square: o 25 per central 2° square o Area: 0.04 mm2 RBC 4° square: o 16 per RBC 3° square o Area: 0.0025 mm2 GENERAL FORMULA • • • PROCEDURE 1. 2. 3. 4. 5. 6. 7. 8. Draw blood up to the 0.5 mark of the thoma pipette (RBC/WBC) Wipe the tip of the thoma pipette o To prevent blood contamination of diluting fluid Draw diluting fluid up to the mark above the bulb of the pipette (RBC: 101; WBC: 11) Shake the pipette for 5 minutes Discard few drops (1-2 drops) o To remove fluid in the shaft o Fluid in the shaft may be pure diluting fluid Charge the counting chamber Stand for 3 minutes o Ensures that blood cells are no longer moving Count cells on both counting area o RBC count: 5 tertiary RBC squares (4 corners, 1 central) o WBC count: 4 secondary WBC squares (4 corners) • Cell count = # of cell counted x dilution factor Depth x Area o Dilution factor - reciprocal of dilution Computation in RBC pipette 0.5 - blood 101 - total volume 0.5:101 = 1:202 - initial dilution 1:200 - final dilution (due to discarding diluting fluid) Computation in WBC pipette 0.5 - blood 11 - total volume 0.5:11 = 1:22 - initial dilution 1:20 - final dilution (due to discarding diluting fluid) Unit: cells/mm3 Note: o Count WBC or RBC on both counting area (primary square) of the hemacytometer and get the average. o Allowable difference between two chambers: ▪ RBC: 15-16 counts ▪ WBC: 10-12 counts ▪ Exceeding will require repeat procedure ▪ E.g. 1° #1 = 105 RBC (5 RBC 3° sq.) 2° #2 = 85 RBC (5 RBC 3° sq.) Difference = 20 → repeat procedure Sample Problem 1: Compute for the RBC count if an average of 576 RBC is counted in the 5 tertiary RBC squares of the central secondary squares of both counting area of the hemacytometer. The dilution is 1:200. o # of cells counted: 576 o Dilution: 1:200 o Area: 0.04mm x 5 = 0.2mm o Depth: 0.10mm • Sample Problem 2: Compute for the WBC count if an average of 201 WBC is counted in the 4 secondary WBC squares of both counting area of the hemacytometer. The dilution is 1:20. o # of cells counted: 201 o Dilution: 1:20 o Area: 1.0mm x 4 = 4.0mm o Depth: 0.10mm