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Hemacytometer and Manual Cell Counts Kimberly Murray University of Phoenix EDTC 560: Applications of Multimedia and Web Page Design June 4, 2007

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Quote of the Day “Education is a technique employed to open minds so that they may go from profound ignorance to thoughtful uncertainty” (Mitner, 1999).

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Objectives Upon completion of this course, the student will: Identify the dimensions of the Neubauer hemacytometer. Identify sources of error in counting. Calculate manual cell counts.

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Hemacytometer Counting Chamber A precision tool designed to standardize counting. Used to count cells in peripheral blood or other body fluids.

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Basic hemacytometer formula Number of cells counted X dilution factor total volume of the cells counted total volume = # primary squares x 0.1 (depth) This formula can be used with any counting chamber as long as one knows the dimensions for the chamber

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Fill trough Chambers or Counting grid Cover slip –NBS standardized Hemacytometer Components Moat Diagrams by K. P. Murray, 2007

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Moat 0.1 mm Depth Cover slip –NBS standardized The design of the hemocytometer is to have the support for the cover slip at a specific height, resulting in a known depth of exactly 0.1mm. Sample National bureau of standards must be met for both the chamber and cover slip. Sample is placed between cover slip and chamber Diagrams by K. P. Murray, 2007

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Do not use a regular cover slip especially for viscous fluids. The sample will float disposable coverslips and the depth will be greater than 0.1mm. Sample floats coverslip Do Not Use Disposable Cover Slip Diagrams by K. P. Murray, 2007

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Neubauer Hemacytometer Dimensions Each side (chamber) has a ruled area – 3 mm wide and 3 mm long –Divided into nine squares –The large squares are subdivided into 16 –The middle square is subdivide into 25 –Each of the 25 is again subdivided into 16 When the dimensions are known, one will always be able to calculate the area counted.

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Dimensions of the Neubauer Primary squares 1.0 mm Tertiary squares mm SecondarySquaresSecondarySquares 0.25mm 0.2mm Note: Center primary square has different divisions Diagrams by K. P. Murray, 2007

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Hemacytometer Quality Control (QC) Ensures accuracy Performed by duplicating the counting area exactly. For example: –If squares 1,3,5,7,9 are counted. –Then 10,12,14,16,18 are counted on the other side. Diagrams by K. P. Murray, 2007

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Area Counted Depend on Cell Type.... Platelet WBC RBCRBC RBCRBC RBC Diagrams by K. P. Murray, 2007

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RBC Count Method Count the five inner tertiary squares on both sides of the chamber –on Peripheral Blood Check difference in counts Calculate RBC count using the basic hemacytometer formula.... RBCRBC RBCRBC RBC Diagrams by K. P. Murray, 2007

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RBC Count Method If a Body Fluid count is less than 100 cells –count all 9 squares on both sides to provide greater accuracy. Adjust the hemacytometer calculation accordingly.... Diagrams by K. P. Murray, 2007

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WBC Count Method Count the four outer secondary squares on both sides of the chamber Check difference in counts Calculate WBC count using the basic hemacytometer formula WBC Diagrams by K. P. Murray, 2007

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WBC Count Method If WBC count is less than 1000 cells –count all 9 squares on both sides to provide greater accuracy. Adjust the hemacytometer calculation accordingly Diagrams by K. P. Murray, 2007

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Platelet (PLT) Area Counted PLT Diagrams by K. P. Murray, 2007

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Neubauer Hemacytometer Calculation Cells X dilution = cells/ L squares X 0.1 Note: 0.1 mm depth is constant in Neubauer Hemacytometer

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Unopette – used today for manual counts Components –Reservoir –Capillary pipette –Protective sleeve that also is used to open the reservoir. Pre-measured diluents in vials. Capillary pipette is specific for the diluent used. The capillary tubes can not be interchanged. Picture by K. P. Murray, 2007

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Unopette System For RBC, WBC and PLT counts RBC vial –Isotonic or physiologic saline –1/200 dilution WBC only vials –Turk’s solution –1/20 dilution WBC/PLT vials –Ammonium oxalate g –Sorenson’s buffer--1.0g –Thimerosal g –(preservative) –Distilled H 2 O qs to 1 liter –1/100 dilution RBC can not resist osmotic pressures, as the WBC can, therefore RBC counts need isotonic solutions Manual RBC counts in PB rarely done Always performed on other body fluids

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Unopettes Test nameSamplediluentType of diluentFinal dilution WBC 25 L mlTURKS- 3% acetic acid1:20 RBC 10 L 1.99 mlIsotonic saline1:200 PLATELET 20 L 1.98 ml1% ammonium oxalate1:100 Eosinophils 25 L mlPhloxine B1:32 Note: different final dilutions

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Loading Chamber using a Unopette Picture by K. P. Murray, 2007

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“Wet Box” prevents solution from drying out and altering count Settle cells to the same optical plane –WBC 2-3 minutes –PLT 10 minutes (take the longest to settle) –RBC 2-3 minutes Picture by K. P. Murray, 2007

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Acceptable differences between chambers of the hemacytometer No more than 10 cell variation between the 4 squares for the WBC count. The difference between the highest and the lowest number should not be greater than 25 cell for the 10 tertiary squares (5 squares per side) the RBC count. Diagrams by K. P. Murray, 2007

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How to Tell If Sample has Uneven Distribution Difference between individual squares exceeds 10% Difference between each chamber count exceeds 20 % REPEAT COUNT with a freshly loaded hemacytometer, if the count does not match the above criteria.

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WBC Count Reporting Report WBC –Number (period) one decimal X 10 3 / L –Number. decimal X 10 3 / L Example: 10.9 X 10 3 / L WBC has one decimal place, if sample is peripheral blood. For other body fluids (BF), report whole number, no scientific notation or abbreviations.

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RBC Count Reporting Report RBC – Number (period) two decimals X 10 6 / L –Number. two decimals X 10 6 / L Example: 4.13 X 10 3 / L RBC has two decimal places, if peripheral blood. For other body fluids (BF), report whole number, no scientific notation or abbreviations.

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Platelet Count Reporting Report PLT –Whole number (no decimal) X 10 3 / L Example: 428 X 10 3 / L Platelet count does not have a decimal point. Other body fluids do not report platelet counts.

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Platelet Count Reporting Verbal reporting always give the full number. For example, “Doctor Jones, the platelet count on Ms. Smith is 428,000”. –DO NOT just say the count is 428. The doctors or the nurse may think only four hundred (transfusion time!!!), rather than a normal count of 400 thousand.

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Reference Units Conventional units ExampleSI unitsExample WBC X 10 3 / L4.5 X 10 3 / L X 10 9 / L4.5 X 10 9 / L RBC X 10 6 / L5.21 X 10 6 / L X / L5.21 X / L HGBg/dL15.4 g/dLg/L 154 g/L MCVfl88 flfl88 fl MCHpg29 pgpg29 pg MCHC%33 %g/L330 g/L PLT X 10 3 / L182 X 10 3 / L X 10 9 / L182 X 10 9 / L Notice the similarities and differences between units

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Sources of Error Failure to mix sample prior to dilution Pipeting technique Uneven distribution in chamber Failure to remove excess blood from outside of pipette Squeezing out some fluid from reservoir [Unopette]

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Sources of Error Cell aggregates (clots) Bacteria ( may be mistaken for plt) Dirt, talc, debris Drying of the chamber –taking too long to count Falsely increase counts

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Fill problems Air bubbles Correct fill Coverslip Overfill Underfill Diagrams by K. P. Murray, 2007

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Problem 1 A blood sample was diluted1:20. On a Neubauer hemacytometer –140 WBC counted in the four outer squares on side A. –147 WBC counted on the same four squares on side B. What is the WBC count?

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Problem 1 answer WBC 140 and 147 ‘match’ and average Depth = 0.1 (always with a Neubauer) cells x 20 (dilution factor) 4 (squares) x 0.1(depth) WBC = 7,175 WBC =7.2 x 10 3 /dL

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Problem #2 A tech counted both side of the hemacytometer on an undiluted spinal fluid sample. –22 WBC were counted and no RBC. What is the WBC count? How is it reported?

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Problem #2 solution Both side area = 18 mm 2 {(1x1) (9+9)} Depth = 0.1 Both side count = 22 Dilution = 1 Answer: WBC = 12 cells / dL NOTE: no decimal or scientific notation because it is a body fluid and one does not report a fraction of a cell.

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References Barry T. Mitzner, (1999), D.V.M, Southeast Vetlab, Inc., Retrieved on July 12, 2003 from matology%20methods%20for%20the%20offi/ppf rame.htm Murray, K.P.,(2007) Hematology Lab Photographs (personal collection) Murray, K.P.,(2007) Hematology Diagrams and Animations (personal collection) END

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