Wbc method
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Wbc method



by WBC methods by shah sunil kumar (bond king)

by WBC methods by shah sunil kumar (bond king)



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Wbc method Wbc method Presentation Transcript

  • A. WBC count (Hemocytometry method/ Microscopic method/ Manual method) Counting chamber Pipets Diluting fluids1. Counting chamber - improved neubauer
  • W WArea= 1mm square W W
  • 2. Diluting fluids 2-3% glacial acetic acid (gentian violet dye) 1% HCL Turk’s solution (methyl violet dye )- Diluting fluid easy to prepare, must be isotonic, must be carciogenic (most toxic substance in 100 LL the benzene)- Hypotonic soln ( WBC) , Isotonic soln (RBC)- Only hemolyse mature RBC- Disadvantage of diluting fluid – nucleus in cytoplasm.(i.e. immature RBC will not hemolyse so counted WBC)
  • Short stem -WBC bulb is smaller than RBC -Upper calibration = 11bead -Constant volume of pipet = 11-1 = bulb 10 -Volume of bulb is 10 times greater than volume of bulb Long stem
  • Procedure Suck the blood up to 0.5 mark. Suck diluting fluid to 11 mark. Shake the pipet for 2 minute. Discard first few drops. Charge the counting chamber – 3 minute for settling down. Count the WBC in 4 WBC square using LPO. Compute for total WBC count.
  • Computation WBC count = nos. of WBC counted x DCF x VCF DCF = vol. of bulb 10/0.5 =20 amount of blood sucked VCF = volume desired (1) 1/ 1x0.1x4 = 2.5 area x depth of counting chamber x nos. of square used.Normal value 5000 – 10,000 / cumm or 5-10 x 10^9/L.
  • B. Corrected WBC Count Done when there is high WBC count and there are more than 10 nucleated RBC per 100 WBC in the blood smearA = B x C A = corrected WBC count C+D B = uncorrected WBC count C = constant (100) D = nos. of nucleated RBC > 10 Nucleated or immature RBC will not hemolyse by hypotonic used in WBC as diluting fluids. In WBC count ,mistakenly immature RBC is counted thinking WBC.
  • C. Differential Leukocyte Count (DLC) Expression in % the relative number of the types of WBC. 4 general steps - blood smear preparation - staining - counting - reporting
  • 1. Blood smear preparation Wedge method – 2 glass slide method(most commonly used) Beacon’s method – coverslip and glass slide method Erlich’s method- 2 coverslip method
  • Procedure (wedge method) A drop of blood is applied near one end of the slide using capillary tube. The spreader is drawn back into the drop of blood and held until blood has spread across its width. The spreader at an angle of 40- 45 degree is pushed steadly along the slide to produce a thin, even film of blood.
  • Method of drying a smear Air dry Use of low flame Use of oven Immersion in methyl alcohol for 1-2 minutes.- Drying a smear (up – down) i.e. Reverse because in blood smear transaction of blood should be thinner to thicker.
  • Prerequisite for proper blood smear1. Slide and spreader should be clean2. Size of the drop of blood3. Smearing should be done quickly.4. Angle and pressure.Types of smear1. Thick - parasite counting2. Thin – cell counting
  • 2. Staining Romanowsky group of stain (basic stain – methylene blue , acidic stain – eosin, neutral stain – mixture of acid and basic stain – cytoplasm) * eosinophilic as granules found in cytoplasm Wright’s – most common Leishmann’s – urgent Giemsa’s – produce more delicate staining Jenner–giemsa May – Grunwald – giemsa (best method) * Wright stain and 50 drops buffer – Metallic greenish sheen.
  • Proper staining reaction Neutrophils – dark blue nucleus ; lilac pink granules. Eosinophils - dark blue nucleus; blue black granules . Lymphocyte - dark blue nucleus; sky blue cytoplasm. Monocyte – faint blue nucleus ; faint blue gray cytoplasm. * Robin- egg blue – sky - blue cytoplasm. - platelets – pale lilac blue - RBC – pinkish buff to orange. - bacteria – blue
  • Counting (scanning of smear) only used one method for entire Two field meander Four-field meander Strip method Exaggerated battlement Crenellation (most commonly used )
  • ReportingA. Relative count-gives the number of WBC type per 100 WBCs. nos. of specific WBC x 100 = % 100N.R. Neutrophil - 51- 67% (bacteria) Lymphocytes – 25-33% (virus) Monocyte – 2-6% (bacteria) Eosinophil – 1-4% Basophil – 0-1%
  • B. Absolute count – gives the number of specific WBC type per cumm. Relative count x WBC count = / cumm (most informative method)N.R. Neutrophil – 1,600 -7,260 /cumm Lymphocyte – 960 - 4,400/cumm Monocyte – 180 - 800 /cumm Eosinophil – 45 - 440/cumm Basophil – 45 -110/cumm