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Hemoglobin determination Slide 1 Hemoglobin determination Slide 2 Hemoglobin determination Slide 3 Hemoglobin determination Slide 4 Hemoglobin determination Slide 5 Hemoglobin determination Slide 6 Hemoglobin determination Slide 7

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Hemoglobin determination

  1. 1. HEMOGLOBINDETERMINATION BOND KING NITISH RAHUL DEV
  2. 2. I. COLORIMETRICMETHOD A. Direct visual colorimetric Method  Tall quist method  Dare’s Hemoglobinometer  Acid Hematin method  Alkaline Hematin method
  3. 3. B. Photoelectric colorimetric method1. Oxyhemoglobin method Measures normal hemoglobin Used 0.007 N NH4OH or 0.1% Na2Co3 Read with the wavelength at 540 nm
  4. 4. 2. Cyanmethemoglobin (HiCN) Also known as hemiglobin cyanide or ferrihemoglobin cyanide All forms of hemoglobin are measured except sulfohemoglobin Uses Drabkin’s solution Potassium ferricyanide Potassium cyanide Dihydrogen potassium phospate Distilled water PH = 7.0-7.4 ( blood capacity ) Used sahli pipet= (0.02ml or 20 micro liter)
  5. 5. PROCEDURE Place 5ml of Drabkin’s reagent into a testube Get 0.02 ml of whole blood using sahli pipet Place the 0.02 ml of blood in to drabkins reagent through rinsing it. Mix and let it stand for 10 minutes Read in a spectrophotometer at 540 nm.
  6. 6. II. Specific gravity method/Gravitationalmethod CUSO4 method  Specific gravity of copper sulfate = 1.053 with an hemoglobin equivalent of 12.5 gm%  Mass blood Procedure  Collect blood sample  Drop a blood into the solution  Observe the activity of the blood  Within 12 seconds, describe how the drop of blood behaves.
  7. 7. Interpretation :- Maintain = equal to 1.053 = 12.5 gm% Sink = > 1.053 = >12.5 gm% Float = < 1.053 = <12.5 gm%
  8. 8. III. Gasometric Method Indirect method Based on the assumption that 1gm Hb can carry approximately 1.34 ml O2.IV. Chemical Method Indirect method Based on the assumption that 1gm Hb contains approximately 3.47 mg iron.
  9. 9. RETICULOCYTE COUNTINGI. Wet method New methylene blue method Cook, meyer and tureen seiverd’s methodProcedure Get blood sample Secure equal proportion of blood and stain. Mix it and letit stand for 10 minutes Make a smear. Dry the smear Examine under microscope using OIO Count reticulocytes in relation to 1,000 RBC.
  10. 10. II. Dry method Schiling’s rapid method =(BCB method). Sabin’s method = (janus green /neutral red) Seiverd’s method =(BCB method) Osogood- wilhelm method = (new methylene blue method) BCB = Brilliant crystal Blue
  11. 11. PROCEDURE Spread stain thinly on a glass slide and air dry. Place a small drop of blood upon the layer of the dried stain. Place a cover slip on the drop of blood. Allow to stand for 10 minutes Examine under the microscope under OIO Count reticulocytes in relation to 1,000 RBCs.
  12. 12. COMPUTATION% Reticulocytes count = no.of retics.counted X 100 1000 RBCsExample :- 12/1000X 100 = 1.2% normal in adult
  13. 13. RBC COUNTA. Hemocytometry method (microscopic method) (used hemoglobinometer) Diluting fluids Thoma pipets Counting chambers / Hemocytometer
  14. 14. 1.Diluting fluids Hayerm’s Gower’s Toisson’s Bethel’s Formol-citrate/Dacies solutn NSS 3.8 % sodium citrate - easy to prepare - must have preservative method - must be safe - no corrosive, non-caustic - should be isotonic
  15. 15. 2.Thoma pipet Bead - identification of type of pipet - used for mixing - seperating color Upper calibration of RBC pipet = 101 Capacity of bulb is 100 times capacity of stem Constant volume of RBC pipet = 100[ 101-1] RBC thoma- red bead WBC thoma – white bead
  16. 16. Thoma pipet Bulb/ mixing chamber Short stem Bead Long stem
  17. 17. 3. Counting chamber Raised platform Drawing ruled area Counting chamber H-shaped moat
  18. 18. Counting chamber  Improved neubaber- commonly used  Cover slip= depth of the counting chamber (0.1mm)  1 ruled area = 1mm2  1 large square width and length 1mm  Center of large square have 25 small squares and each 25 small square has 16 small square which is used in RBC count.  Total 400 small square are found in center of large square.
  19. 19. WBC WBC R R R R R WBCWBC
  20. 20. Procedure Collect blood Suck blood to 0.5 mark of the pipet. Suck diluting fluid to 101 mark. Shake pipet for 2 minutes. Discard first few drops. Charge the counting chamber at an angle from 30- 35 degree. Count the RBC under HPO using 5 RBC squares of central large square Compute.
  21. 21. Computation RBC count = RBC counted X DCF X VCF DCF = Volume of blood / amt of blood sucked VCF = volume desired / area x depth of the counting chamber x nos of squares used. DCF= diluting correction factor VCF = volume correction factor For RBC pipet DCF = 200 and VCF is 50 VCF = 1/ 0.04x 0.1x 5 =50
  22. 22. ErrorsTechnical error Pipetting Shaking the pipet Charging the counting chamber Application of cover slip Counting of the cells Computation Reporting of results.
  23. 23. Never leave that till tomorrow which you can dotoday. Thank you.
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