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RBC Count, Hb, Hct, Blood Indices WBC Count & Plt Count

RBC Count, Hb, Hct, Blood Indices WBC Count & Plt Count. RBC Count. Number of red blood cells per a unit volume (x million / cu.mm.) (x 10 6 / cu.mm.) (x 10 6 / mm 3 .) (x 10 6 / u L.) (x 10 12 / L.). RBC Count (cont.). General Equipment 1. Rubber glove 2. Gown 3. Mask

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RBC Count, Hb, Hct, Blood Indices WBC Count & Plt Count

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  1. RBC Count, Hb, Hct, Blood Indices WBC Count &Plt Count

  2. RBC Count Number of red blood cells per a unit volume (x million / cu.mm.) (x 106 / cu.mm.) (x 106 / mm3.) (x 106 / uL.) (x 1012 / L.)

  3. RBC Count (cont.) General Equipment 1. Rubber glove 2. Gown 3. Mask 4. Goggle 5. Tissue paper Specific Equipment 1. Red pipette (Thoma type) 2. Rubber pipe 3. Plastic syringe 4. Pipette vibrator 5. Hemacytometer & cover slip (Counting chamber) 6. Microscope 7. Counter (hand tally) 8. Report form General Requirement 1. Knowledge 2. Skill

  4. RBC Count (cont.) Hayam’s solution Sodium Sulphate Sodium Chloride Mercuric Chloride Distilled Water Gower’s solution Sodium Sulphate Glacial acetic acid Distilled water Citrate-formalin solution Tri-sodium Citrate Formalin Reagent Red cell diluting fluid must fulfill the following Anti-coagulant Anti-hemolysis Anti-aggregation Anti-rouleaux Preserve RBC shape Lysis WBC

  5. RBC Count (cont.) Procedure 1.Mix the blood sample thoroughly. 2. Fill blood into red pipette at 0.5 mark. 3. Fill reagent add up into the pipette to 101 mark. 4. Shake the pipette on the vibrator for 1 min. 5. Discarded the first 3-4 drops. 6. Fill in the hemacytometer nicely. 7. Allow RBC to set down for 2-3 min. 8. Count 5 red squares under microscope (x400). 9. Calculate the RBC concentration. • Specimens • Fresh blood • 2. Anticoagulated blood • EDTA • Double Oxalate • Citrate • Heparin • Etc.

  6. RBC Count (cont.) Counting 1. Count in 5 red cell squares in the middle (In 4 squares at corners and another 1 in themiddle). 2. Top-left touching count 3. Using counter hand tally or hand tally

  7. RBC Count (cont.) W W High 0.1 mm. 1 mm. R R 1 mm. R R R W W 3 mm.

  8. RBC Count (cont.) Top Left

  9. RBC Count Calculation 1. Volume of 1 red square = 0.2 x 0.2 x 0.1 cu.mm. = 0.004 cu.mm. 2. Volume of 5 red square = 0.004 x 5 cu.mm. = 0.02 cu.mm. 3. In 0.02 cu.mm. the RBC count = N (counted No.) 4. In 1 cu.mm. the RBC count = N x 1 / 0.02 = N x 50 5. The dilution for RBC = 0.5 / 100 = 200 6. The final RBC count = N x 50 x 200 = 10,000 N (/cu.mm.)

  10. RBC Count (cont.) Calculation Red cell count = number of cells counted (N) x volume factor (=50) x dilution factor (=200) = N x 10,000 Normal range = 3.8 - 6.0 x 106 / cu.mm.

  11. Hemoglobin Concentration (Hb) Weight of hemoglobin per a unit volume of blood (gm / dL) (gm%) (g / L)

  12. Hb (cont.) Specific Equipment 1. Sahli (Hb) pipette 2. Rubber pipe 3. Plastic syringe 4. Test tube 5. Volumetric Dispenser 6. Standard Hb conc. 7. Spectrophotometer 8. Double log paper Cyanmethemoglobin Method (Drabkin’s solution pH 7.0 - 7.4) Potassium Fericyanide Potassium Cyanide Sodium bicarbonate Specimens 1. Fresh blood 2. Anticoagulated blood

  13. Hb (cont.) Procedure 1. Mix the blood sample thoroughly. 2. Fill blood into Sahli pipette at the mark (20 uL). 3. Clean outside the pipette nicely. 4. Blow out the blood into a tube containing 5 ml of Drabkin’s solution wash inside the pipette thoroughly. 5. Allow all Hb to convert to Cyanmeth-Hb for 10 min. 6. Read the percent transmittance at 540 nm using pure Drabkin’s solution as a blank. 7. Calculate the Hb concentration from standard curve.

  14. Hb (cont.) Standard Curve Preparation 1. Make various concentration of Hb solutions (0, 4, 8, 12, 16, 20 g/dL). 2. Use them as samples for the procedure as described above. 3. Do concentration plot against percent transmittance on log-log paper. 4. Make a curve most fit with all points. 5. That is a standard curve for further measurement. Normal range male = 14 - 18 g/dL Female = 12 - 16 g/dL

  15. Hematocrit (Hct) or Packed Cell Volume (PCV) Ratio (in percent) of packed red cells in total whole blood (%) (decimal ratio)

  16. Hct (cont.) Specific Equipment 1. Capillary tubes (1 x 75 mm) red & blue tip 2. Oily-clay sealer 3. Hematocrit centrifuge (12,000 rpm) 4. Hematocrit reader 5. Ruler Specimens 1. Fresh blood (red tip) 2. Anticoagulated blood (blue tip)

  17. Hct (cont.) Procedure 1. Mix the blood sample thoroughly. 2. Fill blood into capillary tubes for up to 4/5 of its length. 3. Seal bottom of the tube with oily-clay sealer. 4. Clean outside the tube with tissue paper nicely. 5. Place the tubes in to the rotor, adjust the bottom of the tube to close to the outer edge of the rotor. 6. Close inner lid tightly, then close the outer lid. 7. Centrifuge for 5 minutes. 8. Open the lids after the roter was completely stopped. 9. Read the value with Hct reader or ruler.

  18. Hct (cont.) Normal range male = 40 - 52 % (0.40 - 0.52) Female = 37 - 47 (0.37 - 0.47)

  19. Blood Indices Mean Corpuscular Volume (MCV) (fL) Mean Corpuscular Hemoglobin (MCH) (pg) Mean Corpuscular Hemoglobin Concentration (MCHC) (% or g/dL)

  20. Blood Indices (cont.) MCV = Hct (%) x 10 / RBC (in millions / cu.mm.) normal range = 80 - 97 fL. MCH = Hb (g/dL) x 10 / RBC (in million / cu.mm.) normal range = 27 - 31 (pg) MCHC = Hb (g/dL) x 100 / Hct (%) normal range = 32 - 36 (% or g/dL)

  21. White Blood Cell (WBC) Count Number of white blood cells per a unit volume (x 103 / cu.mm.) (x 103 / mm3.) (x 103 / uL.) (x 109 / L.)

  22. WBC Count (Cont.) Specific Equipment 1. White pipette (Thoma type) 2. Rubber pipe 3. Plastic syringe 4. Pipette vibrator 5. Hemacytometer & cover slip (Counting chamber) 6. Microscope 7. Counter (hand tally) 8. Report form Reagent WBC diluting fluids 1. Turk’s solution Glacial acetic acid Gential violet Distilled water 2. Two % acetic acid 3. One % hydrochloric acid

  23. WBC Count (Cont.) Procedure 1. Mix the blood sample thoroughly. 2. Fill blood into white pipette at 0.5 mark. 3. Fill reagent add up into the pipette to 11 mark. 4. Shake the pipette on the vibrator for 1 min. 5. Discarded the first 3-4 drops. 6. Fill in the hemacytometer nicely. 7. Allow WBC to set down for 2-3 min. 8. Count 4 white squares under microscope (x100). 9. Calculate the WBC concentration. Specimens 1. Fresh blood 2. Anticoagulated blood EDTA Double Oxalate Citrate Heparin Etc.

  24. WBC Count (Cont.) 1 mm. W W 1 mm. R R 1 mm. R R R W W High 0.1 mm. 3 mm.

  25. WBC Count (Cont.) W 1 mm. High 0.1 mm. 1 mm.

  26. WBC Count Calculation 1. Volume of 1 white square = 1 x 1 x 0.1 cu.mm. = 0.1 cu.mm. 2. Volume of 4 white square = 0.1 x 4 cu.mm. = 0.4 cu.mm. 3. In 0.4 cu.mm. the WBC count = N (counted No.) 4. In 1 cu.mm. the WBC count = N x 1 / 0.4 = N x 2.5 5. The dilution for WBC = 0.5 / 10 = 20 6. The final WBC count = N x 2.5 x 20 = 50 N (/cu.mm.)

  27. WBC Count (Cont.) Calculation White cell count = number of cells counted (N) x volume factor (=2.5) x dilution factor (=20) = N x 50 Normal range = 5,000 - 10,000 / cu.mm. 5 - 10 x 103 / cu.mm.

  28. Blood Platelet (Plt) Count Number of platelets per a unit volume (x 103 / cu.mm.) (x 103 / mm3.) (x 103 / uL.) (x 109 / L.)

  29. Plt Count (cont.) Specific Equipment 1. Red pipette (Thoma type) 2. Rubber pipe 3. Plastic syringe 4. Pipette vibrator 5. Hemacytometer & cover slip (Counting chamber) 6. Microscope 7. Counter (hand tally) 8. Report form Reagents Platelet diluting fluids 1. Rees-Ecker Solution - Brilliant cresyl blue - Sodium citrate - Formaldehyde 2. Brecher-Cronchite Solution - Ammonium oxalate (1%)

  30. Plt Count (Cont.) Procedure 1. Mix the blood sample thoroughly. 2. Fill blood into red pipette at 0.5 mark. 3. Fill reagent add up into the pipette to 101 mark. 4. Shake the pipette on the vibrator for 1 min. 5. Discarded the first 3-4 drops. 6. Fill in the hemacytometer nicely. 7. Allow Plt. to set down in moisture chamber for 15 min. 8. Count 4 white squares under microscope (x400). 9. Calculate the Plt. concentration. Specimens 1. Fresh blood 2. Anticoagulated blood EDTA Double Oxalate Citrate Heparin Etc.

  31. Plt Count (cont.) 1 mm. Plt. Plt. 1 mm. R R 1 mm. R R R Plt. Plt. High 0.1 mm. 3 mm.

  32. Plt Count (cont.) Plt. 1 mm. High 0.1 mm. 1 mm.

  33. Platelet Count Calculation 1. Volume of 1 white square = 1 x 1 x 0.1 cu.mm. = 0.1 cu.mm. 2. Volume of 4 white square = 0.1 x 4 cu.mm. = 0.4 cu.mm. 3. In 0.4 cu.mm. the Plt count = N (counted No.) 4. In 1 cu.mm. the Plt count = N x 1 / 0.4 = N x 2.5 5. The dilution for Plt count = 0.5 / 100 = 200 6. The final Plt count = N x 2.5 x 200 = 500 N (/cu.mm.)

  34. Plt Count (Cont.) Calculation Platelet count = number of Platelets counted (N) x volume factor (=2.5) x dilution factor (=200) = N x 500 Normal range = 140,000 - 440,000 / cu.mm. 140 - 440 x 103 / cu.mm.

  35. Human Blood Normal Values Value Male Female Unit SI Unit RBC 4.5-6.0 3.8-5.2 106/uL Hb 13-18 12-16 g/dL Hct 40-52 37-47 % MCV 80-97 fL MCH 27-31 pg MCHC 32-36 % ESR 1-7 3-9 mm/hr WBC 4-11 103/uL Plt 140-440 103/uL

  36. Thank You For Your Attention

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