SCBM343- Complete Blood Count

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1 SCBM343- Complete Blood Count Associate Professor Dr. Wannee Jiraungkoorskul Department of Pathobiology, Faculty of Science, Mahidol University Tel: , 1

2 Objectives 1. Explain the composition of blood and normal blood values for the complete blood count 2. Explain laboratory procedures of CBC including Hematocrit Hemoglobin White blood cell count Differential white blood cell Red blood cell count Blood cell morphology Red blood cell indices (MCV, MCH, MCHC) Platelet count 3. Discuss cause and implications of increased and decreased values. 2

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4 Blood to which an anticoagulant has been added will not clot. Blood cells will settle to the bottom of the tube leaving plasma at the top of the tube. Blood to which no anticoagulant has been added will clot. Blood cells get caught in the clot leaving serum behind. 4

5 Complete blood count: Routine Complete Blood Count (CBC) : Hematocrit Hemoglobin White blood cell count White blood cell Differential Blood cell morphology Red blood cell count Red blood cell indices (MCV, MCH, MCHC) Platelet count 5

6 Skin puncture or Venipuncture (Phlebotomy) Capillary blood Suitable for infant or baby No edema, congestion and cyanosis at the area to be punctured Sites of the puncture: Tip of ring or great finger, ear lobe, lateral portion of the heel or great toe 6

7 Collecting capillary blood into a capillary tube 7

8 Blood tube color EDTA (1-2 mg/ml blood) is the best. Hct : Microcentrifugation (15000 rpm, 5 min) Hb : Cyanmethemoglobin method WBC count : Turk s solution (3% Glac HOAC) Blood film staining : Wright-Giemsa 8

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10 Hematocrit (Hct) or Packed cell volume (PCV) The Hct is the percentage of total volume occupied by packed red blood cells when a given volume of whole blood is centrifuged at a constant speed for constant period of time. The HCT is one of the most precise methods of determining the degree of anemia or polycythemia. 10

11 Hematocrit: 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 rotor was completely stopped. 9. Read the value with Hct reader or ruler. 11

12 Filling a capillary tube from a tube Filling a capillary tube from a capillart puncture Microhematocrit centrifuge 12

13 Diagram of packed cell column in a microhematocrit tube Normal range Male = % ( ) Female = ( ) 13

14 In order to obtain a value of hematocrit from the 3 How to read a hematocrit 4 1 about 18% 2 centrifuged blood sample in the capillary tube, one must refer to a scale plate. The bottom of the packed red cell column is first lined up with the "0" line on the scale plate, and then the scale is moved under the sample until the top of the plasma column lines up with the"100%" line. 14

15 Hemoglobin (Hb) concentration: 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. Hb (Fe ++ ) K Fe (CN)6 3 methemoglobin (Fe 3+ ) KCN Cyanmethemoglobin Normal range Male = g/dl Female = g/dl 15

16 Wbc count: 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 (x400). 9. Calculate the WBC concentration. 16

17 Diluting pipette- WBC, RBC Hemacytometer 17

18 Total areas 18

19 The grids for WBC counts 19

20 The grids for RBC counts 20

21 A B C D 21

22 Using dilution pipette with the white mixer, draw blood up to the 0.5 mark. Dab with piece of paper towel if needed to adjust volume. 22

23 Fill the pipette the rest the 11 mark with WBC diluent. Shake well to mix with the hose end sealed with your finger. WBC diluent : - 10 mg crystal violet ml glacial acetic acid ml with d H

24 Empty ~2-3 drops of pipette into waste container 24

25 Add a small amount of the diluted blood to just fill the first chamber of the hemacytometer. It should flow in to fill the chamber by capillary action. Do not over fill. Let the preparation sit for a minute (for cells to settle). 25

26 To improve your skill, repeat the dilution a second time and fill the second chamber. After completing the counts of each, compare the numbers you have generated. 26

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29 Calculation Blood is drawn to the 0.5 mark and diluted to the 11 mark with WBC diluting fluid. All the blood is washed into the bulb of the pipet (which has a volume of 10). Therefore, 0.5 volumes of blood are contained in 10 volumes of diluting fluid. The resulting dilution is 1:20. 29

30 1 mm. W W 1 mm. R R R R R High 0.1 mm. W W 30

31 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 cells 4. In 1 cu.mm. the WBC count = N cells The dilution for WBC = 1:20 6. The final WBC count = N x cells/cu.mm. 31

32 For example Calculate the average number of WBCs per chamber: Calculate the number of WBCs per cubic mm: 32

33 Rbc count Reagent Red cell diluting fluid Anti-coagulant Anti-hemolysis Anti-aggregation Anti-rouleaux Preserve RBC shape Lysis WBC 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 33

34 Rbc count: 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. 34

35 0.2 mm. W W R R 1 mm. R R R 0.2 mm. W W High 0.1 mm. 3 mm. 35

36 Calculation 1. Volume of 1 red square = 0.2 x 0.2 x 0.1 cu.mm. = cu.mm. 2. Volume of 5 red square = 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 The dilution for RBC = 0.5 / 100 = The final RBC count = N x 50 x 200 = 10,000 N (/cu.mm.) 36

37 Calculation Red cell count = number of cells counted (N) x volume factor (=50) x dilution factor (=200) = N x 10,000 Normal range = x 10 6 / cu.mm. 37

38 Platelet count Reagents Platelet diluting fluids 1. Rees-Ecker Solution - Brilliant cresyl blue - Sodium citrate - Formaldehyde 2. Brecher-Cronchite Solution - Ammonium oxalate (1%) 38

39 Platelet (Plt.) count: 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. 39

40 1 mm. 1 mm. Plt. Plt. R R R 3 mm. R R Plt. Plt. 1 mm. High 0.1 mm. 40

41 Plt. 1 mm. 1 mm. High 0.1 mm. 41

42 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 The dilution for Plt count = 0.5 / 100 = The final Plt count = N x 2.5 x 200 = 500 N (/cu.mm.) 42

43 Calculation Platelet count = number of Platelets counted (N) x volume factor (=2.5) x dilution factor (=200) = N x 500 Normal range = 140, ,000 / cu.mm x 10 3 / cu.mm. 43

44 Red blood cell indices Mean Corpuscular Volume is an average red blood cell size MCV = Hct (%) x 10 / RBC (in millions / cu.mm.) = fl (femtoliter) Mean Corpuscular Hemoglobin is the amount of hemoglobin per red blood cell MCH = Hb (g/dl) x 10 / RBC (in million / cu.mm.) = pg (picogram) Mean Corpuscular Hemoglobin Concentration is the amount of hemoglobin relative to the size of the cell (hemoglobin concentration) per red blood cell. MCHC = Hb (g/dl) x 100 / Hct (%) = (% or g/dl) 44

45 Microcytic Anemia 45

46 Megaloblastic Anemia 46

47 References Williams Hematology. 9 th ed. by Kenneth Kaushansky et al Wintrobe s Clinical Hematology. 13 th ed. by Daniel A. Arber et al Essential Haematology 7 th ed. by Victor Hoffbrand Paul A. H. Moss A. 2015

48 SCBM343 Complete Blood Count 48