RBC Count, Hb, Hct, Blood Indices
WBC Count & Plt Count
1
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.)
2
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
3
RBC Count (cont.)
Reagent
Red cell diluting fluid must fulfill the
following
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
4
RBC Count (cont.)
Specimens
1. Fresh blood
2. Anticoagulated blood
EDTA
Double Oxalate
Citrate
Heparin
Etc.
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
4. Shake the pipette on the vibrator for 1
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.
101 mark.
min.
5
RBC Count (cont.)
Counting
1. Count in 5 red cell squares in the middle
(In 4 squares at corners and another 1 in the middle).
2. Top-left touching count
3. Using counter hand tally or hand tally
6
RBC Count (cont.)
High 0.1 mm.
W
W
1 mm.
R
R
1 mm.
R
R
R
W
W
3 mm.
7
RBC Count (cont.)
Top
Left
8
RBC Count Calculation
1. Volume of 1 red square
2. Volume of 5 red square
3. In 0.02 cu.mm. the RBC count
4. In 1 cu.mm. the RBC count
5. The dilution for RBC
6. The final RBC count
=N
= 0.2 x 0.2 x 0.1 cu.mm.
= 0.004 cu.mm.
= 0.004 x 5
cu.mm.
= 0.02
cu.mm.
(counted No.)
= N x 1 / 0.02
= N x 50
= 0.5 / 100
= 200
= N x 50 x 200
= 10,000 N (/cu.mm.)
9
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.
10
Hemoglobin Concentration (Hb)
Weight of hemoglobin per a unit volume of blood
(gm / dL)
(gm%)
(g / L)
11
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
12
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.
13
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
14
Hematocrit (Hct)
or
Packed Cell Volume (PCV)
Ratio (in percent) of packed red cells in total whole blood
(%)
(decimal ratio)
15
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)
16
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.
17
Hct (cont.)
Normal range
male = 40 - 52 %
(0.40 - 0.52)
Female = 37 - 47
(0.37 - 0.47)
18
Blood Indices
Mean Corpuscular Volume (MCV)
(fL)
Mean Corpuscular Hemoglobin (MCH)
(pg)
Mean Corpuscular Hemoglobin Concentration (MCHC)
(% or g/dL)
19
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)
20
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.)
21
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
22
WBC Count (Cont.)
Specimens
1. Fresh blood
2. Anticoagulated blood
EDTA
Double Oxalate
Citrate
Heparin
Etc.
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.
23
WBC Count (Cont.)
W
W
R
1 mm.
1 mm.
1 mm.
R
R
R
R
W
W
High 0.1 mm.
3 mm.
24
WBC Count (Cont.)
W
1 mm.
1
mm.
High 0.1 mm.
25
WBC Count Calculation
1. Volume of 1 white square
2. Volume of 4 white square
3. In 0.4 cu.mm. the WBC count
4. In 1 cu.mm. the WBC count
5. The dilution for WBC
6. The final WBC count
= 1 x 1 x 0.1 cu.mm.
= 0.1
cu.mm.
= 0.1 x 4 cu.mm.
= 0.4
cu.mm.
=N
(counted No.)
= N x 1 / 0.4
= N x 2.5
= 0.5 / 10
= 20
= N x 2.5 x 20
= 50 N (/cu.mm.)
26
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.
27
Blood Platelet (Plt) Count
Number of platelets per a unit volume
(x 103 / cu.mm.)
(x 103 / mm3.)
(x 103 / uL.)
(x 109 / L.)
28
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%)
29
Plt Count (Cont.)
Specimens
1. Fresh blood
2. Anticoagulated blood
EDTA
Double Oxalate
Citrate
Heparin
Etc.
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.
30
Plt Count (cont.)
Plt.
Plt.
R
1 mm.
1 mm.
1 mm.
R
R
R
R
Plt.
Plt.
3 mm.
High 0.1 mm.
31
Plt Count (cont.)
Plt.
1 mm.
1
mm.
High 0.1 mm.
32
Platelet Count Calculation
1. Volume of 1 white square
= 1 x 1 x 0.1 cu.mm.
2. Volume of 4 white square
= 0.1 x 4 cu.mm.
3. In 0.4 cu.mm. the Plt count
4. In 1 cu.mm. the Plt count
5. The dilution for Plt count
6. The final Plt count
= 0.1
= 0.4
=N
(counted No.)
= N x 1 / 0.4
= N x 2.5
= 0.5 / 100
= 200
= N x 2.5 x 200
= 500 N (/cu.mm.)
cu.mm.
cu.mm.
33
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.
34
Human Blood Normal Values
Value
RBC
Hb
Hct
MCV
MCH
MCHC
ESR
WBC
Plt
Male
4.5-6.0
13-18
40-52
1-7
80-97
27-31
32-36
4-11
140-440
Female
Unit
3.8-5.2
12-16
37-47
3-9
103/uL
SI Unit
106/uL
g/dL
%
fL
pg
%
mm/hr
103/uL
35
Thank You For Your Attention
36