RBC Count, Hb, Hct, Blood Indices

WBC Count & Plt Count

Yuttana Mundee
Clinical Microscopy
Associated Medical Sciences
Chiang Mai University
yuttana@mail.ams.cmu.ac.th
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 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.)

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 RBC Count (cont.)
General Equipment Specific Equipment
1. Rubber glove 1. Red pipette (Thoma type)
2. Gown 2. Rubber pipe
3. Mask 3. Plastic syringe
4. Goggle 4. Pipette vibrator
5. Tissue paper 5. Hemacytometer & cover slip
(Counting chamber)
General Requirement 6. Microscope
1. Knowledge 7. Counter (hand tally)
2. Skill 8. Report form
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RBC Count (cont.) Hayam’s solution
Sodium Sulphate
Reagent Sodium Chloride
Red cell diluting fluid Mercuric Chloride
Anti-coagulant Distilled Water
Anti-hemolysis Gower’s solution
Anti-aggregation Sodium Sulphate
Anti-rouleaux Glacial acetic acid
Preserve RBC shape Distilled water
Lysis WBC Citrate-formalin solution
Tri-sodium Citrate
Formalin
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 RBC Count (cont.
Specimens ) Procedure
1. Fresh blood 1.Mix the blood sample thoroughly.
2. Anticoagulated blood 2. Fill blood into red pipette at 0.5 mark.
EDTA 3. Fill reagent add up into the pipette
Double Oxalate to 101 mark.
Citrate 4. Shake the pipette on the vibrator for 1 min.
Heparin 5. Discarded the first 3-4 drops.
Etc. 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. 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

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 RBC Count (cont.) 0.2 mm.

W W

R R
1 mm. R 0.2 mm.
R R

W W

High 0.1 mm.

3 mm. 7
 RBC Count (cont.)
Top
Left

8
 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.)
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 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 10 / cu.mm.

6

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
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 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.

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 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
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 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 Specimens
2. Oily-clay sealer 1. Fresh blood (red tip)
3. Hematocrit centrifuge 2. Anticoagulated blood
(12,000 rpm) (blue tip)
4. Hematicrit reader
5. Ruler

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 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.
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 Hct (cont.)
Normal range

male = 40 - 52 %
(0.40 - 0.52)

Female = 37 - 47
(0.37 - 0.47)

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 Blood Indices
Mean Corpuscular Volume (MCV)
(fL)
Mean Corpuscular Hemoglobin (MCH)
(pg)
Mean Corpuscular Hemoglobin Concentration (MCHC)
(% or g/dL)
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 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)
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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
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 WBC Count (Cont.)
Procedure
Specimens 1. Mix the blood sample thoroughly.
1. Fresh blood 2. Fill blood into white pipette at 0.5 mark.
2. Anticoagulated blood 3. Fill reagent add up into the pipette
EDTA to 11 mark.
Double Oxalate 4. Shake the pipette on the vibrator for 1 min.
Citrate 5. Discarded the first 3-4 drops.
Heparin 6. Fill in the hemacytometer nicely.
Etc. 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.) 1 mm.

W W 1 mm.

R R
1 mm. R
R R

W W

High 0.1 mm.

3 mm. 24
WBC Count (Cont.)

W
1 mm.

High 0.1 mm.

1 mm.
25
 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.)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.)

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

Plt. Plt.
1 mm.
R R
1 mm. R
R R

Plt. Plt.

High 0.1 mm.

3 mm. 31
Plt Count (cont.)

Plt.
1 mm.

High 0.1 mm.

1 mm.
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 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.)
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 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 Male Female Unit SI Unit
RBC 4.5-6.0 3.8-5.2 106/uL 1012/L
Hb 13-18 12-16 g/dL
130-180 120-160 g/L
Hct 40-52 37-47 %
0.40-0.52 0.37-0.47 ratio
MCV 80-97 fL fL
MCH 27-31 pg pg
MCHC 32-36 % %
Retic 0.2-2.0 % %
20-100 103/uL 109/L
ESR 1-7 3-9 mm/hr
WBC 4-11 103/uL 109/L
Plt 140-440 103/uL 109/L
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Thank You For Your Attention
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