SCBM343

Complete Blood Count

Assoc. Prof. Dr. Wannee Jiraungkoorskul

Department of Pathobiology,
Faculty of Science, Mahidol University
wannee.jir@mahidol.ac.th

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 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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http://fat.surin.rmuti.ac.th/teacher/songchai/bloodweb/blood%20composition.htm
Blood to which an anticoagulant has Blood to which no anticoagulant
been added will not clot. Blood cells has been added will clot. Blood
will settle to the bottom of the tube cells get caught in the clot leaving
leaving plasma at the top of the tube. serum behind.

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 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
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 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

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Collecting capillary blood
into a capillary tube
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 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% glacial acetic acid)
• Blood film staining : Wright-Giemsa

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http://4.imimg.com/data4/QQ/HS/MY-2776055/selling-of-colour-coded-vacuum-blood-collection-tube-500x500.jpg
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 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.

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 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.

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 Filling a capillary tube from a tube

Filling a capillary tube from

a capillart puncture

Microhematocrit centrifuge

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 Diagram of packed cell column
in a microhematocrit tube

Normal range
Male = 40 - 52 % (0.40 - 0.52)
Female = 37 - 47 (0.37 - 0.47)

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 • In order to obtain a value
of hematocrit from the
centrifuged blood sample
3 in the capillary tube, one

How to read a must refer to a scale plate.

hematocrit The bottom of the packed

red cell column is first
lined up with the "0" line
on the scale plate, and then
about
the scale is moved under
18%
the sample until the top of
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2 the plasma column lines up

1 with the"100%" line. 14

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 (Fe3+) KCN Cyanmethemoglobin

Normal range Male = 14 - 18 g/dL

Female = 12 - 16 g/dL 15
 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.

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Diluting pipette- WBC, RBC

Hemacytometer

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Total
areas

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The grids
for WBC
counts

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The grids
for RBC
counts

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A B
1 2

3 4

C D 21
• 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.

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• 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
- 3.0 ml glacial acetic acid
- 100 ml with d H20

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

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• 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).

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• 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.

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 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.

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 1 mm.

W W 1 mm.

R R
R High 0.1 mm.
R R

W W

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 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
0.4
5. The dilution for WBC = 1:20
6. The final WBC count = N x 20
0.4 cells/cu.mm.
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 • For example

Calculate the average number of WBCs per chamber:

Calculate the number of WBCs per cubic mm:

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 Rbc count Hayam’s solution
Sodium Sulphate
Reagent Sodium Chloride
Red cell diluting fluid Mercuric Chloride
Anti-coagulant Distilled Water
Gower’s solution
Anti-hemolysis
Sodium Sulphate
Anti-aggregation
Glacial acetic acid
Anti-rouleaux Distilled water
Preserve RBC shape Citrate-formalin solution
Lysis WBC Tri-sodium Citrate
Formalin
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 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.

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 0.2 mm.

W W

R R
1 mm. R 0.2 mm.
R R

W W

High 0.1 mm.

3 mm.
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 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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 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.

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 Red blood cell indices
• Mean Corpuscular Volume is an average red blood cell size
MCV = Hct (%) x 10 / RBC (in millions / cu.mm.) = 80-97 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.) = 27-31 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 (%) = 32-36 (% or g/dL)

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Microcytic Anemia

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Megaloblastic Anemia

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 References

Williams Hematology. Wintrobe’s Clinical Essential Haematology

9th ed. 7th ed. by
by Hematology. Victor Hoffbrand
Kenneth Kaushansky et al. 13th ed. Paul A. H. Moss
2015 A. 2015
by Daniel A. Arber et al.
2013
 • Blood samples for cell counts must be
• The purpose of doing a differential
thoroughly mixed immediately before
is to:
testing to:
A. determine the proportion of RBC
A. prevent the clumping of platelets
in whole blood
B. prevent the formation of small clots
B. count the number of WBC’s in
C. oxygenate the sample
whole blood
D. ensure even distribution of all blood
C. determine the proportions of
components
WBC’s in whole blood
E. mix anticoagulant with the blood
D. diagnose anemia
• The first drop of blood is wiped away after performing a
skin puncture to:

A. remove any pathogens that are present

B. increase blood flow to the area

C. remove the last traces of alcohol

D. remove any excess tissue fluid

E. C and D