scbm343- complete blood count - mahidol university

Associate Professor Dr. Wannee Jiraungkoorskul

Department of Pathobiology, Faculty of Science, Mahidol University

Tel: 02-201-5563, E-mail: [email protected]

SCBM343- Complete Blood Count

1

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

Objectives

3 http://fat.surin.rmuti.ac.th/teacher/songchai/bloodweb/blood%20composition.htm

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.

• 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

Complete blood count: Routine

• 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

Skin puncture or Venipuncture (Phlebotomy)

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Collecting capillary blood

into a capillary tube

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

Blood tube color

• 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 (Hct) or Packed cell volume (PCV)

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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.

Filling a capillary tube from

a capillart puncture

Filling a capillary tube from a tube

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Microhematocrit centrifuge

Diagram of packed cell column

in a microhematocrit tube

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Normal range

Male = 40 - 52 % (0.40 - 0.52)

Female = 37 - 47 (0.37 - 0.47)

How to read a hematocrit

4

1

3

2

about 18%

• In order to obtain a value

of hematocrit from the

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

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Hemoglobin (Hb) concentration: Procedure


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++) K3Fe (CN)6 methemoglobin (Fe3+) Cyanmethemoglobin KCN

Normal range Male = 14 - 18 g/dL

Female = 12 - 16 g/dL


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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Wbc count: Procedure

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Diluting pipette- WBC, RBC

Hemacytometer

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Total

areas

19

The grids

for WBC

counts

20

The grids

for RBC

counts

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A B

C D

1 2

5

4 3

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• 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

- 1.0 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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• 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.

Calculation

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R R

R R

W W

W W

1 mm.

R High 0.1 mm.

1 mm.

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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.

Calculation

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• For example

Calculate the average number of WBCs per chamber:

Calculate the number of WBCs per cubic mm:

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

Rbc count


2. Fill blood into red pipette at 0.5 mark.





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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Rbc count: Procedure

R R

R R

W W

W W

1 mm.

3 mm.

0.2 mm.

0.2 mm.

R

High 0.1 mm.

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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.) 36

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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Calculation

Reagents

Platelet diluting fluids

1. Rees-Ecker Solution

- Brilliant cresyl blue

- Sodium citrate

- Formaldehyde

2. Brecher-Cronchite Solution

- Ammonium oxalate (1%)

38

Platelet count


2. Fill blood into red pipette at 0.5 mark.





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

Platelet (Plt.) count: Procedure

R R

R R

Plt. Plt.

Plt. Plt.

1 mm.

3 mm.

1 mm.

1 mm.

R

High 0.1 mm. 40

Plt.

1 mm.

1 mm.

High 0.1 mm.

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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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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.

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Calculation

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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45

Microcytic Anemia

46

Megaloblastic Anemia

References

Williams Hematology.

9th ed.

by

Kenneth Kaushansky et al.

2015

Wintrobe’s Clinical Hematology.

13th ed. by Daniel A. Arber et al.

2013

Essential Haematology

7th ed. by

Victor Hoffbrand

Paul A. H. Moss

A. 2015

SCBM343

Complete Blood Count 48

scbm343- complete blood count - mahidol university

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