CHAPTER 7

MANUAL BLOOD CELL COUNT

HEMOCYTOMETRY
By: Dereje M
2024

2/27/2024 Hematology and Immunohematology module 1

Learning Objectives
At the end of this chapter, the students will be able to:

• Discuss the general principles of manual hemocytometer

• List the materials that are basically required in manual hemocytometer

• Mention the diluting fluid, dilution factor and areas of counting on the chamber for RBC,
WBC, platelet and eosinophil counts

• Indicate the area of counting in the Fuchs-Rosenthal counting chamber for Eosinophil
count

2/27/2024 Hematology and Immunohematology module 2

Learning Objectives
• Perform WBC, RBC, platelet and eosinophil counts

• Discuss the clinical significance and normal values of each of the cell Counts

• Identify the sources of error in manual hemocytometery

• Perform quality control procedures in manual hemocytometry

2/27/2024 Hematology and Immunohematology module 3

Outline
• Introduction to manual Hemocytometry

• White Blood Cell (WBC) Count

• The corrected Leukocyte count

• Red Blood Cell (RBC) count

• Platelet Count

• Eosinophil Count

 Source of errors in hemocytometry

2/27/2024 Hematology and Immunohematology module 4

Introduction to Hemocytometry
• The enumeration of blood cells is a fundamental examination in the clinical laboratory

• Cell counts are nowadays performed by automated procedures/instruments

• The classical manual procedures are still used in many laboratories

• Also used as a back up & QC for automated methods

2/27/2024 Hematology and Immunohematology module 5

Introduction to Hemocytometry cont’d
• The manual counting involves:

• Diluting the blood specimen

• Loading it into a special counting chamber

• Counting the cells

• Calculating the number of cells/μl or per liter of blood

2/27/2024 Hematology and Immunohematology module 6

Hemocytometry cont’d
• The cells counted in routine practice are white cells, red cells, and platelets.

• Cell counts are expressed as the number of cells or formed elements (e.g. PLTs, WBCs,
RBCs) per liter of blood (ICSH recommendation).

• The traditional unit of reporting was cubic millimeters (cu mm or mm3)

• 1 cu mm = 1.00003 µL is felt to be insignificant, 1 µL is considered equivalent to 1

cu mm. Thus,1 cu mm = 1 µL = 10-6 liters

2/27/2024 Hematology and Immunohematology module 7

Hemocytometry cont’d
• A hemocytometer consists of a thick rectangular glass slide

• In the center of the upper surface there are :

• Ruled areas separated by moats/channels from the rest of the slide
• Two raised transverse bars one of which is present on each side of the ruled area

• The ruled portion may be:

• In the center of the chamber (single chamber) or
• An upper and lower ruled portion (double chamber)

• The double chamber is to be recommended

• It enables duplicate counts to be made rapidly
2/27/2024 Hematology and Immunohematology module 8
Hemocytometry cont’d
• When an optically plane cover glass is rested on the raised bars there is a predetermined
gap (depth)

• Depth varies with the type of chamber

• The ruled area itself is divided by microscopic lines into a pattern that varies again with
the type of the chamber

• The Improved Neubauer ruled chamber is recommended for cell counts (WBC, RBC,
PLT)

Improved Neubauer
2/27/2024 Hematology and Immunohematology module 9
Types of Hemocytometer
1. Improved Neubauer counting chamber

2. Fuchs-Rosenthal Counting Chamber

3. Bürker ruled counting chamber

2/27/2024 Hematology and Immunohematology module 10

Hemocytometry cont’d
Improved Neubauer counting chamber

• Each counting chamber has two ruled areas

• Each ruled area is 3mm x 3mm (area of 9mm2).

• Each chamber is divided into 9 large squares

• Each square is 1mm x 1mm (area of 1mm2)

• Each of the four corner squares are divided into 16 smaller squares.

2/27/2024 Hematology and Immunohematology module 11

Hemocytometry cont’d

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Hemocytometry cont’d
• The central square (1 mm2) is divided into 25 smaller squares each bordered by double-
ruled lines

• Each of the 25 small squares in the central square is 1/5mm per side (area of
1/25mm2=0.04mm2)

• Each of these 25 small squares is further subdivided into 16 smaller squares (400
tiny squares of 0.0025 mm2)

2/27/2024 Hematology and Immunohematology module 13

Hemocytometry cont’d

2/27/2024 Hematology and Immunohematology module 14

Types of Hemocytometers

2/27/2024 Hematology and Immunohematology module 15

Hemocytometry cont’d
Fuchs-Rosenthal Counting Chamber:

• Designed for counting eosinophils from whole blood

• Also for counting leucocytes and cells in body fluids including CSF

• It has 16mm2 area divided by 16 smaller squares of 1mm2 area.

• The depth is 0.2mm

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Hemocytometry cont’d

2/27/2024 Hematology and Immunohematology module 17

Hemocytometry cont’d
• Bürker ruled counting chamber

• This chamber is occasionally found in laboratories

• The 4 large corner squares are used (4 mm2) to count white 1 2

cells using a Bürker Chamber

• The depth of Bürker ruling chamber is 0.1mm 3 4

• The same calculation as described for the Improved
Neubauer ruled chamber is used.

2/27/2024 Hematology and Immunohematology module 18

Hemocytometry cont’d
• Counting chamber cover glasses

• Special optically plane cover glasses of defined

thickness Cover Glass (20x26x0.5mm) designed Use
Special
for use with hemocytometers are required. Cover
Glasses
• Other cover glasses give incorrect calculations
only!!!!!
(due to variability of thickness)

2/27/2024 Hematology and Immunohematology module 19

Hemocytometry cont’d
Dilution of the Sample

• Accomplished by using

1. Thomma pipet
• Small calibrated diluting pipettes designed for WBC or RBC count

• The WBC pipet has an upper numerical value of 11 while that of the RBC pipet marked by 101.

2. Tube dilution method

• Larger volumes of blood and diluting fluid are used

• Greater accuracy compared with the smaller volumes used in the thomma pipette techniques.

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Hemocytometry cont’d

2/27/2024 Hematology and Immunohematology module 21

Hemocytometer (complete set)

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Hemocytometry cont’d
Counting and Calculation

• The diluted cells are introduced into the counting chamber and
allowed to settle
Follow
• Counting in the designated area (s) counting
direction/
• Cells lying on or touching the upper or left boundary lines are pattern
included in the count while those on the lower and right
boundary lines are disregarded (or vice versa)

• Count cells in a systematic manner (zigzag pattern)

2/27/2024 Hematology and Immunohematology module 23

Hemocytometry cont’d

2/27/2024 Hematology and Immunohematology module 24

White Blood Cell (WBC) Count
WBC count

• Is the number of white cells in 1 liter (L) of whole blood

• Normal range: 4.0 -11.0 x 109/L (Ethiopian: 3.0-10.0 x 109/L, Tsegaye et al)

• Count varies with age and other population parameters

• New born: 10.0 - 30.0 x 109/L decreases to 6.0 -17.0 x 109/L at 1 year of age and drops to
normal level by the age of 21

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White Blood Cell (WBC) Count cont’d
Significance of WBC count:

• To investigate infections and unexplained fever

• To follow prognosis and

• To monitor treatments, which can cause leukopenia

2/27/2024 Hematology and Immunohematology module 26

WBC Count cont’d
Principle

• Whole blood is diluted 1 in 20 in an acid reagent, which hemolyzes mature red cells,
leaving the white cells to be counted. Then number of WBCs per liter or per microlitre
of blood is calculated and reported.

2/27/2024 Hematology and Immunohematology module 27

WBC Count cont’d
Reagents and Equipments

• Equipment for dilution: NEVER

• Thomma white cell pipet
Mouth
pipette!!
• 20 µL micropipet (or sahli pipet) and 10 x 75 mm test tubes
!
• Microscope

• Improved Neubauer hemocytometer

• Syringe fitted with rubber tubing for aspiration of specimen and

diluting fluid (home made)

2/27/2024 Hematology and Immunohematology module 28

Reagents and Equipments cont’d
• Diluting fluid:

• 2% Acetic acid, v/v, in distilled water

• 1% HCl or

• Turk’s diluting fluid (3 ml acetic acid, 1ml aqueous gentian violet, 100 mL dH2O)

• Type of specimen: EDTA whole blood or capillary blood

2/27/2024 Hematology and Immunohematology module 29

Method
A. Test tube dilution method

1. Dispense 0.38 ml of diluting fluid to a test tube (you can take 0.4 ml and discard 20
μl of diluting fluid).

2. Take 20 µl (0.02 ml, 20 cu mm) of well-mixed EDTA anticoagulated venous blood or

free-flowing capillary blood

3. Clean the outside of the micropipet tip or Sahli pipette with dry cotton/gauze with out
touching the tip

2/27/2024 Hematology and Immunohematology module 30

Method cont’d
4. Dispense the blood to the diluent in the tube

5. Rinse by sucking and expelling 3-4 times to remove blood clinging in the inside wall of
the pipette

6. Mix by tapping (will become a 1:20 dilution)

• Avoid formation of bubbles while mixing

7. Allow about 5 minutes for red cells to lyse

2/27/2024 Hematology and Immunohematology module 31

WBC Count cont’d
B. Thomma pipette dilution method
Inspect the
1. Suck blood up to the 0.5 mark; wipe the outside pipette to
ensure
with clean gauze without touching the tip
that it is
2. Take diluting fluid up to 11 mark clean, dry
and has no
3. Detach the aspirator from the Thomma pipet by chipped
tip!
sealing the open tip by your index finger (hold
horizontally)

4. Mix systematically (like figure of 8 manually or

using a mixer)
2/27/2024 Hematology and Immunohematology module 32
WBC Count cont’d
5. The volume contained between 1 and 11 mark will be 10 units out of
which 0.5 is blood and this will make a 1:20 dilution.
6. Wait at least 5 min

7. Clean chambers and cover slip with alcohol and dry well with lint free
cloth

8. Place cover slip on hemocytometer (press cover slip on both corners

until Newton’s ring (rain bow) formation is observed)

2/27/2024 Hematology and Immunohematology module 33

Method cont’d
7. Re-mix blood with diluent by inverting several times before charging on
hemocytometer to ensure even distribution of cells

8. Discard the first four drops (unmixed diluting fluid) from the pipette and plate one
dilution on each side of hemocytometer (to ensure quality)*

*For the test tube method, remix the tube by tapping and charge the
hemocytometer using pasteur pipet or micropipet held at an angle of
about 45o

2/27/2024 Hematology and Immunohematology module 34

Method cont’d
11. Fill the chamber smoothly and don't overfill or under fill it; there should be no bubbles

N.B. if overflow cells will spill into the moat, thus falsely reducing the cell count. Under
filling also gives a falsely lower cell count

12. Allow cells to settle for 3 minutes

A
2/27/2024 Hematology and Immunohematology module 35
Method cont’d
13. Use 10x (low power) objective with low light by lowering the condenser

14. Check for even distribution of cells

• N.B. Difference in the number of cells among squares should not exceed 10%

15. Count WBCs in the four large corner squares

2/27/2024 Hematology and Immunohematology module 36

Method cont’d

2/27/2024 Hematology and Immunohematology module 37

Method cont’d
Calculation:

• The white cell count is reported as the number of white cells in 1cubic millimeter (l µl)
of undiluted blood (1 mm3= 1µl)

• However we diluted the blood and we count the cells in less than 1 mm3of blood!
• Therefore we must multiply our total counted cells by two correction factors:
• Dilution correction factor (DCF) and
• Volume correction factor (VCF)

• DCF compensates for dilution of blood. Since we diluted the blood 1:20 the dilution
factor is 20

• VCF compensates for the volumeHematology

2/27/2024
in which the cells were counted
and Immunohematology module 38
Method cont’d
• Total number of White cells/ µL = No. WBCs counted x VCF x DCF
• VCF=1/V (=volume desired/volume counted)
• V= L x W X h (h=depth of the chamber)

• Dilution factor = invert dilution used (if 1:20, then DCF is 20; if 1:10, DCF is10)

Alternative formula:

Number of cells X dilution factor X 10*

Area counted

• *invert 0.1 depth of chamber

• Report WBC counts to nearest hundred or nearest tenth, depending on units.

2/27/2024 Hematology and Immunohematology module 39
Technical Tips
• Avoid formation of bubbles in the bulb of the pipette as this affects volume

• The long stem of the pipette should be filled with clear fluid

• The final volume should exactly be on the 11 mark

2/27/2024 Hematology and Immunohematology module 40

Exercise 1: WBC Calculation Formula
Question:

Using the formula, calculate the WBC for the following: 2 minutes!
• WBC dilution using traditional pipet (1:20)

•Diluent 380 L to Sample 20L

• WBC counting area(4 corner squares on each side)

• Counted 100 cells on one side of counting chamber, 110

cells on the other side

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Exercise1 Answer: WBC Calculation Formula
Question:

Using the formula, calculate the WBC for the

Number of cells X dilution factor X 10
following:
Area counted
• WBC dilution using traditional pipette
(1:20)

• WBC counting area (4 corner squares)

• Counted 100 cells on one side of

Hemacytometer, 110 cells on other side

Answer: 210 X 20 X 10 =5250/μl

8
2/27/2024
5.3 X
= 9/L
Hematology and Immunohematology module 42
Case Study 2: WBC Calculation Formula
Question:

Using the formula, calculate the WBC for

the following: Number of cells X
dilution factor X 10
• WBC dilution: 1:20
Area counted
• Cells counted WBC area (4 corner squares)
=160 total of both sides

2 minutes!

2/27/2024 Hematology and Immunohematology module 43

Case Study 2 Answer: WBC Calculation Formula
Question:

Using the formula, calculate the WBC for the

following:
Number of cells X
• WBC dilution: 1:20 dilution factor X 10
• Cells counted WBC area (4 corner squares)
Area counted
=160 total of both sides

Answer:
160 X 20 X 10 = 4000/μl
8 = 4.0 X 109/L
2/27/2024 Hematology and Immunohematology module 44
The corrected Leukocyte count
• The white cell diluting fluids destroy only the non-nucleated red blood cells

• In certain disease states, NRBCs are present in the peripheral blood

• When there are more than 10 NRBCs per 100 WBC in the blood film, correct the
WBC count as follows:

2/27/2024 Hematology and Immunohematology module 45

The corrected Leukocyte count
Corrected WBC Count = Uncorrected WBC count x 100
100 + Nucleated RBCs*

*Number of nucleated RBCs per 100 WBC as seen in stained blood film.

E.g. if 15 NRBC/100 WBC; total WBC= 15 x 103/L (15 x 109/L )

Corrected WBC = 13.0 x 103/L (13.0 x 109/L)

2/27/2024 Hematology and Immunohematology module 46

Quality control of WBC counts
• Cell counts are performed in duplicate using two pipettes and counting both sides of the
counting chamber.

• The difference between the two counts should not be more than 10%.

• Additional squares should be counted if the cell count is low /lower dilutions could be
used.

2/27/2024 Hematology and Immunohematology module 47

Quality control cont’d
• When there are decreased and increased WBC counts, alter the dilution

• If WBC>50 x 109/L, increase dilution e.g. 1:40 (0.02 ml (20 L) blood & 0.78 mL
diluting fluid)

• If WBC< 2 x 109/L, decrease dilution e.g. 1:10 (0.04 ml (40 L) blood & 0.36 mL
diluting fluid)

• Verifying count with WBC estimate from May Grunwald-Giemsa or Wright’s stained
smear

2/27/2024 Hematology and Immunohematology module 48

Sources of error in manual WBC count
• Not checking for clots in sample

• Inadequate mixing of anicoagulated blood specimen

• Incorrect measurement (unclean, wet, or chipped pipette)

• Not mixing blood with diluting fluid

• Use of dirty chamber or cover glass

• Not using the right cover glass

• Dilution, charging, counting, calculation error

2/27/2024 Hematology and Immunohematology module 49

Sources of error cont’d
• Air bubbles in the filled chamber

• Not allowing cells to settle

• Having too intensive light source

• Drying of the loaded chamber before completing counting

• Counting too few cells (e.g not to decrease dilution in leukopenia)

• Not correcting for NRBC

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Interpretation of WBC count
• Reference range vary with age, by gender, race, etc

Children at 1yr 6.0-18.0 x 109/L

Children 4-7 yr 5.0-15.0 x 109/L

Adults 4.0-10.0 x 109/L

Adults of African origin 2.6-8.3 x 109/L

Pregnant women up to 15 x 109/L

• Ethiopian adult WBC Reference range: 3.0-10.2 x 109/L (Tsegaye A et al Clin Diagn
Lab Immunol 1999; p410-414)
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Interpretation of WBC count cont’d
• Leukocytosis

-Acute infections -Inflammation and tissue necrosis

-Metabolic disorders -Poisoning

-Acute hemmorhage -Leukemias

-Stress -Menstruation strenuous exercise

-Pregnancy -Hemolytic disease of the new born

-ulcers

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Interpretation of WBC count cont’d
Leukopenia

• The WBC may drop below normal in:

• Viral, bacterial, parasitic infections

• Drugs

• Rheumatoid arthritis, cirrhosis of the liver, and lupus erythematosus

• Radiation and certain drug therapy
• Hypersplenism
• Aplastic anemia and megaloblastic anemia
• Bone marrow infiltration
• Anaphylactic shock
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Red Blood Cell (RBC) count
o RBC count: is the total number of red cells in 1L of whole blood

o Significance of RBC count

• Is used to diagnose anemia

• To know the number of RBCs in other pathological conditions and during normal
physiological conditions

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RBC count cont’d
Principle

• A sample of blood is diluted with a diluent that maintains (preserves) the disc-
like shape of the red cells and prevents agglutination

• The diluted specimen is loaded in a counting chamber and the cells are
counted, calculated and reported

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RBC count cont’d
Equipment and Reagents

• Red cell Thomma pipet (1:200 dilution)

• Improved Neubauer counting chamber

• Test tubes

• Micropipette

• Microscope

• RBC diluting fluid

• Isotonic with red cells, that is it protect red cells from swelling & shrinking

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RBC count cont’d
RBC diluting fluid

• 1% formal citrate
• Dilute 10 ml of 40% formaldehyde solution in 100 ml of trisodium citrate (31.3 g/l)
• Filter and store in a clean glass container

• Gower’s solution
• Dilute 62.5g crystalline sodium sulfate in 500 ml of distilled water
• Add 167 ml of glacial acetic acid
• Dilute to the 1 litre mark with distilled water & Mix.

• Hayem’s solution
• Not recommended because conditions such as hyperglobulinemia cause rouleaux and clumping
of red cells.
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RBC count cont’d
 Type of specimen: EDTA anticoagulated blood or capillary blood

 Method

• Dilution and counting techniques are similar to WBC count (differences are in the
dilution fluid, dilution factor and area of counting)

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RBC Count cont’d
1. Dilute 1 in 200 using the RBC Thomma pipet (blood
to the 0.5 mark, diluting fluid to the 101 mark)

• Tube dilution (0.02 ml blood, 3.98 ml diluting fluid)

R R
2. Fill chamber smoothly and don't overfill or under fill;
R
there should be no bubbles
R R
3. Allow cells to settle for 3 minutes

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RBC count cont’d
4. Use 40x (low power) objective

5. Check for even distribution of cells

6. Count RBCs in the five small squares in the central 1 mm2 area

7. Report the number of Red cells per liter of blood using the following simple
calculation:

• DCF compensates for the dilution that we made i.e. 0.5:100 which is 1 in 200
• Therefore DCF =200

• VCF compensates for volume of blood used for reporting.

• The volume, which is used for counting, is 0.02mm3, and the report is in 1mm3?
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RBC count cont’d
Area of 1 R square = 0.2mx 0.2mm = 0.04mm2
Volume = 0.04mm2x 0.1mm
= 0.004mm3
the volume in 5 R square = 5x0.004 mm3
R R
= 0.02 mm3
R

VCF= Volume desired = 1mm3 = 50 R R

Volume used 0.02mm3

The over all correction factor = DCF x VCF

= 200 x 50
= 10,000

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RBC count cont’d
• Finally multiply the number of RBCs counted in 5R squares by 10000 and this will give
RBC count per mm3 of undiluted blood

• Final report should be given per liter

• Increase or decrease dilution depending on cell number

 QC: Similar to manual WBC count

 Sources of errors: similar to manual WBC count

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RBC count cont’d
 Disadvantage of Manual RBC count

• Since the over all correction factor is 10,000 the possible error is very large, due to this,
Manual RBC count is not recommended.

• The estimated range of error for a manual RBC is about 10-20%.

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Interpretation of RBC count
• Normal values

• Females 3.6 – 5.6 x 1012/L

• Males 4.2 – 6.0 x 1012/L

• The newborn shows an RBC of 5.0 – 6.5 x 1012/L at birth which gradually decreases to
3.5 to 5.1 x 1012/L at 1 year of age

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Interpretation of RBC count

• RBC count is increased in:

• Polycythemia vera

• Secondary polycythemia due to other causes such as dehydration and the effect of
altitude

• RBC counts below Normal in:

• Anemia

• Secondary to other disorders

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Exercise
• A Laboratory technologist counted 440 and 460 red cells in both sides of the
hemocytometer after the usual 1:200 dilution. The total RBC per liter of blood will be:

• Ans. 4.5 x 1012/L

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Platelet count
 Platelet count
• Is the total number of platelets per liter of whole blood

 Clinical significance of Platelet count

• To investigate bleeding disorders,

 Principle

• Blood is diluted 1 in 100 in a filtered solution of 1% ammonium oxalate reagent which

lyses the red cells.

• Then platelets are counted microscopically using an Improved Neubauer counting

chamber and reported per liter of blood
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PLT count cont’d
 Equipment

• Bright field microscope/use ordinary microscope, Preferably phase contrast

microscope

• An Improved Neubauer ruled, described previously for WBC counting are required
for counting platelets

• Note: clean the hemocytometer thoroughly

• It should be completely free from dirt and lint

• The use of 95% ethyl alcohol and lint free cloth is recommended.

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PLT count cont’d
• Reagent (diluting fluid): Brecker-Cronkite method
• Ammonium oxalate 10 g/l (1% W/V)
• Dissolve 1g ammonium oxalate in 100ml fresh distilled water in a scrupulously
clean volumetric flask.

 Types of specimen

Blood sample: EDTA anti-coagulated venous blood

• Capillary blood should not be used because there is a tendency for the platelets to
clump as the blood is being collected (but quick capillary blood is venous blood
impossible)
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PLT count cont’d
 Method

• Perform a platelet count within 2 hours of collecting the blood (venous sample).

• Make a 1 in 100 dilution (0.02 ml blood and 1.98 ml diluent; for Thomma pipet, blood to
the 1 mark and diluent up to the 101 mark)

• Mix well and charge the counting chamber

• Place the chamber in a petri dish with wet cotton and cover with a lid (to prevent drying)

• Leave the chamber undisturbed for 15-20 minutes (allows time for platelets to settle).

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PLT count cont’d
• Using the 10×objective focus the ruling of the grid and focus
the central square of the chamber in to view

• Change into 40x and focus the small platelets

• They will be seen as small bright fragments (refractile)

against a dark background

• Dirt and debris are distinguishable because of their high

refractility

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PLT count cont’d
• Count the platelets in the 5 small squares marked as P

• If PLT count is < 100, 000 count all 25 small squares

• If the 5 middle squares are counted,

you multiply the No. of platelets x 5000

• If the 25 of the middle squares are counted,

you multiply the No. of platelets x 1000

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PLT count cont’d
• Exercise 1; PLT count ( when counting 5 squares and 25 squares)
• If a technologist counted 60 cells using the 1:100 dilution
• Calculate VCF and total number of platelets
• if 5 squares counted;
• if 25 squares counted (assignment).
• VCF=
• Total Platelet count=

• Report the platelet count per 1cubic millimeter (and per 1 liter) undiluted blood

• Interpret the result

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VCF = volume desired
Volume used
Volume used = Area of 5 P sections X height
= (0.04mm2 X 5) X 0.1 mm
= 0.02mm3
VCF = 1 mm3
0.02mm3
= 50

Total no of PLT/μl = No of PLTs counted X DCF X

VCF
= 60 X 100 X 50
= 300,000/μl (3 X 1011/l)

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PLT count cont’d
 Quality control

• Count in duplicate (both sides of the hemocytometer); duplicate counts should agree
within 10%.
• If they do not agree, repeat counts.

• Results are double-checked by examination of the platelets on a Wright-stained smear

• Trained lab professional

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PLT count cont’d
 Sources of error

• All sources of error discussed for manual WBC using hemocytometry (under or
overloading of the counting chamber, calculation, etc) also observed

• Unfiltered and non-refrigerated diluting fluid; debris and bacteria can be mistaken for
platelets

• Platelet clumps

• Counting before platelets settle

• Light adjustment
2/27/2024 Hematology and Immunohematology module 76
Interpretation of platelet count
• Reference range

• 150-400 x 109 platelets per liter of blood

• Ethiopian: 98 – 337 x 109/L ((Tsegaye A et al Clin Diagn Lab Immunol 1999;

p410-414)

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Clinical significance
Thrombocytosis

• Chronic myeloproliferative diseases:

• Essential thrombocythemia
• Polycythaemia vera
• Chronic myelogenous leukemia
• Myelofibrosis.

• Following splenoctomy

• Chronic inflammatory disease

• Hemorrhage

• Sickle cell disease associated.

• Iron deficiency anemia, associated with active bleeding

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cont’d
 Thrombocytopenia

• Thrombocytopenic purpura

• Aplastic anemia

• Acute leukemia

• Infections

• Deficiency of folate or vitamin B12

• Drugs

• Hereditary thrombocytopenia (rare condition)

• Following to chemotherapy and radiation therapy

• Increased destruction or consumption of platelets

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Eosinophil count
 Eosinophil count

• The number of eosinophils per liter of blood

• It is determined when a more accurate count is required

 Clinical Significance

• Eosinophil count is used for the assessment of conditions, which can cause Eosinophilia or
Eosinopenia

• Eosinophilia is common in allergic conditions (e.g., asthma) and in parasitic infections

 Principle

• Blood is diluted with a fluid that causes lysis of erythrocytes and stains eosinophils rendering
them readily visible
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Eosinophil count cont’d
 Equipment and reagents

• Microscope

• Fuchs Rosenthal counting

chamber
• Has a depth of 0.2 mm

 Type of specimen: EDTA whole

blood or capillary blood

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Eosinophil count cont’d
Diluting Fluid:

• Hinkleman’s fluid
• Has the advantage of keeping well at room temperature and not needing filtering
before use

• Hinkleman's Solution
• Eosin yellow …………………….. 0.5g
• Formaldehyde (40%)…..………… 0.5ml
• Phenol (95% aq. solution…………..0.5ml
• Distilled water …………………… to 100ml
• Filter after preparation
• The solution will keep indefinitely
2/27/2024
at 40C
Hematology and Immunohematology module 82
Eosinophil count cont’d
 Method:
• Make a dilution of blood using Thomma pipette or tube dilution as described for
the white cell count

• A Fuchs-Rosenthal chamber (with a total area of 16mm2 and depth of 0.2mm) is

used for counting

• Counting is carried out as soon as the cells are settled (10 minutes)

• All the cells in the ruled area are counted

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Eosinophil count cont’d
• Calculation: If E is the number of eosinophils in 16 large squares (in 3.2l volume), then the
absolute eosinophil count per l of blood is:
E  20 = 6.25E
3.2
• i. e. The overall correction factor is 6.25

vii. Quality control and sources of error

• To increase the accuracy at least 100 cells should be counted, i.e., both ruled areas should be
counted and if the count is low, the chamber should be cleaned and refilled

• QC measures for manual WBC count also apply here

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Eosinophil count cont’d
• Additional QC measures and the sources or errors for manual WBC count also apply
here
 Interpretation:
Normal Range: 40-440  106/l

Eosinophlia: increased Eosinophils

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 Thanks for Your
patience!!!

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 Review Questions
7. Indicate the diluting fluid, dilution factor, and areas
of counting on the chamber (in mm2) for each of the
following cell counts:
a) WBC count
b) RBC count
c) Platelet count
d) Eosinophil count (here indicate the area of count in
mm2 and the volume of diluted sample in mm3 or l in
the Fuchs-Rosenthal counting chamber)

8. Briefly state the clinical implications of each of the

cell counts in question 7

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