Learning Objectives

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

Discuss the general principles of manual hemocytometery

List the materials that are basically required in manual

hemocytometery
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

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

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

7.1. 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
 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
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
 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

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
Hemocytometry cont’d

Improved Neubauer
Hemocytometry cont’d
Improved Neubauer counting chamber
Each counting chamber has two ruled areas

Each ruled area is 3mm x 3mm (area of 9mm 2).

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.
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)
E.g., Types of Hemocytometers
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
 Hemocytometry cont’d

Bürker ruled counting chamber

This chamber is occasionally found in

laboratories
The 4 large corner squares are used (4 mm2)
1 2
to count white 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.

Hemocytometry cont’d
Counting chamber cover glasses

Special optically plane cover glasses

of defined thickness Cover Glass

(20x26x0.5mm) designed for use with Use
Special
hemocytometers are required. Cover
Glasses
Other cover glasses give incorrect
only!!!!!
calculations (due to variability of
thickness)
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.
Hemocytometer (complete set)
 Hemocytometry cont’d
Counting and Calculation
The diluted cells are introduced into
the counting chamber and allowed to
settle
Counting in the designated area (s)
Cells lying on or touching the upper or
left boundary lines are included in the
count while those on the lower and Follow
right boundary lines are disregarded counting
(or vice versa) direction/
Count cells in a systematic pattern
manner (zigzag pattern)
7.2. White Blood Cell (WBC) Count
i. WBC count
Is the number of white cells in 1 liter (L) of whole blood

NR: 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
ii. Significance of WBC count:
to investigate infections and unexplained fever

to follow prognosis and

to monitor treatments, which can cause leukopenia

WBC Count cont’d
iii. 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.
Note: When examining a stained blood film, if many nucleated

red cells are present (more than 10%), the WBC count should
be corrected and reported as corrected white cell count
(diluting fluid will not lyse nucleated red cells)
 WBC Count cont’d
iv. Reagents and Equipments NEVER
Equipment for dilution: Mouth
 Thomma white cell pipet pipette
 20 µL micropipet (or sahli pipet) and 10 x !!!
75 mm test tubes or
 WBC unopette

Microscope

Improved Neubauer hemocytometer

Syringe fitted with rubber tubing for

aspiration of specimen and diluting fluid
(home made)
 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)

(Note: if WBC unopette is used, it contained premeasured

amount of diluting fluid)

Lint free cloth

v. Type of specimen: EDTA whole blood or capillary blood

 vi. 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
 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

WBC Count cont’d
B. Thomma pipette dilution Inspect the
method pipette to
1. Suck blood up to the 0.5 mark; wipe ensure that
the outside with clean gauze it is clean,
without touching the tip dry and has
2. Take diluting fluid up to 11 mark no chipped
3. Detach the aspirator from the tip!
Thomma pipet by sealing the open
tip by your index finger (hold
horizontally)
4. Mix systematically (like figure of 8
manually or using a mixer)
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)
Method cont’d
9. Re-mix blood with diluent by inverting several times before

charging on hemocytometer to ensure even distribution of

cells

10. 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
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
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
16. Average the number of WBCs counted on the two sides.
Using the average, calculate the WBC count using formula
 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 mm3 of 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 volume in which the cells were
counted
 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 is 10)
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.
 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

 Exercise 1: WBC Calculation Formula
Question:

Using the formula, calculate the WBC for the 2 minutes!

following:
 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

 Exercise1 Answer: WBC Calculation Formula
Question:
Using the formula, calculate Number of cells X dilution factor X 10
the WBC for the 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

5.3 X 9/L
=
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!
Case Study 2 Answer: 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

160 X 20 X 10 = 4000/μl
Answer: 8

= 4.0 X 109/L
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:

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)

 vii. 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.

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

viii. 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

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
ix. Interpretation of WBC count
Reference range vary with age, by gender, race, etc

Children at 1y 6.0-18.0 x 109/L

Children 4-7 y 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)

Interpretation of WBC count cont’d
Leukocytosis

 Acute infections (e.g. pneumonia, abscess, whooping cough,

tonsillitis, appendicitis
 acute rheumatic fever, septicemia, gonorrhoea, cholera, septic

abortion, etc)
 Note: Acute infections in children can cause a sharp rise in

WBC count (than in adults to a corresponding infection).

Metabolic disorders (e.g eclampsia, uremia, Diabetic coma,
acidosis)
Inflammation and tissue necrosis (burns, gangrene, fractures and
trauma, arthritis, tumors, acute myocardial infarction)
Interpretation of WBC count cont’d
Poisoning (e.g. chemicals, drugs, snake venoms)

Acute hemmorhage

Leukemias and myeloproliferative disorders

Stress, Menstruation strenuous exercise

Pregnancy

Hemolytic disease of the new born

ulcers
Interpretation of WBC count cont’d
Leukopenia
The WBC may drop below normal in:
 Viral, bacterial, parasitic infections

 e.g. HIV/AIDS, viral hepatitis, measles, rubella, influenza,

rickettsial infections, overwhelming bacterial infections
such as miliary tuberculosis, relapsing fever, typhoid,
paratyphoid, brucellosis, parasitic infections including
leishmaniasis and malaria.
  Drugs (e.g. Cloroamphenicol, phenlybutazone)

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

Leukopenia cont’d

Radiation and certain drug therapy (e.g. cytotoxic) and

reactions to chemicals
Hypersplenism

Production failure as in Aplastic anemia and megaloblastic

anemia (Folate and vitamin B12 deficiencies)

Bone marrow infiltration (e.g. lymphomas, myelofibrosis,

myeloma)
Anaphylactic shock
7.3. Red Blood Cell (RBC) count
i. RBC count: is the total number of red cells in 1Litre of whole blood

ii. Significance of RBC count

 is used to diagnose anemia

 to know the number of RBCs in other pathological conditions and

 during normal physiological conditions

iii. 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 and reported
RBC count cont’d
iv. Equipment and Reagents
Red cell Thomma pipet (also RBC Unopipet; 1:200 dilution)

Improved Neubauer counting chamber

Test tubes

Micropipette

Microscope

RBC diluting fluid – is isotonic with red cells, that is it

protect red cells from swelling & shrinking

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.

 RBC count cont’d
v. Type of specimen: EDTA anti-coagulated blood or capillary
blood

vi: Method
Dilution and counting techniques are similar to WBC count

(differences are in the dilution fluid, dilution factor and area of

counting)

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)
RBC Count cont’d
2. Fill chamber smoothly and don't
overfill or under fill; there should be
no bubbles
3. Allow cells to settle for 3 minutes

4. Use 40x (low power) objective R R

R
5. Check for even distribution of cells
R R
6. Count RBCs in the five small squares
in the central 1 mm2 area
N.B. for QC purpose you need to increase
counting areas
 RBC count cont’d
7. Report the number of Red cells per litre 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 how?
 RBC count cont’d
Area of 1 R square = 0.2mm x 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

  R R

VCF= Volume desired = 1mm3 = 50

Volume used 0.02mm3

The over all correction factor = DCF x VCF

= 200 x 50
= 10,000
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 litre

Increase or decrease dilution depending on cell number

vii. QC: similar to manual WBC count

viii. sources of errors: similar to manual WBC count

 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%

ix. 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
 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

Exercise
A technician 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

7.4. Platelet count
i. Platelet count
 Is the total number of platelets per liter of whole blood

ii. Clinical significance of Platelet count

 to investigate bleeding disorders

iii. 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
PLT count cont’d
iv. Equipment
 Bright field microscope fitted with dark field condenser

 Or use ordinary microscope with lowered condenser or iris

diaphragm partially closed

 Preferably phase contrast microscope

 An Improved Neubauer ruled Bright-line counting chamber and

other equipment as 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.
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. The solution should be
filtered and kept at 4oC. For use, a small part of the stock should
be refiltered

v. Types of specimen

Blood sample:
 Whenever possible use 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

PLT count cont’d
Check tube for clots; if any (even tiny clot), a new specimen

should be obtained
If it is not feasible to draw venous blood, wipe away the first

drop of blood from the finger or heel after the puncture

Blood should enter the diluting pipet as quickly as it flows

from the wound in order to prevent platelet clumping or

adhesion to the puncture site
PLT count cont’d
vi. 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)
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

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

 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
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)
PLT count cont’d
vii. 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
PLT count cont’d
viii. 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
ix. 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)

 
Clinical significance
Thrombocytosis
The main causes for an increase in PLT numbers include:
 Chronic myeloproliferative diseases:
• essential thrombocythemia

• polycythaemia vera

• chronic myelogenous leukemia

• myelofibrosis.

 Following splenoctomy
 Chronic inflammatory disease, e.g. tuberculosis
 Hemorrhage
 Sickle cell disease associated with a non-functioning spleen or after
splenectomy.
 Iron deficiency anemia, associated with active bleeding
 cont’d
Thrombocytopenia
The main causes for a reduction in platelet numbers are:
 Thrombocytopenia purpura

 Aplastic anemia

 Acute leukemia

 Gaucher’s disease

 Infections, e.g. typhoid and other septicemias

 Deficiency of folate or vitamin B12

 Drugs (e.g. cytotoxic, quinine, aspirin), chemicals (e.g. benzene), some

herbal remedies
 Hereditary thrombocytopenia (rare condition).

 Following chemotherapy and radiation

 cont’d
Increased destruction or consumption of platelets:
Infections, e.g. acute malaria, dengue, trypanosomiasis,

visceral leishmaniasis
Disseminated intravascular coagulation (DIC)

Hypersplenism

Immune destruction of platelets, e.g. idiopathic

thrombocytopenic purpura (ITP), systemic lupus

erythematosus (SLE), etc.
Unopette WBC/Platelet count
 The Unopette system has become a valuable method:

 for standardizing the pipetting and diluting of blood and other body

fluids
 for increased safety, accuracy, and precision.

 The standard Unopette is made up of:

 a reservoir which contains a premeasured volume of diluting fluid

which is sealed by a thin covering plastic (diaphragm) located in the

neck
 a self filling pipette which is available in various sizes (3 µL, 3.3µL, 10

µL, 20 µL, 25 µL, 44.7 µL) depending on the procedure to be performed.

 Each pipette is color- coded according to size
Unopette cont’d

Principle: The Unopette system is a system of prefilled blood

dilution vials containing solutions that will preserve certain

cell types while lysing others. Capillary pipettes are available

to draw up different volumes of blood. The dilution is

determined by the type of capillary used. The diluted blood is

added to a counting chamber.

7.5. Eosinophil count
i. Eosinophil count
 The number of eosinophils per liter of blood
 It is determined when a more accurate count is required
ii. 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
iii. Principle
 Blood is diluted with a fluid that causes lysis of erythrocytes and
stains eosinophils rendering them readily visible.
Eosinophil count cont’d
iv. Equipment and
reagents
Microscope

Fuchs Rosenthal

counting chamber
N.B. has a depth of

0.2 mm
V. Type of specimen:
EDTA whole blood or
capillary blood
 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 at 4 0C.

Eosinophil count cont’d
vi. 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. Usually

10 minutes in a moist atmosphere petri dish will suffice.

All the cells in the ruled area are counted (i.e., in 3.2l volume).
 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
 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

Review Questions
 
1. What are the main principles of manual hemocytometery?
 2. List the items that are generally required in manual
hemocytometery.
 3. How do you calculate the number of cells per unit volume of blood
after you count the cells in a sample of diluted blood?
4. How do errors in hemocytometery arise? How do you reduce the
introduction of such errors in your count?
5. What are the QC measures to be taken in manual cell counting?
6. Describe the difference between Impvoved Neubauer and Fuchs
Rosenthal counting chambers
 
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