Hematology

1. MANUAL HEMOCYTOMETRY
Pre-Internship Training 2023
by RIS NRH

2. 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

3. 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

4. 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

5. 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

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

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

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

9. 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.

10. 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)

11. E.g., Types of Hemocytometers

12. 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

13. 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 cells using a Bürker
Chamber
 the depth of Bürker ruling chamber is 0.1mm
 The same calculation as described for the
Improved Neubauer ruled chamber is used.
1
4
3
2

14. Hemocytometry cont’d
 Counting chamber cover glasses
 Special optically plane cover
glasses of defined thickness Cover
Glass (20x26x0.5mm) designed for
use with hemocytometers are
required.
 Other cover glasses give incorrect
calculations (due to variability of
thickness)
Use
Special
Cover
Glasses
only!!!!!

15. 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.

16. Hemocytometer (complete set)

17. 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 right boundary lines are
disregarded (or vice versa)
 Count cells in a systematic
manner (zigzag pattern)
Follow
counting
direction/
pattern

18. 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

19. 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)

20. WBC Count cont’d
iv. Reagents and Equipments
 Equipment for dilution:
 Thomma white cell pipet
 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)
NEVER
Mouth
pipette!
!!

21. 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

22. 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

23. 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

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

25. 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

26. Method cont’d
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)
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

27. 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

28. 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

29. 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 volume in which the cells
were counted

30. 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.

31. 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

32. Exercise 1: WBC Calculation Formula
Question:
Using the formula, calculate the
WBC for the 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
2 minutes!

33. Exercise1 Answer:
WBC Calculation Formula
Question:
Using the formula, calculate
the WBC for the following:
 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
Number of cells X dilution factor X 10
Area counted
Answer: 210 X 20 X 10 =5250/μl
8
=
5.3 X 9/L

34. Case Study 2: WBC
Calculation Formula
Question:
Using the formula,
calculate the WBC for the
following:
 WBC dilution: 1:20
 Cells counted WBC area
(4 corner squares) =160
total of both sides
Number of cells X
dilution factor X 10
Area counted
2 minutes!

35. Case Study 2 Answer: WBC
Calculation Formula
Question:
Using the formula, calculate the
WBC for the following:
 WBC dilution: 1:20
 Cells counted WBC area (4
corner squares) =160 total of
both sides
Number of cells X
dilution factor X 10
Area counted
Answer:
160 X 20 X 10 = 4000/μl
8 = 4.0 X 109/L

36. 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:

37. 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)

38. 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.

39. 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

40. 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

41. 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

42. 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)

43. 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)

44. 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

45. 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

46. 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

47. 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

48. 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

49. 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.

50. RBC count cont’d
v. Type of specimen: EDTA anticoagulated 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)

51. 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
5. Check for even distribution of cells
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
R
R R
R
R

52. 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?

53. 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
= 0.02 mm3
VCF= Volume desired = 1mm3= 50
Volume used 0.02mm3
The over all correction factor = DCF x VCF
= 200 x 50
= 10,000
R
R R
R
R

54. 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

55. 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%.

56. 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

57. 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

58. 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.

59. 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.

60. 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

61. 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.

62. 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).

63. 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

64. 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

65. 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

66. 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)

67. 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

68. 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

69. 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)

70. 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

71. 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

72. 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.

73. 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.

74. 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.

75. 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.

76. 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

77. 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 40C.

78. 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).

79. 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

80. 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

81. 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

82. 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

83. Acknowledgements
 Addisa Ababa University
 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-Ethiopia