Useful for MBBS students

1. TOTAL LEUCOCYTE COUNT
TOTAL DIFFERENTIAL COUNT
Dr Neha Mahajan
MD Pathology

2. Haematopoeisis

3. G
R
A
N
U
L
O
P
O
E
I
S
I
S

4. Type of WBC’s
 Granulocytes—have large
granules in their cytoplasm
 Neutrophils( 40 to 75%)
 Eosinophils(1 to 6%)
 Basophils(0 to 1%)

6. Types of WBC’s
 Agranulocytes—do not have
granules in their cytoplasm
 Lymphocytes(20 to 40%)
 Monocytes( 2 to 10%)

8. Granuloctyes
 Neutrophils
 Stain light purple with neutral dyes
 Granules are small and numerous—course
appearance
 Several lobes in nucleus
 65% of WBC count
 Diapedesis,inflammation

10. Granulocytes
 Eosinophils or Acidophils:
 Large, numerous granules
 Nuclei with two lobes
 2-5% of WBC count
 Found in lining of respiratory and digestive tracts
 Protections against infections caused by parasitic
worms and involvement in allergic reactions
 Secrete anti-inflammatory substances in allergic
reactions

12.  Basophils
 Least found- 0.5 to 1%
 Contain histamine,serotonin,heparin—
inflammatory chemical

14. Agranulocytes
 Lymphocytes
 Smallest WBC
 Large nuclei/small amount of cytoplasm
 Account for 25% of WBC count
 Two types—T lymphocytes—attack an
infect or cancerous cell,
B lymphocytes—produce antibodies
against specific antigens (foreign body)

16. Agranulocytes
 Monocytes
 Largest of WBCs
 Dark kidney bean shaped nuclei
 Highly phagocytic

18. TOTAL LEUCOCYTE COUNT

19. WHITE CELL COUNT (WBC)
 White cell count (WBC) is the total number of leukocytes in a
volume of blood, expressed as thousands/µl.
 WBC can be done by manual methods or by automated cell
counters.
 Normal Values:
• Newborn 9.0-30.0 x 103/μl
• 1 week 5.0-21.0 x 103/μl
• 1 month 5.0-19.5 x 103/μl
• 6-12 months 6.0-17.5 x 103/μl
• 2 years 6.2-17.0 x 103/μl
• Child/adult 4.8-10.8 x 103/μl

20. PRINCIPLE OF WBCS COUNT TEST
 Free-flowing capillary or well-mixed anticoagulated
venous blood is added to a diluent) at a specific volume in
the thoma pipette.
 The diluent lyses the erythrocytes but preserves
leukocytes and stains the nuceli.
 The diluted blood is added to the hemacytometer
chamber.

21.  Specimen:
 EDTA- anticoagulated blood or capillary blood is
preferred.
 Reagents, supplies and equipment:
 White blood cells count diluting fluid
Turks' solution which is formed of:
 Glacial acetic acid 3 ml
 Crystal violet 1 ml
 100 ml distilled water.

22. EQUIPMENT
1. White blood cells count diluting fluid
2. Thoma white pipette
3. Hemacytometer and coverslip
4. Microscope
5. Lint-free wipe
6. Alcohol pads

23. haemocytometer chamber
Thoma white pipette
Rubber sucking tube

24. HEMACYTOMETER
 The hemacytometer counting chamber is used for cell
counting.
 It is constructed so that the distance between the bottom of
the coverslip and the surface of the counting area of the
chamber is 0.1 mm.
 The surface of the chamber contains two square ruled areas
separated by an H-shaped moat.

25. HEMACYTOMETER

26. PROCEDURE
1. Draw the blood up to 0.5 mark in the thoma
pipette.
2. Wipe the outside of the capillary pipette to
remove excess blood that would interfere with
the dilution factor.
3. Holding the pipette almost vertical place into the
fluid. Draw the diluting fluid into the pipette slowly
until the mixture reaches the 11 mark, while
gently rotating the pipette to ensure a proper
amount of mixing.
4. Place the pipette in a horizontal position and
firmly hold the index finger of either hand over
the opening in the tip of the pipette, detach the
aspirator from the other end of the pipette now
the dilution of the blood is completed

27. PROCEDURE
5. Mix the sample for at least 3 minutes to facilitate
hemolysis of RBCs.
6. Clean the hemacytometer and its coverslip with an
alcohol pad and then dry with a wipe.
7. Before filling the chamber, discard the first four to
five drops of the mixture on apiece of gauze to expel
the diluent from the stem.

28. PROCEDURE
8. Carefully charge hemacytometer with diluted
blood by gently squeezing sides of reservoir to
expel contents until chamber is properly filled.

29. PROCEDURE FOR COUNTING WBC’S
1. Under 10 x magnifications, scan to ensure even
distribution. Leukocytes are counted in all 4 large
squares of counting chamber.
2. Count cells starting in the upper left large corner
square. Move to the upper right corner square,
bottom right corner square, bottom left corner square
and end in the middle square.
3. Count all cells that touch any of the upper and left
lines, do not count any cell that touches a lower or
right line.

31. CALCULATIONS
Dilution factor 20
Volume= Area x depth
TLC/uL= No of WBCx Correction for Volume X
dilution
No of large squares (4)
= N x 20 x10
4
= N x 50

32. Corrected TLC
Diluting fluid does not lyse nucleated
RBC`s/erythroblasts
Falsely counted as WBC
Corrected TLC/uL= TLC x 100
NRbc/100 Wbc+ 100

33. WHITE BLOOD CELL
DIFFERENTIAL COUNT

34. DEFINITION
The relative percentage of each
type of white blood cells in
peripheral blood.
This experiment is a part of blood
routine test.

35. PERIPHERAL BLOOD SMEAR
 A properly prepared blood smear is
essential to accurate assessment of cellular
morphology
 The wedge smear is the most convenient
and commonly used technique for making
PBS

36. PERIPHERAL BLOOD SMEAR
Wedge technique of making
PBS
A. Correct angle to hold
spreader slide
B. Blood spread across width
of slide
C. Completed wedge smear

37. PERIPHERAL BLOOD SMEAR
 Characteristics:
 It is smooth without
irregularities, holes, or
streaks
 When the slide is held
up to light, the
featheredge of the
smear should have a
“rainbow” appearance
 The whole drop is
picked up and spread
Well-made PBS

38. tail body head

39. PERIPHERAL BLOOD SMEAR
 Examples of unacceptable smears

40. PERIPHERAL BLOOD SMEAR
Examples of unacceptable smears

41. Observing direction:
Observe one field and record the number of WBC according to
the different type then turn to another field in the snake-liked
direction
*avoid repeat or miss some cells

42. ROMANOWSKY STAINING
Leishman's stain : a polychromatic stain
• Methanol : fixes cells to slide
• methylene blue stains RNA,DNA
 blue-grey color
• Eosin stains hemoglobin, eosin granules
 orange-red color
• pH value of phosphate buffer is very important
42

43. PROCEDURE
• Thin smear are air dried.
• Flood the smear with stain.
• Stain for 1-5 min.
• Experience will indicate the optimum time.
• Add an equal amount of buffer solution and mix the stain by blowing
an eddy in the fluid.
• Leave the mixture on the slide for 10-15 min.
• Wash off by running water directly to the centre of the slide to prevent
a residue of precipitated stain.
• Stand slide on end, and let dry in air.
43

44. FEATURES OF A WELL-STAINED PBS
 Macroscopically: color should be pink to purple
 Microscopically:
RCS: orange to salmon pink
WBC: nuclei is purple to blue
cytoplasm is pink to tan
granules is lilac to violet
Eosinophil: granules orange
Basophil: granules dark blue to black
44

45.  Optimal Assessment Area:
1. RBCs are uniformly and singly distributed
2. Few RBC are touching or overlapping
3. Normal biconcave appearance
45

46. PRINCIPLE
 White Blood Cells
1. Check for even distribution and estimate the number
present (also, look for any gross abnormalities present on
the smear).
2. Perform the differential count.
3. Examine for morphologic abnormalities.
46
MANUAL DIFFERENTIAL

47.  Red Blood Cells, Examine for :
1. Size and shape ( Anisocytosis,Poikilocytosis
2. Relative hemoglobin content.
3. Polychromatophilia.
4. Inclusions.
5. Rouleaux formation or agglutination
47

48. WBC ESTIMATION UNDER 40X
• Using the × 40 high dry with no oil.
• Choose a portion of the peripheral smear where there is
only slight overlapping of the RBCs.
• To do a WBC estimate by taking the average number of
white cells and multiplying by 2000.
48

49. PLATELET ESTIMATION UNDER 100X
1. Use the oil immersion lens estimate the number of
platelets per field.
2. Look at 5-6 fields and take an average.
3. Multiply the average by 20,000.
4. Note any macroplatelets.
49

50.  Platelets per oil immersion field (OIF)
1) <8 platelets/OIF = decreased
2) 8 to 20 platelets/OIF = adequate
3) >20 platelets/OIF = increased
50

51. MANUAL DIFFERENTIAL COUNTS
• These counts are done in the same area as WBC and platelet
estimates with the red cells barely touching.
• This takes place under × 100 (oil) using the zigzag method.
• Count 100 WBCs including all cell lines from immature to
mature.
 Reporting results
• Absolute number of cells/µl = % of cell type in differential x
white cell count
51

52. MICROSCOPIC EXAM
 10× (low fold): overall smear quality,
rouleaux, agglutination or parasites
 100× (oil Len): WBC Diff, RBC
morphology
 RBC Morphology
 WBC- Total count
Differential count
 Platelets
 Abnormal cells
 Parasites
Peripheral smear reporting

53.  Normal blood smear
53

54. NORMAL NEUTROPHIL COUNT (40 TO 75%)
NEUTROPHILIA
54
 Absolute neutrophil count greater than 7500/uL
 Causes
Acute bacterial infections-
abscesses,pneumonia,meningitis,UTI
Tissue necrosis- burns,injury,MI
Acute blood loss
Acute haemorrhage
Myeloproliferative disorders
Posisoning

55. NEUTROPENIA
 Absolute netrophil count less than 2000/uL
 Mild- 2000 to 1000/uL
 Moderate-1000 to 500/uL
 Severe- < 500/uL
CAUSES
I )Decreased or ineffective production in bone marrow
 Infections- bacterial,protozoal,viral
 Haematologic disorders- megaloblastic
anemia,aplastic anemia,aleukemic leukemia
 Drugs
 Ionising radiation
 Congenital disorders

56. II) Increased destruction in peripheral blood
Neonatal isoimmune neutropenia
Systemic lupus erythematosus
Felty`s syndrome
III) Increased sequestration in spleen
Hypersplenism

57. EOSINOPHILA
 Absolute eosinophil count greater than 600/uL
CAUSES
1.Allergic diseases-Asthma,rhinitis,urticaria
2.Skin diseases-Eczema,pemphigus,Dermatitis herpetiformis
3.Parasitic infections with tissue invasion-
filariasis,trichinosis,echinoccocoosis
4.Hematologic disorders-MPD,Hodgkins disease,Peripheral T
cell lymphoma

58. 5.Carcinoma with necrosis
6.Radiation therapy
7.Lung diseases- loefflors syndrome,tropical
eosinophilia
8.Hypereosinophilia syndrome

59. MONOCYTOSIS
 Increase in absolute monocyte count > 1000/uL
Causes
 Infections-TB,SABE,Malaria,Kala Azar
 Recovery from neutropenia
 Autoimmmune disorders
 Hematologic diseases- MPD,Monocytic
leulemia,hodgkins disease
 Others-ulcerative colitis,chron`s
disease,sarcoidosis

60. LYMPHOCYTOSIS
 Absolute lymphocyte count more than upper limit of
normal for age( 4000/uL in adults,>7200/uL in
adolescents,>9000/uL in children and infants)
Causes
Infections-
Viral- acute infectious
lymphocytosis,neoatitis,CMV,rubella,mumps,varicell
a
Bacterial- pertussis,TB
Protozoal- toxoplasmosis

61.  Hematological disorders-
ALL, CLL, Multiple myeloma, Lymphoma
 Others-
Serum sickness, post vaccination, drug reactions

62. PLATELETS
 Small,1 to 3 um in diameter,purple structures with
tiny irregular projections on surface
 Occur in clumps
 Pseudothrombocytopenia
 Platelet sateletism

63. SUMMARY
 Normal haematopoeisis
 Total leucocyte Count
 Manual differential count
Peripheral smear
Interpretation

64. THANK YOU