This document provides an overview of blood, including its composition, functions, and key components like red blood cells and white blood cells. It discusses the properties of red blood cells in depth, covering their morphology, membrane structure, biconcave shape, and the process of erythropoiesis. Key aspects of hemoglobin such as its structure, function in oxygen transport, derivatives, and synthesis within red blood cells are also summarized.

1. Blood
Done By
A Mahesh Kumar
1st
Year Pg student
Dept of Pedodontics
Drs S&N SIDS
1

2. CONTENTS
1.INTRODUCTION
2.PROPERTIES
3.FUNCTIONS
4.COMPOSITION
5.RBC
6.WBC
7.REFERENCES
2

3. REFERENCES :
1.Text book of Medical Physiology- 10th
edition- Guyton & Hall
2.Concise Medical Physiology- 4th
edition- Chaudhuri
3.Essentials of Medical physiology-4th
edition-Sembulingam
4.Per haavardsholm finne and Sverre halvorsen. Regulation of
Erythropoiesis in the Fetus and Newborn. Archives of Disease in
Childhood 1972; 47: 683-687
5.Narla Mohandas and Patrick G. Gallagher. Red cell membrane:
past, present, and future. Blood 2008; 112: 3939-3948
3

4. 6.Michael Fo¨ller1, Stephan M. Huber2 and Florian Lang.
Erythrocyte Programmed Cell Death. Life 2008; 60(10): 661–8
7.Petra Kleinbongard et all. Red blood cells express a functional
endothelial nitric oxide synthase. Blood 2006; 107: 2943-51
8. Veronique Witko-Sarsat et al. Neutrophils: Molecules, Functions
and Pathophysiological Aspects. Laboratory investigations 2000;
80(5): 617-653
9. SM Rashmi et al. Neutrophils in health and disease: An overview.
J Oral Maxillofac Pathol 2006; 10: 3-8
4

5. INTRODUCTION
• Specialized connective tissue
• Contains cells suspended in a fluid matrix
• Fluid of life
• Fluid of growth
• Fluid of health
5

6. PROPERTIES
Colour : Red
Volume : In adults 5 ltrs
New born 450 ml
pH : slightly alkaline 7.4
Specific gravity : 1.052 – 1.061 total blood
1.092 – 1.101 blood cells
1.022 – 1.026 plasma
Viscosity : five times more viscous than water
6

7. FUNCTIONS:
1.Respiratory function
2.Excretory function
3.Nutrient function
4.Role of blood in various homeostatic processes
a)Body temperature and blood
b)pH and blood
7

8. 5. Role of blood in various defence mechanism
6. Transport of hormones and enzymes
7. Storage function
8. Regulation of water balance
8

9. COMPOSITION OF BLOOD
1.Plasma - 55%
2.Formed elements - 45%
a) RBC or Erythrocytes
b) WBC or Leukocytes
c) Platelets or Thrombocytes
9

10. 10

11. 11

12. CONTENTS
1.MORPHOLOGY
2.PROPERTIES
3.ERYTHROPOIESIS
4.HEMOGLOBIN
5.IRON METABOLISM
6.LAB INVESTIGATIONS
12

13. MORPHOLOGY
• Circular, biconcave cell without a nucleus
• Diameter - 7.5µ
• Thickness at the periphery - 2µ
• Thickness at the centre - 1µ
13

14. Advantages of biconcave shape
 Thickness of an RBC in its central part is not great,
so oxygen doesnot have to travel a great distance for the
diffusion
 Biconcavity increases the surface area of the RBC,
so oxygen gets a bigger area for diffusion
 The erythrocyte can squeeze itself through a
capillary more easily.
14

15. Red cell membrane
It is a tri laminar structure having a bimolecular lipid layer
interposed between the two layers of proteins.
Lipids:
Glycolipids,
Phospholipids,
Cholesterol.
15

16. The proteins comprising the erythrocyte cytoskeleton
include
1.Ankyrin
2.spectrin
3.Band 4.1
4.Band 3
5.Actin filaments
16

17. Protein 4.1R-dependent multiprotein complex: New
insights into the structural organization of the red
blood cell membrane
17

18. PROPERTIES OF RED BLOOD CELLS
1.Rouleaux formation
2.Specific gravity : 1.092 to 1.101
3.Packed cell volume
4.Suspension stability
18

19. ERYTHROPOIESIS
Erythropoiesis is the process by which the origin,
development and maturation of erythrocytes occur.
Hemopoiesis is the process which includes origin,
development and maturation of all the blood cells.
19

20.  Blood forming tissues
Myeloid tissue
Lymphoid tissue
 Myleloid tissue
means RBM
produces RBCs, granulocytes, monocytes and platelets.
 Lymphoid
lymphnode, thymus, and spleen
Produce lymphocytes.
20

21. Site of Erythropoiesis
In fetal life
21

22. Age Site
Upto 5-6 years RBM of all bones
6-20 years RBM of long bones & all
membranous (flat) bones
After 20 years All membranous bones &
ends of long bones
In Post natal life and in adults
22

23. Bone marrow
2 types
• Red bone marrow
• Yellow bone marrow
RBM – found in flat bones (cranial, ribs, sternum ,
pelvic bones)
YBM – shafts of long bones in fully grown adults.
23

24. Extra medullary hemopoiesis
when there is necessity of increased erythropoiesis the
YBM is converted into RBM. If it is more intense liver
and spleen also start producing RBC.
(Medulla = bone marrow)
24

25. Histology of RBM
Consists of
1.Large no of sinusoids
2.Adventitious cells outside the sinusoids
3. Blood forming cells in between adventitious cells
25

26. • Sinusoids are basically capillaries with larger
diameter.
• Their walls contain big pores – big molecules,
blood cells pass.
• Adventitious cells – become fat cells
• The blood cells – Precursors of erythrocytes,
leucocytes, and platelets
• Normally – blood cells : fat cells – 1:1
26

27. RBM blood picture
Cells Percentage
Granulocytes & their precursors 60%
Erythrocytes & their precursors 20%
Lymphocytes, monocytes &
precursors
10%
Others ( non identifiable,
degenerated cells)
10%
27

28. Stages of development
28

29. 29

30. 30

31. Different stages of RBC development
Progenitor cells – BFU-E (Burst forming unit)
CFU-E
Blast cells – Pronormoblast (E1)
Early normoblast(E2)
Intermediate normoblast(E4)
Late normoblast (E5)
Reticulo cyte (E6)
Matured RBC
31

32. 1. Pronormoblast : (proerythroblast)
 It is a large cell - deeply basophilic
cytoplasm with a large central
nucleus.
 The deep blue colour - high content
of RNA associated with protein
synthesis.
32

33. Basophilic Normoblast
•This is a round cell having a
diameter of 12-16 um with a
large nucleus.
•The nucleus is more condensed
than the pronormoblast and
contains basophilic cytoplasm.
•This cell undergoes rapid
proliferation.
33

34. Polychromatic normoblast
(Intermediate)
•The nucleus - coarse and deeply
basophilic.
•The cytoplasm is polychromatic with
mixture of basophilic RNA and
acidophilic hemoglobin.
34

35. Orthochromatic (late) normoblast
•This is the final stage in the
maturation of nucleated red cells.
•The cell is smaller - small pyknotic
nucleus with dark nuclear chromatin.
•The cytoplasm - acidophilic due to
large amounts of hemoglobin.
35

36. Reticulocytes
•Reticulocytes - devoid of nucleus but
contain RNA
•These cells in the peripheral blood
smear - slightly basophilic hue.
36

37. Precursors of RBC
37

38. Factors influencing erythropoiesis:
1. Hemopoietic growth factor
Erythropoietin
Stem Cell Factor (SCF)
Interleukins
(GM-CSF)
Colony Stimulating Factor (CSF)
Thrombopoietin
2. Vitamin B12, Folic Acid, Pyridoxine, Vitamin C
3. Iron and Copper
38

39. Erythropoietin (EPO)
39

40. Applied physiology
•COPD – high RBC count due to high production of
EPO
•Chronic damage of kidney – anemia due to lower
levels of EPO
40

41. Interleukins (IL)
•IL-1: Stimulates granulocytic cells. Induces
expression of GM-CSF
•IL-6 & IL-1: Stimulates proliferation of progenitor
cells.
•IL-3: Stimulates growth in all phagocytes.
41

42. INTERLEUKINS:
• Involved in erythropoiesis are
• IL-3
• IL-6
• IL-11
42

43. Stem Cell Factor (SCF):
Stimulates early stem cells to differentiate.
(TGF-beta)
Inhibits hematopoieses.
Directly inhibits proliferation of progenitor
cells.
43

44. Role of Vit B12
44

45. Folic acid
intestine
polyglutamate monoglutamate
methylmonoglutamate &
absorbed & enter blood
reduced to become
methyltetrahydrofolate
this form reaches tissues
they loose methyl group & become
tetrahydrofolate
accepted by Vit B12 methylcobalamin
45

46. THF (tetrahydrofolate)
formyl THF (folinic acid )
5,10 methylene THF (active form of folate )
dihydrofolate(FH2 )
46

47. Hemoglobin: (Hb )
• Present inside the RBC.
• Chromoprotein specialized for transport of oxygen
and carbon dioxide
• Conjugated protein with mol wt 68,000 daltons
• Forms 95% dry weight and 35 – 40% of volume of
RBC
47

48. NORMAL VALUES
AGE NORMAL VALUE
At birth 25gm%
After 3rd
month 20gm%
After 1 year 17gm%
In adult males 15gm%
In adult females 14.5gm%
48

49. DERIVATIVES OF HEMOGLOBIN
1.OXYHEMOGLOBIN
2.REDUCED HEMOGLOBIN OR FERROHEMOGLOBIN
3.CARBHEMOGLOBIN
4.CARBOXYHEMOGLOBIN
5.SULFHEMOGLOBIN
6.NITROUS OXIDE HEMOGLOBIN
7.METHEMOGLOBIN OR FERRIHEMOGLOBIN
49

50. FUNCTIONS OF HEMOGLOBIN
1.Transport of O2
2.Transport of CO2
3.Acts as blood buffer
50

51. Hb Chemistry &
synthesis:
Hb molecule contains
two ingredients:
•Haem ( iron &
protoporphyrin )
•Globin
51

52. Heme formation
• Heme formation takes place in the mitochondria then
the cytoplasm of erythroid precursors through the
reticulocyte stage.
• It begins with production of a protoporphyrin ring.
• Iron then incorporates with protoporphyrin to form
heme.
• Heme synthesis is stimulated by erythropoietin
52

53. Heme structure:
4 pyrrole structures
They are linked up with one another by methine ( = CH - )
53

54. Globin formation
•The polypeptide chains of globin are produced on the
ribosomes.
•The four most common chain types are alpha, beta,
gamma and delta chains.
•Each of these chains differs from the others in their
amino acid sequence.
•Each globin molecule is made of 2 pairs of chains and
each chain is made of 141-146 amino acids.
54

55. Globin structure:
•contains 4 polypeptide chains
2 alpha - containing 141 amino acids
2 beta - containing 146 amino acids
•with each polypeptide chain 1 molecule of haem is
attached.
55

56. 56

57. 57

58. Synthesis of Hb
•Hb is synthesized in the erythroid series in the red bone
marrow
•Hb first appears at the intermediate normoblast
•Protoporphyrin can be synthesized by normoblasts from
products of metabolism and aminoacids like succinyl Co
A , glycine etc
•Iron has to be obtained from food or iron contained in
the hemoglobin of dead RBC’s
58

59. Basic Chemical Steps : (synthesis of Hb )
1.2 succinyl-CoA + 2 glycine ---------------> pyrrole
molecule
2.4 pyrrole -----------------> protoporphyrin IX
3.Protoporphyrin + ferrous iron -------> heme
4.Heme + polypeptide (globin) ---------> hemoglobin
chain
5.2 alpha +2 beta ------------- > hemoglobin
59

60. Factors necessary for Hb maturation
1.Proteins and aminoacids
2.Iron
3.Copper – necessary for absorption of iron from gut
4.Cobalt and nickel for utilization of iron during Hb
formation
60

61. Iron metabolism
•Imp for oxygen transport
•Imp for formation of Hb and myoglobin
•Also necessary for formation of other substances
like cytochrome, cytochrome oxidase, peroxidase,
and catalase
61

62. Normal values
Total quantity of iron in the body is 4gms
Hb 65-68%
Muscle as myoglobin 4%
Intracellular oxidative
heme compound
1%
In the plasma as
transferrin
0.1%
Reticuloendothelial
system
25-30%
62

63. •Dietary iron available 2 forms
•Heme iron
•Non heme iron
•Heme iron- RBC , absorbs easily
•Non heme – vegetables, grains, cereals. not absorbed
easily
63

64. Absorption of iron
Iron is absorbed mainly from the small intestine.
Bile is essential for absorption
Immediately after absorption into the blood, iron
combines with a β globulin called – apotransferrin.
Which results in formation of transferrin
64

65. 65

66. Fate Of RBC
•Life span of RBC is about 120 days.
• As age of the RBC increases, the enzymes which
protect the erythrocyte from the damaging effects of
oxygen begin to lose their efficiency, therefore oxidative
damages begin to appear & the RBC becomes fragile.
•Now the cell ruptures , self-destruct in spleen
66

67. LAB INVESTIGATIONS
67

68. RED CELL COUNT:
Equipment
• Red cell pipette with a red bead in the bulb & markings of
0.5, 1 & 101
• Improved Neubauer chamber
68

69. Diluting Fluid
1.Hayem’s fluid
Mercuric chloride 0.5 g
Sodium chloride 1.0 g
Sodium sulfate 5.0 g
Distilled water 200 ml
69

70. RBC count = N
1/5 x 1/10 x 1/200
= N x 10000
Normal Range :
Adult male 4.5-5.5 million/ cumm
Adult female 3.8-5.2 million/cumm
At birth 4.0-6.0 million/cumm
70

71. VARIATIONS IN NUMBER OF RED BLOOD CELLS
Physiological variations
1.Increase in RBC
a)Age
b)Sex
c)High altitudes
d)Muscular exercise
e)Emotional conditions
f)Increased environmental temperature
g)After meals
71

72. 2. Decrease in RBC
a)High barometric pressures
b)After sleep
c)Pregnancy
72

73. Pathological Variations
1.Primary Polycythemia – Polycythemia Vera
Myeloproliferative disorders like malignancy of red
bone marrow
2.Secondary Polycythemia
a)Respiratory disorders like emphysema
b)Congenital heart disease
c)Ayerza’s disease
d)Chronic carbon monoxide poisoning
e)Poisoning by chemicals like P & As
f)Repeated mild hemorrhages
73

74. VARIATIONS IN SIZE OF RBC
1.Microcytes
a)Iron deficiency anemia
b)Prolonged forced breathing
c)Increased osmotic pressure in blood
2.Macrocytes
a)Megaloblastic anemia
b)Muscular exercise
c)Decreased osmotic pressure in blood
3.Anisocytes
a)Pernicious anemia
74

75. VARIATIONS IN SHAPE OF RED BLOOD CELLS
1.Crenation
2.Spherocytosis
3.Elliptocytosis
4.Sickle cell
5.Poikilocytosis
75

76. VARIATIONS IN STRUCTURE OF RED BLOOD CELLS
1.Punctate Basophilism:
•Dots of basophilic materials (porphyrin) appear in RBC
•Lead poisoning
2. Ring:
•Twisted strands of basophilic materials appear in the
periphery
•Goblet ring
3.Howell – Jolly bodies:
•Nuclear fragments in the RBC
76

77. Hb estimation methods
1.Calorimetric Methods
a)Visual Methods
b)Methods using photoelectric colorimeter
2. Gasometric Method
3.Chemical Method
4.Specific Gravity Method
77

78. Visual
1.Tall Quist paper method - inaccurate
2.Sahlis Acid hematin
3.WHO Hb colour scale – simple, reliable,
inexpensive, suitable for where calorimeter is not
available.
78

79. Sahli’s Method
Tube with marking
Principle :
Blood is added to N/10 Hcl – Acid
Hematin.
Acid hematin is matched against the
brown colour.
79

80. • Add N/10 Hcl – into Hb meter tube
• Fill the Hb pipette with 0.02ml of blood
• Acid act on the RBC for 10min to lyse the cells and
convert Hb to acid hematin.
• Match colour of the solution with comparator in
natural light.
• Darker – add distilled water
• Continue till the colour of solution matches
80

81. Disadvantages:
• Visual error
• After 10min acid hematin starts fading
81

82. Tall Quist Paper Method
• Includes a color chart
and 150 test papers
• Allow blood to absorb
into one of the test
papers and compare the
color scale.
82

83. WHO Hb colour scale
• Place a drop of blood on the
test strip provided
• Wait about 30 seconds
• Match immediately the colour
• simple, reliable, inexpensive,
suitable for where colorimeter
is not available.
83

84. Methods using photoelectric colorimeter
1.Cyanmet haemoglobin method
2.Oxyhemoglobin method
3.Alkaline haematin Method
84

85. Cyanmethemoglobin Method
Principle :
Hb is oxidized to - Met Hb by potassium ferricyanide
Met Hb – Cyanmet Hb
Technique
Take 5ml of Drabkin’s solution
( Drabkin’s reagent contains sodium bicarbonate, potassium
ferricyanide, and potassium cyanide)
Add 0.02ml of blood
Wait for 10min
Hb = OD of test sample x Conc. Standardx250
OD of standard x1000

86. Oxyhemoglobin Method
• 0.04% of ammonia is used
• Which lyses the RBC
• Colour is compared to standard
Alkaline Hb method
• N/10 NaOH is used.
• Colour of alkaline hematin is compared with standard
86

87. Gasometric Method – oxygen carrying capacity of blood
is measured in van slyke like apparatus.
Not suitable for routine use
Specific Gravity Method – simple , rapid & inexpensive.
Rough estimation of Hb is obtained from specific gravity
of blood, used for mass screening
87

88. PCV
•Hematocrit is the % volume of red cells in a given sample
of blood.
•It gives an estimate of relative volume of cells and plasma.
•It is useful for evaluating absolute values like MCV and
MCHC
88

89. Wintrobe’s method :
•Collect 2ml of blood
•Take wintrobe’s tube
• 0-100 – ESR
• 100-0 – PCV
•Take the blood in pasteur’s pippete and withdraw the
blood into the tube, till it reaches 100 marking.
89

90. normal range
Males – 40- 55%
Females – 35- 48 %
90

91. MCV: ( normal value 80 – 100 fl )
It is the volume of an average RBC (single RBC)
expressed in femotoliters or fl
PCV X 1000
MCV = -----------------------------------------
RBC in millions / cmm.
Normal : 82-98 fl
91

92. MCH - Amount of Hb in the red cell
Hb x10
MCH = ----------------------- pg
RBC count
Normal = 28-32pg
92

93. MCHC: ( normal value 31 – 36% )
represents the average concentration of Hb in a given volume
of packed red cells.
Hb
MCHC = -------------------------------------------
PCV ( % )
93

94. Erythrocyte Sedementation Rate : (ESR)
It is a measure of the settling of red blood cells in a tube
of blood during one hour.
normal value - 0-20mm in female in 1st
hr
- 0-10mm in male in 1st
hr
Stages of sedimentation :
•Rouleaux formation – first 15 min
•Formation of fine threads – next 15 mins
•Rapid fall – next 15min
•Packing phase – packing of RBC next 15min
94

95. • Two methods to determine ESR.
Westergrens Method:-Westergrens tube - 300mm long(30cm),
2.5mm internal diameter & opened on both ends, calibrated
from 0-200.
Procedure
•1.6 -2ml of blood mixed with 0.4-0.5ml of 3.8% sodium
citrate - (anticoagulant) and mix well, loaded in westergrens
tube up to zero mark.
95

96. • The tube fitted to the stand vertically and left
undisturbed at room temperature.
• Reading taken at the end of 1 hour (Distance from 0
mark to top of RBC column is recorded as ESR)
96

97. Anticoagulated whole
blood has just been added.
(Time: 0)
Red blood cells have settled, leaving
plasma at the top of the tube. Reading:
18 mm/hour
(Time: one hour)
WESTERGREN’S TUBES
97

98. Modified Westergrens method
•EDTA instead of citrate as anticoagualant
•2ml of EDTA diluted with 0.5ml of 3.8% sodium
citrate or 0.5ml of 0.85% sodium chloride
•Undiluted EDTA blood gives poor precision
98

99. Procedure
•Collect 2ml of blood
• Blood loaded in the tube up to’0’mark,
tube is placed vertically on the Wintrobe’s
stand.
•The reading taken after one hour
Wintrobes Method
•Wintrobe’s tube short tube (110mm long,
diameter 2.5mm) opened on only one end.
99

100. NORMAL VALUES OF ESR
Westergren’s method
•Males – 0-15mm 1st
hr
•Females - 0-20mm 1st
hr
Wintrobe’s method
•Males – 0-7mm 1st
hr
•Females - 0-14mm 1st
hr
100

101. Factors That May Influence ESR
Factors that increase
ESR
Old age
Female
Pregnancy
Anemia
Macrocytosis
Factors that decrease ESR
Polycythemia
Red blood cell abnormalities
Spherocytosis
Microcytosis
101

102. LEUCOCYTES
102

103. Introduction :
• Leukos = white, cytes = cells
• Mobile units of the body’s protective system.
• White blood cells are the colorless and nucleated formed
elements of blood.
• These cells are larger in size and their number is less
compared to that of RBC’s.
• They play a very important role in defense mechanism
103

104. 104

105. Type of leucocyte Normal range Lifespan
Neutrophil 50-75 % 2-5 days
Eosinophil 1-6 % 7-12 days
Basophil 0-1% 12-15 days
Lymphocyte 25-45% ½-1 day
Monocyte 3-8 % 2-5 days
105

106. NEUTROPHILS
•Diameter : 10-14µ
•Nucleus :
Young neutrophil : horse-shoe shaped nucleus
Mature neutrophil : multilobed (2-6 lobes)
lobes connected by chromatin filaments
•Cytoplasm : pale bluish in colour & full of fine granules
Granules take both acidic & basic stain & look violet-
pink in colour
106

107. •Condensed chromatin along the inner surface of nuclear
envelope
•More central region of each lobe appears paler
•Cytoplasm contains few mitochondria & a small Golgi
complex
•Azurophilic granules are round or oval & more electron
dense than specific granules.
107

108. NEUTROPHILS - DEVELOPMENT
MYELOBLAST :
• Earliest recognizable cell in the granulocytic maturation
process.
• 15-20µm in diameter
• Large round to oval nucleus
• Small amount of basophilic cytoplasm
• Nucleus contains 2 to 5 nucleoli
• Nuclear chromatin is fine and reticular
108

109. PROMYELOCYTE :
• Slightly larger in size than myeloblast.
• Primary or azurophilic granules appear at the
promyelocyte stage.
• Nucleus contains nucleoli as in myeloblast stage
• But nuclear chromatin shows slight condensation.
109

110. MYELOCYTE :
• Characterized by the appearance of secondary or specific
granules.
• Smaller cell with round to oval eccentrically placed
nucleus.
• More condensation of chromatin than in promyelocyte
stage and absence of nucleoli.
110

111. • Cytoplasm is relatively greater in amount than in
promyelocyte stage.
• Contains both primary and secondary granules.
• Last cell capable of mitotic division
111

112. METAMYELOCYTE :
• Nucleus becomes indented and kidney shaped.
• Nuclear chromatin becomes moderately coarse
• Cytoplasm contains both primary and secondary
granules
112

113. BAND STAGE ( STAB FORM ) :
• Characterized by band-like shape of the nucleus
with constant diameter throughout
• Condensed nuclear chromatin
113

114. SEGMENTED NEUTROPHIL:
• With Leishman’s stain,
nucleus appears deep purple
with 2-5 lobes joined by thin
filamentous strands.
• Nuclear chromatin pattern is
coarse.
• Cytoplasm stains light pink
and has small, specific granules
114

115. 115

116. Granules
•Number of granules : 500-1500/granulocyte
•Large amount of protein, traces of lipids & nucleic acids
1.Primary or azurophilic granules
2.Secondary or specific granules
3.Tertiary or gelatinase granules
4.Secretory vesicles
116

117. Azurophilic granules
•Myeloperoxidase, defensins, lysozyme, azurocidin etc
•These granules fuse with phagocytes vesicles resulting in
the delivery of their contents to the ingested organism
•Greenish coloration to pus is imparted by
myeloperoxidase
117

118. Secondary or specific granules :
• 3times more common in cytoplasm
• Lysozyme, Lactoferrin, collagenase, histaminase may
modify the inflammatory process
Tertiary granules :
• Gelatinases
118

119. Life history of neutrophils :
•Released from the bone marrow
•Exist in two populations
•Circulating pool
•Marginal pool
•Rapid exchange between the two pools
• Activated by numerous stimuli
119

120.  Disposed of internally by cells of reticuloendothelial
system
 External loss : emigration through gingiva into the
saliva & excretion in urine ( common)
 Neutrophils occur in the secretions of uterus during
second half of menstrual cycle
 Dead neutrophils – granulocyte-inducing factor
120

121. Old senile neutrophils are characterized by:
•Loss of motility
•Poorly stained granules
•Increased nuclear lobulation
•Easy breakability while making blood smear
121

122. Nitroblue tetrazolium reduction test :
•Ability of neutrophils to destroy micoorganisms with
intracellular enzymes can be evaluated by NBT
•Normal neutrophils contain enzymes that convert colorless
NBT to dark blue granules within the cell
•When dark blue granules are not seen, neutrophils will not
destroy bacteria
122

123. FUNCTIONS :
First line of defence
1.PHAGOCYTOSIS
2.REACTION OF INFLAMMATION
3.FEBRILE RESPONSE
123

124. VARIATION IN COUNTS
Neutrophilia - >1000 cumm
Physiological causes
•New born babies
•After exercise
•After meals
•Pregnancy
•Menstruation
•Parturition
•Lactation
•Mental & emotional stress
124

125. Pathological causes
•Acute bacterial infections
Lobar pneumonia
Bronchopneumonia
Pyogenic meningitis
Cellulitis
Infected burns
Diphtheria
125

126. •Acute inflammatory diseases like
Acute rheumatic fever
Acute appendicitis
•Acute stress states like
Post surgery
Post haemorrhage
Myocardial infarction
•Chronic myeloproliferative disorders like
CML
Polycythemia vera
126

127. NEUTROPENIA:
CAUSES
•Typhoid and paratyphoid fever
•Physical agents like radiation
•Chemicals : Benzene
•Antimetabolite drugs : Cyclophosphamide, Methotrexate
•Hematologic disorders like megaloblastic anemia, aplastic
anemia, subleukemic leukemia, cyclic neutropenia,
127

128. COOKE’S ARNETH COUNT
Stage Nuclear lobes Normal count
Stage I (N1) 1 lobe 5-10 %
Stage II (N2) 2 lobes 20-30 %
Stage III (N3) 3 lobes 40-50 %
Stage IV (N4) 4 lobes 10-15 %
Stage V (N5) 5 lobes or more 3-5 %
128

129. Clinical Significance :
1.Left shift
•N1+N2+N3 > 80%
•More younger cells
•Indicates hyperactive bone marrow (high rate of
formation)
2.Right shift
•N4+N5 > 20%
•More mature cells
•Hypoactive bone marrow (slow rate of formation)
129

130. Variation in neutrophil morphology :
1.Variation in granules
2.Formation of vacuoles in cytoplasm
3.Formation of Dohle bodies in cytoplasm
4.Presence of sex chromatin with nuclear lobes
5.Hypersegmented neutrophils
6.Band and straw form neutrophil
7.Pelger – Huet anamoly
130

131. DOHLE BODIES
131

132. SEX CHROMATIN
132

133. HYPERSEGMENTED NEUTROPHILS
133

134. PELGER HUET ANAMOLY
134

135. If most of the neutrophils appear bilobed, this is
indicative of an uncommon condition known as Pelger-
Huet anomaly, an inherited condition.
This is the heterozygous form.
The homozygous form is fatal. Just be aware of this
condition when you get back a manual differential count
with mostly bands, but the WBC count is normal or the
patient shows no signs of infection or inflammation.
135

136. EOSINOPHILS :
• Forms via same stages as the neutrophil.
• Diameter = 12µ - 17µ
• Nucleus: bilobed, purple colored, spectacle shaped, nucleoli
– absent
Condensed chromatin stains less intensely than that of
neutrophil
136

137. Cytoplasm: acidophilic and appears bright pink
Contains deep red staining granules which do not cover the
nucleus.
•Condensed chromatin is peripherally distributed along the
inner surface of nuclear envelope.
•Specific granules : ovoid with electron dense crystalloid
cores that consist chiefly of basic proteins.
•Only other prominent organelles : Golgi apparatus,
mitochondria
137

138. In RBM ( 5-6 days)
Circulatory pool (8-12 hours)
Emigrates into tissue
Death and removed
138

139. Cytoplasm contains two types of granules
1. Specific granules
Large numerous elongated granules
Contain Crystalloid bodies
- Contain four major proteins
1. Major basic protein
2. Eosinophil cationic protein
3. Eosinophil peroxidase
4. Eosinophil derived neurotoxin
Also contains histaminase, arylsulfatase, collagenase,
cathepsins
139

140. Azurophilic granules
•Lysosomes, contains acid hydrolases
•Function in the destruction of parasites
•Hydrolysis of antigen – antibody complexes.
140

141. FUNCTIONS OF EOSINOPHILS :
1.ROLE IN PARASITIC INFESTATIONS:
•Major basic protein (MBP) : damage the parasites by
causing distension and detachment of the tegumental
sheath of these organisms.
•Eosinophil cationic protein : major destroyer of
helminths.
10 times more toxic than MBP
Destroys parasites by complete disintegration
141

142. • Eosinophil peroxidase : capable of destroying helminths,
bacteria and tumor cells
• Eosinophilic derived neurotoxin : destroys the nerve
fibers particularly myelinated nerve fibers.
2. ROLE IN ALLERGIC REACTION
• Capable of destroying inflammation inducing substances
like histamines and bradykinin.
142

143. • Destroy antigen-antibody complexes & thus prevent
spread of local inflammatory process.
• Eosinophil arylsulfatase can degrade SRS-A liberated
by mast cells and basophils
• Produces histaminase that can inactivate histamine.
143

144. Eosinophilia :
•Allergic conditions like hay fever, bronchial asthma
•Parasitic infestations
•Skin diseases like urticaria
•Scarlet fever
Eosinopenia :
•ACTH & steroid therapy
•Stressful conditions
•Acute pyogenic infections
144

145. BASOPHILS :
•Diameter = 10-12µ
•Nucleus – bilobed , stains less deeply than that of
neutrophil
•Cytoplasm : basophilic, full of granules
granules : deep purple or blue
145

146. • Membrane bound granules – 0.5µ
• metachromatic & similar to mast cell granules.
• Chemical mediators liberated when basophils
degranulate include histamine, SRS-A, ECF-A
• The cell surface receptors on basophils closely
resemble those on mast cells.
146

147. FUNCTIONS
1.ROLE IN ALLERGIC REACTION :
release histamine, bradykinin, SRSA, seratonin.
2. ROLE IN PREVENTING SPREAD OF ALLERGIC
INFLAMMATORY PROCESS :
•releases eosinophil chemotactic factor
•eosinophils then phagocytose & destroy antigen – antibody
complexes & prevent spread of local inflammatory process
147

148. 3. RELEASE OF HEPARIN
• Prevents clotting of the blood.
• Activates the enzyme lipoprotein lipase which
removes fat particles from the blood after a fatty meal
148

149. BASOPHILIA : >100 cumm
•Viral infections
•Allergic diseases
•Chronic myeloid leukemia
BASOPENIA :
•Corticosteroid therapy
•Drug-induced reactions
•Acute pyogenic infections
149

150. MONOCYTES
•12-20µ in diameter, Largest leukocytes
CFU-M
Monoblast
Promonocyte
Monocyte
150

151. • Nucleus : variable in appearance
ranging from indented ovoid or
roughly kidney shaped to wide horse
shoe
Chromatin less condensed
• Cytoplasm : pale grayish-blue color
Fine pinkish-purple granules can
also sometimes be seen
Specific granules lacking
151

152. • 2 or more nucleoli
• Fairly prominent Golgi apparatus, moderate amount of
ribosomes, small amount of rER, no. of small
mitochondria
• Small dense granules – 0.3-0.6µ
• Surface of monocytes is irregular
152

153. 153

154. Functions
1.Phagocytosis :
second line of defence
2.Immediate precursors of macrophages
3.Releases IL-1, TNF-α, transferrin, lysozyme,
proteases, acid hydrolase
154

155. MONOCYTOSIS
•Parasitic conditions like malaria and kalaazar
•Infective conditions like subacute bacterial endocarditis,
tuberculosis
•Neoplastic states like acute monocytic leukemia, acute
myelomonocytic leukemia, chronic myelomonocytic
leukemia
155

156. LYMPHOCYTES
•20-50% of blood leukocytes
•Lack prominent cytoplasmic granules when seen in LM. 10%
- may contain reddish purple staining granules
•Based on size
1.Small lymphocyte : 6-9µ
2.Large lymphocyte : 9-15µ
156

157. Committed stem cells
Pre -T cell Pre B cell
T lymphocytes Blymphocytes
157

158. Small lymphocyte :
•Small spherical nucleus with a small indentation on one side.
•Condensed chromatin
•Remarkably little cytoplasm
•Cytoplasm is hardly seen in LM
158

159. •Cytoplamic organelles are
scant
•Few mitochondria, sparse
cisternae of rER, small Golgi
apparatus
159

160. Large lymphocyte :
•Nucleus slightly larger than small lymphocyte
•More cytoplasm
EM :
•Large numbers of mitochondria, free ribosomes,
more cisternae of rER, slightly larger Golgi apparatus
160

161. 161

162. On the basis of functional properties
162

163. LYMPHOCYTOSIS : >45%
•Chronic infective conditions like tuberculosis,
syphilis & brucellosis
•Viral diseases like infectious mononucleosis,
measles, chicken-pox & viral fever
•Neoplastic conditions like CLL
163

164. LAB INVESTIGATIONS
TOTAL LEUCOCYTE COUNT
Principle :
Whole blood is diluted with WBC fluid that hemolyses the
red cells.
Nucleated cells – WBC s stained by Gentian Violet are
counted in a Neubauer chamber
164

165. Equipment:
Hemocytometer set consisting of WBC pipette and
improved Neubauer chamber with a cover slip
WBC pipette has a white bead in the bulb and markings
of 0.5, 1, 11
165

166. WBC Fluid ( Turk’s fluid )
Glacial Acetic Acid 2ml
1 % Gentian violet 5 drops
Add water to 100 ml
166

167. TLC = N x 20
4 x 0.1
= N x 50
Depth of the chamber = 0.1 mm
Dilution = 1 in 20
4 corner squares are counted
N = Number of WBCs in 4 large squares
167

168. Adults 4000 – 11000 cells / cumm
At birth 8000 – 28000 cells / cumm
Childhood 6000 – 15000 cells / cumm
Normal Value
168

169. Leucocytosis Leucopenia
> 11000 / cumm < 4000 / cumm
•Pregnancy
•Exercise
•Leukemoid reaction
•Diabetic and uremic coma
•Infectious mononucleosis
•Bacterial and viral infections
•Leukemias
•Aplastic anemia
•Hypersplenism
•Typhoid, paratyphoid fever
•Drug induced leucopenia
•Radiation & cytotoxic therapy
•Megaloblastic anemia
•Subleukemic leukemia
169

170. ABSOLUTE EOSINOPHIL COUNT
•Accurate assessment of eosinophils in blood & is useful
in diagnosis, treatment & follow-up of cases of
eosinophilia
•EQUIPMENT :
Improved Neubauer chamber, WBC pipette
170

171. Eosin 200mg dissolved in
10ml of distilled
water
Stains the granules
of eosinophils
orange red
Water 80ml Lyses the red cells
& white cells
except eosinophils
Acetone 10ml Fix the eosinophils
Dunger’s fluid
171

172. AEC = N X10
4 X 0.1
= N X 25
Normal Range = 40-450 cells /cumm
172

173. PERIPHERAL SMEAR AND DIFFERENTIAL
LEUCOCYTE COUNT
1.PREPARATION OF SMEAR
2.FIXATION OF THE SMEAR
•Fixed within 4hours to get good staining
•Methanol
3. STAINING OF THE SMEAR
173

174. Commonly used stains :
•Leishman stain
•Giemsa stain
•Wright’s stain
•Jenner’s stain
•Jenner – Giemsa stain
174

175. BLOOD GROUPS
•Discovered by Karl Landsteiner in 1901
•Nobel prize in 1930
•30 blood group systems given by International Society of
blood transfusion
175

176. 176

177. 177

178. International Society of Blood Transfusion. October 2006
178

179. LANDSTEINER’S LAW
1.If a particular antigen is present in the red blood cells,
corresponding antibody must be absent in the serum.
2.If a particular antigen is absent in the red blood cells,
the corresponding antibody must be present in the serum
Second part of law – not applicable to Rh factor
179

180. ABO Blood groups
Group Antigen in RBC Antibody in
serum
A A Anti B
B B Anti A
AB A and B No antibody
O No antigen Anti A and Anti
B
180

181. Population A B AB O
Europeans 42 9 3 46
Asians 25 25 5 45
181

182. DETERMINATION OF THE ABO GROUP
Blood grouping, blood typing or blood matching.
Principle
•Basis of agglutination
•Occurs antigen + antibody
Requisites
•Suspension of patients red blood cell
•Anti serum A & B
182

183. Procedure & Results
Agglutination with antiserum Blood group
Anti serumA A
Anti serum B B
Both A & B AB
No agglutination O
183

184. Universal donors O
Universal recipients AB
Cross matching
Mixing the serum of the recipient and the red blood
cells of donor
184

185. Gene receive from
parents
Group of the
offspring
Genotype
A+A A AA
A+O AO
B+B B B
B+O BO
A+B AB AB
O+O O OO
Inheritance
185

186. Transfusion reactions due to ABO incompatibility
•Jaundice
•Cardiac Shock
•Renal Shut down
186

187. Rh factor
• Antigen present in RBC
• Discovered by Landsteiner and Wiener in Rhesus
monkey
• Many Rh antigens only D is highly antigenic
• Among Asians 85% - Rh +ve
• 15% - Rh –ve
• Rh +ve more among blacks
187

188. Dd Dd Dd
Father
DD
Rh+
Mothe
r
dd
Rh -
Dd
All offsprings are Rh +
188

189. dd dd dd
Father
dd
Rh -
Mothe
r
dd
Rh -
dd
All offsprings are Rh -
189

190. dd Dd dd
Father
Dd
Rh+
Mothe
r
dd
Rh -
Dd
50% offsprings are Rh +
190

191. Transfusion reactions due to incompatibility
•Erythroblastosis Fetalis
•Hydrops Fetalis
•Kernicterus
191

192. REFERENCES :
1.Text book of Medical Physiology- 10th
edition- Guyton & Hall
2.Concise Medical Physiology- 4th
edition- Chaudhuri
3.Essentials of Medical physiology-4th
edition-Sembulingam
4.Per haavardsholm finne and Sverre halvorsen. Regulation of
Erythropoiesis in the Fetus and Newborn. Archives of Disease in
Childhood 1972; 47: 683-687
5.Narla Mohandas and Patrick G. Gallagher. Red cell membrane:
past, present, and future. Blood 2008; 112: 3939-3948
192

193. 6.Michael Fo¨ller1, Stephan M. Huber2 and Florian Lang.
Erythrocyte Programmed Cell Death. Life 2008; 60(10): 661–
668
7.Petra Kleinbongard et all. Red blood cells express a functional
endothelial nitric oxide synthase. Blood 2006; 107: 2943-2951
8. Veronique Witko-Sarsat et al. Neutrophils: Molecules, Functions
and Pathophysiological Aspects. Laboratory investigations 2000;
80(5): 617-653
9. SM Rashmi et al. Neutrophils in health and disease: An overview.
J Oral Maxillofac Pathol 2006; 10: 3-8
193

194. 194