Development anatomy and physiology of haematopoiesis, hematological indices

1. Development , Anatomy and
Physiology of Haematopoiesis,
Hematological Indices
Dr. Sreemayee Kundu
MD Paediatrics

2. Hematopoiesis
• It is a process of formation of blood cellular
components. All cellular blood components
are derived from haematopoietic stem cells
(HSC).
Hemo /Hemato = Blood
Poiesis = Production

5. Stem Cell
Stem Cells – are undifferentiated cells that have
the ability to continuously divide and
differentiate (develop) into various other
kind(s) of cells/tissues.
 has the ability to give rise to new stem cells (Self
Renewal)
 is a source of all kinds of blood cells (stem cell is
pluripotent – has an ability to develop into all kinds
of cells)
 from all the cells, stem cell is most resistant to
damage.

6. Classification
Totipotent
stem cells are
found only in
early embryos.
Each cell can
form a
complete
organism.
Pluripotent
stem cells
Obtained from
the inner cell
mass of the
blastocyst, able
to differentiate
into almost all
cells of the
three germ
layers – but not
into an embryo.
Multipotent
stem cells
Found in most
tissues, produce a
limited range of
differentiated cell
lineages
appropriate to
their location
(e.g.Hematopoieti
c stem cells from
the bone
marrow).
Unipotent
cells capable of
generating only
one cell type
(epidermal stem
cells, adult liver
stem cells).
Stem cells can be classified into four broad categories,
based on their ability to differentiate:
Stem Cells

8. Hematopoietic stem cells (HSCs) are bone marrow cells
that are capable of producing all types of blood cells.
They differentiate into one or another type of committed
stem cells (progenitor cells). These in turn form the various
differentiated types of blood cells.

9. Stages of developement

10. Progenitor cells
• PHSC slowly self replicate differentiating into cells that
are multipotent but have reduced self-renewal
capacity.
• The progenitor cells for blood cells are commonly
called colony-forming units (CFUs), because they give
rise to colonies of only one cell type
 4 major types of progenitor cells/CFUs:
1- Erythroid lineage
2- Thrombocytic lineage
3- Granulocyte-monocyte lineage
4- Lymphoid lineage

11. Precursor and mature cells
• Each progenitor cell/CFU lineage produces
precursor cells (or blasts) that gradually
assume the morphologic characteristics of
the mature, functional cell types.

12. Haematopoeisis

13. Dr Radhakrishnan

14. HEMOPOIETIC GROWTH FACTORS
• Most of them are glycoproteins
• Regulates hematopoiesis
• Acts on specific stem cells, progenitor cells,
and precursor cells
• Induced rapid mitosis, proliferation,
differentiation and maturation

15. Hemopoietic growth factors
• Lymphohaematopoietic cytokines like IL 2 t-
13,15,21,23, GM-CSF, G-CSF, EPO, TPO, TSTP,
LIF, OSM, CT-1, CNTF
•Receptor Tyrosine-kinase ligands
•IL -1 gene family
• Chemokines
• Tumour Necrosis Factors
•Interferons
• Transforming growth factor beta gene family
like- TGFbeta1,2,3, BMPs(2-6), GDF(1-10),
activin,inhibin

18. Figure 19.6
Stages of RBC Maturation

19. Stages of Erythropoiesis Important events
Proerythroblast Synthesis of Hb starts
Early Erythroblast Nucleoli disappear
Intermediate Erythroblast Hb starts appearing
Late Erythroblast Nucleus disappears
Reticulocyte Reticulum formed. Cells enter into
capillary from site of production
Mature RBC Reticulum disappears. Cell attain
Biconcavity
Cells of CFU-E pass through different stages and finally become the matured RBCs.
During these stages four important changes are noticed.
1. Reduction in size of the cell (from the diameter of 25 to 7.2 µ)
2. Disappearance of nucleoli and nucleus
3. Appearance of hemoglobin
4. Change in the staining properties of the cytoplasm. „
CHANGES DURING ERYTHROPOIESIS
Dr Radhakrishnan

20. ERYTHROCYTE(RBC) Biconcave-shape disk
 Without nuclei and
organelles
 Have soluble enzymes
 Filled with Hemoglobin
(Hb)
 Average life span of
erythrocyte : 120 days

21. Factor needed of Erythropoiesis
1. Erythropoietin ( Released in response to Hypoxia)
2. Vitamin B 6 (Pyridoxine)
3. Vitamin B 9 (Folic Acid)
4. Vitamin B 12 (Cobolamin)
Essential for DNA synthesis and RBC
maturation
5. Vitamin C  Helps in iron absorption (Fe+++ 
Fe++)
6. Proteins  Amino Acids for globin synthesis
7. Iron & copper  Heme synthesis
8. Intrinsic factor  Absorption of Vit B 12
9. Hormones
21

22. Role of erythropoietin
• Glycoprotein
• Produces primarily by cells in the kidney that lie
between the kidney tubules (peritubular interstitial
cells) and remainder from liver
• Control erythropoiesis
-Stimulates red cell production & formation in
response to hypoxia

24. GRANULOCYTOPOIESIS
• Formation of the granulocytes (neutrophil,
eosinophil, and basophil)
– CFU-Eo : eosinophil lineage
– CFU-Ba : Basophil lineage
– CFU-GM
• CFH-G : Neutrophil line
• CFU-M : monocyte line
– Influence by G-CSF, GM-CSF

25. Colony forming Unit-GM / Granulopoisis
Myeloblast
Promyelocyte
Early Neutrophillic Myelocyte Early Eosinophillic Early Basophillic
Late Neutrophillic Myelocyte Late Eosinophillic Late Basophillic
Neutrophillic Metamyelocyte Eosinophillic Metamyelocyte
Mature Neutrophil Mature Eosinophil Mature Basophil

27. Neutrophil Eosinophil Basophil
Constitutes in WBC 60-70% 2- 4% 1%
Nucleus consisting of 2-5
lobes linked by
chromatin
Bilobed nucleus Irregular lobes
Cytoplasm Specific granules
and Azurophillic
granules
Weekly stains
Large elongated
refractile specific
granules stained by
Eosin
Granules contains
Heparin, Histamine.
Stains
Metachomatically.
Purple in colour
Life span 1-4 days 8-12 days Few hours to days

28. NEUTROPHILS
•60-70% of total leucocytes
•9-12 µm in diameter
•Multilobe nucleus
• Granules :
- Small specific granules
- Azurophilic granules
(Lysosomes)
- Tertiary granules (gelatine
and cathepsins)
•Function :
Phagocytosis

29. EOSINOPHILS •4% of total leucocyte
• 10 - 14 µm in diameter
•Bilobed nucleus
•Many large specific
granules stained by eosin
• Function :
-Eliminate antibody-
antigen complexes
-Destroy parasitic
worms

30. BASOPHILS •<1% of total leucocyte
• 8-10 µm in diameter
• S-shape nucleus
(irregular lobes)
• Large specific granules
obscured the nucleus
• Granules (dark blue)
contain heparin & histamin
Surface receptor (Ig E
receptors)
• Function :
As initiator of inflamatory
process

31. Colony forming Unit-GM / Monocyte
 Euchromatic nucleus
 Oval, Horeshoe, Kidney shaped. Eccentrically placed
 Cytoplasm basophilic
 In electron microscope many Microvilli seen at the cell surface

32. MONOCYTES
•Largest circulating blood
cells
• 3-8% ot total leucocyte
• Large, acentric, kidney-
shape nucleus
• Numerous azurophlic
granules
• Migrate to the
connective tissue 
MACROPHAGES
(phagocytose antigens
and as APC)

33. PLATELET FORMATION
• CFU-Meg, gives rise the Megakaryoblast
• Megakaryoblast differentiate 
Megakaryocytes (single lobulated nucleus)
• Megakaryocytes protrude  clusters of
proplatelets  platelets

34. Colony forming Unit-M
Megakaryoblast
Megakaryocytes
Platelets
 Anucleate cell fragments
 Also known as thrombocytes
 200000-400000 per micro litre of blood.
 Appears as clumps
 Peripheral light blue stain & central purple
stain

35. PLATELETS
• 2 to 4 µm in diameter
• Display peripheral clear
region (hyalomere ) and
central darker region
(granulomere)
• Function : Blood clott

36. Lymphocytopoiesis
• The first identifiable progenitor of lymphoid cells is the
lymphoblast.
• Lymphoblast is a large cell capable of dividing two or three
times to form lymphocytes
• As lymphocytes develop, their nuclei become smaller,
nucleoli become less visible, and the cells decrease in size
overall.
• In the bone marrow and thymus, these cells synthesize
specific cell surface proteins that characterize B or T
lymphocytes

37. Lymphoid colony forming cell
Nucleus is :
 Round
 Stains densely
 Surrounded by very narrow rim of
cytoplasm, basophilic.
Lymphoblast
Lymphocytes
T- Lymphocyte B- Lymphocyte

38. LYMPHOCYTES
• 20%-25% of total
leucocyte
• 8-10 µm in diameter
• Round nucleus with
slight indented, occupies
most of the cell
• Contain few azurophilic
granules

39. Development Of Hematopoiesis
Physiology Of Marrow Evolution

40. Sites of Hematopoiesis
• Yolk sac
• Liver
• Spleen
• Thymus
• Lymph node
• Bone marrow – long bone (children)
- Axial skeleton (Adult)

41. PRENATAL HEMOPOIESIS
Divided into three phases :
–Mesoblastic
–Hepatic
–Myeloid

42. PRENATAL HEMOPOIESIS
Mesoblastic
• All blood cells derived from embryonic connective
tissue- Mesenchyme
• Blood formation 1st detected 19th day of gestation
• Blood island in yolk sac differentiate into 2 direction
-peripheral cells (wall of blood vessel)
- central cells (primitive blood
cells/hemocytoblast)
• 22nd day scattered
• 6th week
• By end of 3rd month ceased.

43. Sites of Haemopoiesis

44. PRENATAL HEMOPOIESIS
Hepatic
• Begins around 35th gestational day
• Site of pure erythropoeisis - 3rd to 5th month of
gestation
• Continue till 1st postnatal week
• During 3rd month also seen in spleen and thymus
and shortly afterwards in the lymphnode

45. Sites of Haemopoiesis

46. PRENATAL HEMOPOIESIS
Myeloid
• Starts around 4th – 5th fetal months.
• Becomes quantitively important by 6th fetal month
• Last 3months bone marrow is the chief site

47. Sites of Haemopoiesis

48. Postnatal haemopoiesis
Hemopoiesis almost exclusively in BONE MARROW
• Until 5yrs, bone marrow of all bones performs
haemopoiesis.
• By 20 yrs, marrow cavities of the long bones, except
for the upper humerus and femur, has become
inactive
• Haemopoietically active Red marrow is replaced by
inactive-resting- Yellow marrow
• Active haemopoiesis continues throughout life in
epiphysis of long bones and all flat bones; vertebra
sternum, ribs, pelvis and skull

50. Sites of Haemopoiesis

51. BONE MARROW
• Soft, spongy, gelatinous, vascular connective tissue
located in medullary cavity of long bones & small cavities
of cancellous bone
2 types
1- Red bone marrow- Consists of
Stroma, Hemopoitic cord,
Sinusoidal capillaries
2- Yellow bone marrow -
fat ( adipocyte )
red marrow space child>adult

53. BONE MARROW
• Responsible for hematopoiesis
• Structure :
Hemopoietic compartment
Islands of haematopeitic
cells
Adipose cells
Vascular compartment
Extensive network of
sinusoids , arteries and
veins

54. Erythropoeisis in developing embryo
• 1st blood cells produced by embryo
• 2 types of erythrocytes observed
Hemocytoblast(primitive blood cells)
Early embryo later phase
Primitive megaloblastic Definitive normoblastic
erythropoesis erythropoeisis
-begins at 6th week
Megaloblast -by 10th week >90%
large ,irregular shaped,hypochromic RBC
Gradually replaced by normoblastic series

55. Red cell changes
EARLY EMBRYO LATER PHASE
Red cell count Low Increases
Haemoglobin
concentration
Low Increases
PCV Low Increases
Red cell size Very large Decreases
Nucleated cells Mostly decreases gradually

57. Lymphopoiesis
• Generation of lymphocytes
• Begins 8th week of gestation in lymph plexuses
• By 9th week seen in thymus
• 3rd fetal month- lymph glands
• Initially rapidly
• By 20th week of gestation high(10,000per
cu.mm)
• By term (3000 per cu. mm)

58. Myelopoeisis
• Production of leukocytes
• Place- liver ,meninges,mesentery,stroma of
lymph plexus
• Begin on 7th week embryo.
• Significant production occurs in the myeloid
phase
• 1st half of gestation- very few granulocytes
• Last trimester rises rapidly
• At birth, count > adult count

59. Megakaryocytes
• Between 5th – 6th week of gestation seen in
yolk sac , from this time till term seen in liver,
after 3rd month also seen in bone marrow
• Activity observed in blood by 11th gestational
week
• By 30th gestational week activity & count
similar to adult

60. PHYSIOLOGY AND EVOLUTION
OF HAEMOGLOBIN

61. Formation of hemoglobin
Chemical step:
• Succinyl-CoA,formed in the Krebs metabolic cycle.
• Binds with glycine forms pyrrole molecule.
• 4 pyrrole molecule combines to form protoporphyrin
IX
• Protoporphyrin IX combines with iron forms heme
molecule
• Heme combines with Globin forms subunit of
hemoglobin called hemoglobin chain
• 4 hemoglobin chains binds and forms hemoglobin
molecule

64. Types of haemoglobin
RBC mass of an embryo, fetus, child, and adult
6 different hemoglobins normally detected-
• Embryonic hemoglobins: Gower-1, Gower-2,
and Hb Portland
• Fetal hemoglobin : HbF
• Adult hemoglobins : HbA and HbA2

66. Types of haemoglobin
Embryonic hemoglobin :
• Gower-1 : ζ2ε2
• Gower-2 : α2ε2
• Hb Portland : ζ2γ2
• In embryos of 4-8 wk gestation, Gower hemoglobins
predominate
• By 3rd month disappeared.

68. Types of haemoglobin
Fetal Hemoglobin
• HbF - α2γ2
• After 8th wk, HbF - predominant
• 24 wk gestation HbF - 90% of the total hb
• 3rd trimester -
• At birth HbF - 70%
• Postnatally -
• By 6-12 month of age only a trace is present.

69. Types of haemoglobin
Adult Hemoglobins
• Hb A - α2β2 , HbA2 - α2δ2.
• 24th wk of gestation, HbA 5-10%
of total hb
• At term - averages 30%
• By 6-12 month of age, the
normal HbA pattern appears.

70. • Switch Mechanism-
 Transition from Hb F (fetal life and early
childhood) to HbA in later life by 6-12months.
 Mechanism not clear
 Methylation and deacetylation in the DNA
sequence of hemoglobin gene complex

73. Applied anatomy : Hemoglobin
1st wk of life Hb <13 – Anemia
• In utero – fetal 02 saturation 45% EPO level RBC
production
• After birth – O2 saturation 95%- EPO undetectable
hence RBC,Hb
• At 8-12 wks Hb level reaches Nadir, O2 delivery to
tissue impaired renal EPO production + , RBC
production

74. AGE HAEMOGLOBIN
12 wks of gestation 8- 10
20 wks of gestation 11
28 wks of gestation 14.5
34 wks of gestation 15
Cord blood 16.8
Day 1 18.4
Day 3 17.8
Day 7 17
Day 14 16.8
3- 4 weeks 14.2 +/- 2.1
3 month 11.3+/- 0.9

76. Red Blood Cell Indices

77. • The relationships between the hematocrit, the
hemoglobin level, and RBC are converted to red
blood cell indices through mathematical
formulas
• 1st introduced by Wintrobe in 1929
• The indices include :
-Mean corpuscular volume (MCV).
-Mean corpuscular hemoglobin (MCH)
-Mean corpuscular hemoglobin
concentration (MCHC)
-Red cell distribution width (RDW)

78. WHY RBC INDICES REQUIRED?
• To classify the erythrocytes by their volume
and Hemoglobin content
• This indices suggest how the RBC’s appear
microscopically and provide significant
information (most commonly for Anemia
diagnosis)

79. DEFINITIONS:
• Mean Corpuscular Volume (MCV)
– It is the measure of average volume of RBCs
• Mean Corpuscular Hemoglobin (MCH)
– It is a measurement of the average weight of hemoglobin in individual
erythrocytes.
• Mean Corpuscular Hemoglobin Concentration (MCHC)
– It is the average concentration of hemoglobin in erythrocytes
• Red Cell Distribution Width (RDW)
– It is a measure of variability of erythrocyte size

80. MEAN CORPUSCULAR VOLUME
• MCV = Hct(L/L) x 1000
RBC count ( x 1012/L)
• Average volume of the RBC in femtoliters (fL)
• Normocytic: 80-100 fL
• Microcytic: Red cells with reduced
volume(<80fL)
• Macrocytic: Red cells with an increased
volume(>100 fl)

81. MCV Increased
MCV Decreased
Fig. 2
Fig. 1

83. MEAN CORPUSCULAR HEMOGLOBIN
• It is a measurement of the average weight (in
picograms 10 -12 g)of hemoglobin in individual
erythrocytes. It is calculated by:
• MCH = Hb (g/dl) x 10
RBC( x 10 12/L)
• MCH varies in direct linear relationship with
the MCV. Cells with less volume contain less
Hb and vice versa
• Normal value for the MCH : 28 to 34 pg

84. MCH Increase MCH Decrease
• B12 deficiency
• Folic acid deficiency
• Reticulocytosis
• Hemolytic anemia
• Alcoholism
• Iron deficiency anemia
• Thalassemia
• Anemia of chronic
disorder
MEAN CORPUSCULAR HEMOGLOBIN

85. Anisocytosis
Fig. 5

86. MEAN CORPUSCULAR HEMOGLOBIN
CONCENTRATION
• It is the average concentration of hemoglobin in a
deciliter of erythrocytes and expressed in g/dl
• It is the ratio of hemoglobin mass to volume in
which it is contained
• MCHC = Hb (g/dl) x 100
Hct (L/L)
• Normochromic: 32-36g/dl
• Hypochromic: <32g/dl
• Hyperchromic: >36g/dl

87. MEAN CORPUSCULAR HEMOGLOBIN
CONCENTRATION
• Hypochromic: If the area of central pallor is
>1/3rd of the cell size, occur in thalassemia and
iron deficiency.
• Hyperchromic: The only erythrocyte that is
hyperchromic – spherocyte
Apparent hyperchromia ( high MCHC) is usually due
to an artifactual increase in the haemoglobin result
like due to haemolysis or large numbers of Heinz
bodies

88. MCHC Decrease
MCHC Increase.
Fig. 3
Fig. 4

90. RED CELL DISTRIBUTION WIDTH
• RDW is used because MCV is less reliable in
describing the erythrocyte population when
considerable variation in erythrocyte size occurs.
• RDW is a coefficient of variation in size
distribution of RBCs
• Measured as : RDW = Standard deviation of MCV × 100
MCV
• Normal value:11.5-14.5%
• Increased value indicates ANISOCYTOSIS.

91. RDW
• RDW is increased in Iron deficiency anemia.
• While RDW is normal in Thalassaemia minor.
• Combination of low MCV and high RDW is one
of the best screening test for the Iron
deficiency anemia.

92. RETICULOCYTES
• Premature RBC
• They contain remnants of Ribosomal RNA
• Number of reticulocytes in PBS is a fairly accurate
reflection of erythropoietic activity
• It is most useful and cost effective test in monitoring
and response to iron therapy

93. RETCULOCYTES COUNT
• Corrected reticulocyte count
– Used to adjust the reticulocyte count in proportion to the
severity of anemia
– Calculated by = Retic% x patient hematocrit
normal hematocrit
– Its practical importance is to assess the degree of
erythropoiesis in anemic patient.
– In anemic patient <2% of corrected retic count associated
with hypo cellular bone marrow

94. • Reticulocyte production index
– This index is used to correct the time of prolongation of
maturation of reticulocyte due to severe anemia
– Calculated by = patient hematocrit x retic count (%)
normal hematocrit x retic maturation time(days)
– Also known as SHIFT CORRECTION INDEX
PCV% MATURATION DAY(S)
36-45% 1
26-35% 1.5
16-25% 2
15% & below 2.5

95. RETCULOCYTES COUNT
• A higher reticulocytes count may indicate:
– Hemolytic anemia
– Bleeding (GI Bleeding)
– Bleeding disorder in a fetus/newborn
(erythroblastosis fetalis)
– Kidney disease, erythropoietin
– May be high during pregnancy.

96. RETCULOCYTES COUNT
A lower reticulocytes count may indicate:
- deficiency of nutrients required for Hb/RBC
– Bone marrow failure (eg. drug, tumor, radiation Rx
or infection)
– Cirrhosis of the liver
– Untreated patient of pernicious / megaloblastic
anemia
– Chronic kidney disease

97. MCV :
 At birth 104- 118 fl (normal adult 82-92)
 Decrease rapidly
 At 2months achieve adult range
MCH :
 At birth 33.5 -41.4 (normal adult 27- 31)
MCHC
 Value in new born(30-35) similar to adult value (32-36)
Retic count :
 Avg at birth 1.6 – 6.2%
 Premature infant count 6- 16 %
 Values drop by 1% by 7th day of life

101. • WBC Count
 At birth Ranges from 9,000 – 30,000 / cumm
 Mean count in term- 15,000 – 20,000 and
preterm slightly lower
 1st hour of life – slight increase
 End of 1st week – mean level falls to
approximately 12,000

102. REFERENCES
• Hematologic problems in new born – Oski and
Naiman
• Hematology of Infancy and childhood 7th edition –
Naithan and Oski
• Medical Physiology – Guyton and Hall
• Nelson Textbook of Pediatrics 20th edition
• Article in Toxicologic pathology –Feb 2006-Normal
structure,function and histology of the bone marrow
by Gregory S. Travlos

103. THANK
YOU