Blood

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 Blood
 Blood is the “river of life”
 Viscous fluid composed of cells and
plasma
 Blood is a specialized type of
connective tissue in which living blood
cells, (formed elements), are
suspended in a non living fluid matrix
called plasma.
• Cellular Part (Formed Elements)
• Non cellular part (Plasma)
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 Blood
• 1/12th of body weight
• 8 % of total body weight
Color range
 Oxygen-rich blood is scarlet red bright
crimson
 Oxygen-poor blood is dull red
 pH must remain between 7.35–7.45
 Temp 38 c or 100.4 F

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 Blood Composition
 Blood Composition

Cellular Part (Formed Elements)--- 45%
• RBCs, Red blood cells or erythrocytes
• WBCs, white blood cells or Leukocytes
• Platelets (thromobocytes)

Non cellular Portion (plasma)--- 55%
• Fluid part (91-92%)--- water
• Solid part (8%-9%)

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 Composition of plasma
 Straw colourd fluid
 Contains over 100 solutes
 Organic substances
 Plasma Proteins (Approx 7%)
• Albumin
• Globulin
• Fibrinogin
• Prothrombin
• Plasma complement system, approx 20 proteins

Nitrogenous substances
• Urea
• Uric acid
• Ammonia
 Non-nitrogenous substances
• carbohydrates
• Lipids

Enzymes
• Amylase
• Carbonic Anhydrase
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 Pigments (Biluribin)
 Composition of plasma
 Inorganic substances
• Different ions
• Sodium
• Potassium
• Bi-carbonate

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 Functions of plasma
 Helps in transport of substances in the
body
 Maintains colloid osmotic pressure of blood
 Causes blood clotting because it contains
the fibrinogen and prothrombin
 Stores proteins for supply in needs
 Helps in maintaining blood pressure and
blood viscosity
 Contains antibodies and antitoxins

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Physical Properties of Blood and
plasma
 Specific Gravity of plasma is 1.024
 Specific Gravity of blood is 1.055 - 1.062
 Male: 1.057
 Female: 1.053
 Blood is 5 times thicker or viscous than distilled
water.
 Blood----- blood cells
 Plasma----Plasma Proteins
 Relative viscosity of water, plasma and blood are
1, 1.8, 4.7 respectively.
 Plsama-(clotting factor and fibrinogen) = serum

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 Functions of blood
 Blood performs a number of functions.
 Distribution
 Regulation
 Protection
• Distribution Functions
• Nutritive Function:
 Nutrients from GIT to whole body
• Respiratory Function:
 O2 and Co2 Transport
• Excretory Function:

Metabolic Wastes to kidneys
• Transport Function:
 Enzymes
 Hormones
 Vitamins

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 Functions of blood
• Regulation Functions
• Maintainance Functions
 Body Temperature maintenance through skin
 Blood Volume, salts and blood proteins prevent excessive fluid loss.
 Blood Pressure
• Buffering Functions

Maintaining normal pH in body with the help of blood proteins
and other solutes
 Acts as body’s alkaline reserve of HCO3- ions.

• Protection Functions
• Preventing blood loss
 Platelets and plasma proteins initiate clot formation in case of
damage
• Defensive function
 Prevents body from being infected from invaders eg bacteria and
viruses with the help of antibodies, compliment proteins and WBCs

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Blood flow

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 Plasma Proteins
 Most are produced by liver, except for
hormones and gamma globulins
 Not used up by cells as fuels
 Plasma proteins account for almost 7% by
weight of plasma volume
• 6 - 8 grams of protein in a volume of 100 milliliters of
blood (referred to as g/dl)
 The plasma proteins include:
 Albumins
 Globulins
 Fibrinogen & prothrombin
 Regulatory proteins
• Enzymes – coagulation enzymes, complement factors
• C-reactive protein – acute phase reactant
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 Albumins
 Smallest and most abundant of the plasma
proteins almost 58% of total plasma proteins.
 Soluble in distilled water
 Precipitated by saturated ammonium sulphate
 Coagulated by heat
 20-Days half life
 At pH 7.4 it is anionic with 20 negative charges
per molecule
 Highly polar
 Functions:
• Regulate water movement between the blood and
interstitial fluid. (Maintain osmotic pressure)
• Albumins act as transport proteins that carry ions,
hormones, and some lipids in the blood.

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 Regulation of osmotic
pressure
Albumin Structure

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 Causes of decreased plasma
albumin:
Decreased synthesis
A. malnutrtion
B. malabsorption
C. advanced chronic liver disease

Abnormal distribution or dilution

A. overhydration
B. increased capillary permeability like in
septicemia

Abnormal excretion or degradation

A. nephrotic syndrome
B. burns
C. hemorrhage
D. loss of protein from the digestive tract

Rare congenital defects

A. hypoalbuminemia
B. analbuminemia
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 Globulins
 Not soluble in distilled water
 38 % of plasma proteins
 More easily precipitated by saturated ammonium
sulphate
 They are coagulated by heat
 Series of slightly different globulins may be
separated by using different concentrations of
alcohol.
 Electrophoresis can also result in separation and
identification of different globulins (alpha, beta,
gamma)
 Functions:
• Alpha & beta: produced by liver; transport proteins that bind
to lipids, metal ions, and fat – soluble vitamins
• Gamma: Antibodies released primarily by plasma cells during
immune response. 17
 Fibrinogen & prothrombin
 Fibrinogen:
• Produced by liver,
• converted to web like substance of clot
 Prothrombin:
• produced by liver,
• formation requires vitamin K,
• converted to thrombin which enzymatically
converts fibrinogen to fibrin

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 Blood Cells
 RBCs, Red blood
cells or erythrocytes
 WBCs, white blood
cells or Leukocytes

Platelets
(thromobocytes)

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 Erythrocytes
 Cell Type
• Erythrocytes (Red blood cells, RBCs)
 Description
• Bicancavae, anucleate disc, salmon-colored, sacs of
hemoglobin,most organelles ejected, diameter 7-8 µm
 Cells/mm3 (µl) of blood
• 4-6 millions
 Duration of development (D) & Life Span (LS)
• D: 5-7 days
• LS: 100-120 days
 Function
• Transport oxygen bound to hemoglobin and also small
amount of CO2
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 Leuckocytes
 Cell Type
• Leukocytes (lecuko- white) (White blood cells, WBCs)
 Description
• Spherical, nucleated cells
 Cells/mm3 (µl) of blood
• 4800-10,800
 Types
• Granulocytes
 Neutrophils
 Eosinophils
 Basophils
• Agranulocytes
 Lymohocytes
 Monocytes
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 Leukocytes
 General structural and functional characteristics
 Complete cells (nucleus and other organelles)
 < 1 % of total blood volume

They form a mobile army of body’s protective system
 Diapedesis (Leaping Across)
The process of squeezing through the pores of blood vessels.
 Ameboid motion
WBCs move through tissue spaces by Ameboid motion i.e. by forming
flowing cytoplasmic extensions (throwing pseudopodia)
 Chemotaxis
The ability of WBCs to locate areas of tissue damage and infection in
body by responding to certain chemicals.
 Chemotactic substances
• Bacterial toxins
• Degenerative products of inflammed tissues
• Plasma clotting end products

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Genesis of Formed Elements

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 Hematopoiesis
 Hematopoiesis or hemopoiesis (Hemato, hemo = blood, Poiesis = to
make)
 Process occurs in Red bone marrow
 Red bone marrow composition
• It is composed of a soft network of reticular connective tissue
bordering on wide blood capillaries called blood sinusoids. With in
this network are immature red blood cells, fat cells, reticular cells
( secrete the fibers).
• On average, the marrow produces 1 ounce of new blood every day
• Cells produced are about 100 billion
 All cells arise from the same type of stem cells the PHSC or
hemocytobalsts (Cyte = cell , blast = bud) that reside in red
bone marrow.
 But the maturation pathway is different form each other,
once a cell is committed to a specific blood cell pathway, it
can not change
 This commitment is signaled by appearance of membrane
surface receptors that respond to specific hormones or growth
factors, which in turn push the cell towards further
specialization.
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 Production of Leukocytes
 Leukopoiesis
 Hormonally stimulated

T-Lymphocytes

Macrophages
 Hematopoietic Factors
• Glycoproteins
 Interleukins
• IL-3, IL-5
 CSFs (colony stimulating factors)
• Leukocyte population stimulated eg G-CSFs

Functions
• Stimulation of WBCs precursors to divide and mature
• Enhance protective potency of mature leukocytes
• Clinically used (EPO) and other CSFs
 Stimulation of bone marrow in cancer patients

Marrow transplants
 AIDS patients
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Production of Leukocytes

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 Production of Leukocytes
 Pluripotential hemopoietic stem cell (PHSC)
(Hemocytoblasts)
• A stem cell derived from the embryonic mesenchyme and
considered to be capable of developing into any type of blood cell.
 Myeloid Stem cells
 Lymphoid Stem cells
 Myeloid Stem cells
 Committed cells
• Myeloblast
• Monoblast
 Lymphoid Stem cells
 Committed cells
• Lymphoblast

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 Production of Granulocytes
 Myeloblasts accumulate lysosomes to become
promyelocytes
 Distinctive granules of each granulocyte appear in
myelocyte stage cell division stops
 Band cells nuclei become arc- like
 Nuclear constriction & segmentation just before
leaving bone marrow
 Mature granulocytes are stored in bone marrow,
10-20 times more than in blood
 Production ratio 3:1 (erythrocytes : granulocytes)
 Shorter life span 0.5-9 days

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Production of Agranulocytes
 Monocytes diverge from pleuripotent myeloid stem
cells  Monoblast promonocyte  Monocytes 
some cells form macrophages (in tissues)
 Lymphocytes diverge from pleuripotent lymphoid
stem cells  Lymphoblast  prolymphocyte 
Lymphocytes  Plasma cells
 Promonocytes and Prolymphocytes leave the bone
marrow and travel to lymphoid tissue, where there
further differentiation occur
 Monocytes live for months
 Lymphocytes  days to years

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 Leukocyte Disorders
 Leukocytosis
 Physiological cause
• Newborn
• Pregnancy
• Emotion
• Stress

Pathological Causes
• Infections
• Burns
• Malignancy
• Allergic Reactions

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 Leukocyte Disorders
 Leukopenia
• Exposure to Rays, e.g. Gamma rays
• Chemicals e.g. Benzene
• Drugs e.g. Chloramphenicol
 Leukemias
An increased number of abnormal
circulating WBCs due to uncontrolled over
production as a result of mutation of myeloid
or lymphoid cells.
• Lymphocytic Leukemia
• Myelocytic Leukemia

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 Platelets (Thrombocytes)
 Not cells
 Cytoplasmic fragments of extraordinary large cells
(60µm)  Megakaryocytes
 Cytoplasm stain blue, granules Stain Purple
 Essential for the clotting process when blood
vessels are ruptured or their lining is injured.
 Components of Granules
• Seortonin
• Ca 2+
• Different Enzymes
• ADP
• Platelets derived Growth Factors (PDGF)
 When not involved in clotting mechanism, they are
kept inactive by molecules (NO, PG I2) secreted by
endothelial cells lining blood vessels.
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 Genesis of Platelets

 Platelets formation is regulated by a Hormone Thrombopoietin1

 Hemocytoblasts (PHSC) --> Myeloid Stem cells --> Megakaryoblasts
Megakaryoblasts under go repeated mitosis but cytokinesis does
not occur, final result is MEGAKARYOCYTE. (A cell with a huge
nucleus)
 When formed the megakaryocyte presses up against a sinusoid (a
specialized type of capillary in marrow) and sends cytoplasmic
extensions through sinusoid wall into blood stream.
 These extensions rupture, releasing the platelet fragment in blood
stream.
 150,000-400,000
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 Platelets Disorders
 A number of factors can cause thrombocytopenia
(a low platelet count).
• Inherited (passed from parents to children), or it can develop
at any age.
• Sometimes the cause isn't known
 Causes: (See Notes)
• The body's bone marrow doesn't make enough platelets.

Cancers

Aplastic Anemia

Toxic Chemicals - pesticides
 Medicines – Chloramphenicol, Sulpha drugs
 Viruses- Dengue
• The bone marrow makes enough platelets, but the body
destroys them or uses them up.
 Autoimmune Disease
 Surgery
 Pregnancy- 5%
• The spleen holds onto too many platelets.
 Enlarged Spleen
• Cirrhosis
• Liver Cancer
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 Erythrocytes (RBCs)
 Red, oxygen carrying, hemoglobin containing, non-
nucleated cells, present in the blood
 Shape  Bi-concave Discs
 Size:
• Dia  7.5 - 7.8 µm
• Thickness:
 Thickest  2.5 µm
 Thinnest  1 µm or <1 µm
• Thin centers appear lighter in colour than edges
 Volume: 90-95 µm 3

 Life Span:
• Adults: 100-120 Days
• Neonates: 70-90 Days
 Count:
• Males: 5.2 million + 3,00,000 cells/mm3
• Females: 4.7 million + 3,00,000 cells/mm3
• Newborn: 6 – 6.5 million cells/mm3
• Fetus: 7.8 million cells/mm3
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 Why count is different? 1
 Erythrocytes (RBCs)
 Composition of RBCs:
The composition of RBCs is same as that of a normal cell
except that mature RBCs contain Hb and don’t contain
nucleus, mitochondria, and other important organelles.
– Water = 65 %
– Solid and semisolids = 35 %
 Hb (33 %)

 Organic and inorganic substances (2%)

(Amino Acids, Cholesterol, Creatinine, Proteins, Phospholipids, Urea)

 How RBCs Change and Maintain Shape:
• Main protein – Hb - 97 %
• Other Proteins
 Anti-Oxidant Enzymes (Get rid body of harmful O2 radicals)
 Maintenance proteins
Bi-concave shape of RBCs is maintained by network of proteins,
especially one called spectrin, it is attached to the cytoplasmic side
of the plasma membrane, as spectrin net is deformable, it gives
erythrocytes the flexibility to change their shape as necessary- to
twist, turn and become cup shaped when pass through small
capillaries – and then resume their normal shape. 40
 Erythrocytes (RBCs)
 Energy Production:
 For energy RBCs depend on plasma glucose, metabolic
break down takes place through
• Embden Meyerhof Glcolytic pathway
• Pentose phosphate Pathway (PPP) or (Hexose Monophosphate shunt)
 Structural Characterstics VS Function
• Small size and Biconcave shape provides huge surface area (about 30
% more area than comparable spherical cells).
• Excluding water content RBC is 97 % Hb that transports resp. gases.
• Don’t use oxygen themselves as produce energy by anaerobic
mechanisms.
 Functions or RBCs:
• O2 Transport:
 Contains Hb, that carries oxygen bound to ‘Heme’ portion
• CO2 Transport:
 CO2 Transport takes place in combination with ‘globin’ protion. (20%)
• Acid-Base balance
 By buffering action of Hb
• Blood Viscosity
• Ionic balance
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Erythrocytes Production (Erythropoiesis)
PHSC
Hemocytoblasts:
•Cell size large 20-25 mircon
Myeloid •Nucelus large
Stem cells •Less cytoplasm
•Mitosis present

Proerythroblast:
•Cell size decrease 15-17 mircon

Basophilic 1
Erythroblast:
•Cell size 12-15 mircon
•Nucelus Condensed
•Mitosis present
•Nucleoli Rudimentary
•Produces huge number of Ribosomes
•Hb synthesis starts

Polychromatophil 2
Erythroblast:
•Cell size 10-12 mircon
•Nucelus Condensed
•Mitosis Absent

Orhochromatic 3
Erythroblast:
•Cell size 8-10 mircon
•Nucelus More Condensed

Reticulocyte:
•Young Erythrocytes
•Cell size 7-8 mircon
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Erythrocytes Production (Erythropoiesis)

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 Erythrocytes Production (Erythropoiesis)
1. PHSC

3. Myeloid stem cells

5. Proerythroblast (Megaloblasts)

7. Basophilic Erythoroblasts (Early erythroblasts) (early Normoblast)

9. Polychromatophil Erythroblasts (Intermediate erythroblast or Normoblast)

11. Orhochromatic Erythroblasts (Late Erythroblast or Normoblasts)

13. Reticulocytes
• Young erythrocytes
• Contain a short network of clumped ribosomes and RER.
• Enter the blood stream
• Fully mature with in 2 days as their contents are degraded by
intracellular enzymes.
• Count = 1-2% of red cells
• Provide an index of rate of RBC formation

14. Erythrocytes
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 Basophilic Polychromatophilic Dividing
Proerythroblast erythroblast Polychromatophilic
(or intermediate)
or or Erythroblast or
Erythroblast or
pronormoblast Early Normoblast
Normoblast
Normoblast

Orthochromatic
Orthochromatic (Acidophilic)
Reticulocyte erythroblast erythroblast
(brilliant cresyl Reticulocyte Extruding Or
blue dye) 1 Nucleus Late
Erythroblast

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 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
 Vitamin C  Helps in iron absorption (Fe+++  Fe++)
 Proteins  Amino Acids for globin synthesis
 Iron & copper  Heme synthesis
 Intrinsic factor  Absorption of Vit B 12
 Hormones
Physiological Variations in RBC count
1. Diurnal Variation (During 24 hours)
• 5%
• Lowest - Sleep and early morning hours
• Highest - Evening
2. Temperature
3. High Altitude
4. Hypoxia
5. Radiations
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• X-rays
 Fate and destruction of RBCs 1

 Anucleate  certain limitations.

• No synthesis of new proteins, No growth, No division.
 However they do have Cytoplasmic enzymes (hexokinase, Glu-6-
phosphate dehydrogenase) that are capable of metabolizing glucose
and forming small amounts of ATP. These enzymes also perform
following actions
• maintain pliability of the cell membrane,
• maintain membrane transport of ions,
• keep the iron of the cells’ hemoglobin in the ferrous form rather than ferric
• Prevent oxidation of the proteins in the red cells.
 Erythrocytes become “old” as they lose their flexibility and become
pikilocytes (spherical), increasingly rigid and fragile. Once the cell
become fragile, they easily destruct during passage through tight
circulation spots, especially in spleen, where the intra-capillary
space is about 3 micron as compared to 8 micron of cell size
RBCs useful life span is 100 to 120 days,After which they become trapped
and fragment in smaller circulatory channels, particularly in those of the
spleen. For this reason, the spleen is sometimes called the “red blood cell
graveyard.”
 Dying erythrocytes are engulfed and destroyed by macrophages.
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 Fate
and
destr-
uction
of
RBCs

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 Regulation of RBCs production
 Control of rate of erythropoiesis is based on ability of RBCs to
transport sufficient oxygen to tissues as per demand, not the
number
 Tissue Oxygenation
– Drop in normal blood oxygen levels may result due to
• Reduced number of RBCs
 Hemorrhage
 Excess RBC Destruction
• Reduced Availability of Oxygen
 High Altitude
 Lung Diseases
• Increase Tissue demands of Oxygen
 Aerobic Exercises
 Erythropoietin (Formation & role)1
 Glycoprotein, Mol wt= 34,000.
 Erythropoietin, a hormone, produced mainly by the kidneys(90%) and also
by liver(10%), stimulates erythropoiesis  by  acting  on committed stem cells
to induce proliferation and differentiation of erythrocytes in bone marrow.
 Site of Action: BONE Marrow

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 Regulation of RBC production

A negative Feed back mechanism

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 Hemoglobin (Hb)
 Red, oxygen carrying pigment present in RBCs.
• Heme (4%)
• Globin (96%)
 Quantity
• 700-900g in body
• 29-32 peco gram/RBC
 RBCs
• Male= 36g/100ml
• Female = 34g/100ml
 Whole Blood
• Newborn = 14-20g/100ml
• Male= 14-16g/100ml
• Female = 12-14g/100ml
 Molecular Weight
• 64,450
 Types
• 4 types of poly peptide chains based on amino acid composition and sequence.
• alpha, beta, gamma, delta

Adult Hb
• Hb A = 2 alpha (141 AA)+ 2 beta (146 AA) chains (α2β2 )
• Hb A2 = 2 alpha (141 AA)+ 2 delta (146 AA) chains (2.5%) 1 (α2δ2) (10 AA differ)

Fetal Hb
• Hb F = 2 alpha (141 AA)+ 2 gamma (146 AA) chains 2 ( α2γ2) (37 AA differ)53
 Hemoglobin (Hb)
 250 million Hb molecules / RBC
 So carry 1 billion oxygen molecules / RBC
 Synthesis of Hb
• Starts at proerythroblastic stage
 Synthesis steps:
• Heme is made from acetic acid and glycine in mitochondria
• Acetic Acid  α-ketoglutaric Acid  Succinyl Co A (Krebs Cycle)
• Globin (polypeptide chain) is synthesized by Ribosomes

 Reactions of Hb:
• Oxyhemoglobin (oxygen + Hb) Ruby Red (in lungs) (Co-ordination
bonds)
• Deoxyhemoglobin (Reduced Hb) Dark Red (in tissues)
• Carbaminohemoglobin (Co2 + Hb) (Globin’s amino acids) (20 %)
• Caroboxyhemoglobin (Co + Hb)
• Methemoglobin (Fe+++ instead of Fe++) 54
 Reactions of Hb:
 Hemoglobin binds O2 to form oxyhemoglobin, O2 attaching to the
Fe2+ in the heme. The affinity of hemoglobin for O2 is affected by
• pH,
• Temperature,
• The concentration of 2,3-diphosphoglycerate (2,3-DPG) in the red cells.
 2,3-DPG and H+ compete with O2 for binding to deoxygenated
hemoglobin, decreasing the affinity of hemoglobin for O2 by shifting
the positions of the four peptide chains (quaternary structure).
 Each of the four iron atoms can bind reversibly to one O2 molecule.
The iron stays in the ferrous state, so that the reaction is an
oxygenation, not an oxidation. It has been customary to write the
reaction of hemoglobin with O2 as
Hb + O2 ↔ HbO2
 Since it contains four Hb units, the hemoglobin molecule can also
be represented as Hb4, and it actually reacts with four molecules of
O2 to form Hb4O8 as following.

 The reaction is rapid, requiring less than 0.01 s.

 The deoxygenation (reduction) of Hb4O8 is also very rapid.

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 Hb Abnormalities
 Globin Genes1 determine the AA sequence in Hb.
 Two types of Abnormalities:
 Hemoglobinopathy

• Abnormal polypeptide chains are produced

 Sickle cell disease due to Hb-S
 Thalassemia
• In which the chains are normal in structure but produced in
decreased amounts or absent because of defects in the
regulatory portion of the globin genes.
 The α and β thalassemias are defined by decreased or absent α
and β polypeptides, respectively.
 1000 Abnormal Hbs due to mutant genes in humans.
usually identified by letter—Hb-C, E, I, J, S, etc.
 Mostly, the abnormal Hbs differ from normal Hb-A in the
structure of the polypeptide chains.
 For example, In hemoglobin S,
• α chains normal
• β chains abnormal, among the 146 AA residues in each β
polypeptide chain, one glutamic acid residue has been
replaced by a valine residue. 56
 Hb Abnormalities
 Heterozygous Half the circulating hemoglobin is abnormal and half
is normal.
• Have sickle cell trait
 Homozygous  all of the hemoglobin is abnormal.
• Develop the full blown disease
 Results of abnormality
 Many of the abnormal hemoglobins are harmless.
 Abnormal O2 equilibriums.
 Anemia.
• Hb-S polymerizes at low O2 tensions, and this causes the red cells to become
sickle-shaped, hemolyze, and form aggregates that block blood vessels.
• The result is the severe hemolytic anemia known as sickle cell anemia.
 The sickle cell gene is an example of a gene that has persisted and
spread in the population.
 It originated in the black population in Africa, and it confers
resistance to one type of malaria.
 Africa = 40% of the black population have the sickle cell trait.
 In United States 10 %
 Treatment:
• Bone marrow Transplatation
• Hb-F production by hydroxyurea.
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Hb Abnormalities

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 Hemoglobin Metabolism
 The heme of the hemoglobin is split off from globin.

Its core of iron is saved, bound to protein (as ferritin or
hemosiderin), and stored for reuse.

The heme is converted to biliverdin. In humans, most of the
biliverdin is converted to bilirubin, a yellow pigment that is
released to the blood and binds to albumin for transport.

Bilirubin is picked up by liver cells, which in turn secrete it
(in bile) into the intestine, where it is metabolized to
urobilinogen.

Most of this degraded pigment leaves the body in feces, as a
brown pigment called stercobilin.

Exposure of the skin to white light converts bilirubin to
lumirubin, which has a shorter half-life than bilirubin.
 Phototherapy (exposure to light) is of value in treating infants

with jaundice due to hemolysis.

 The protein (globin) part of hemoglobin is metabolized or broken
down to amino acids, which are released to the circulation.

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 Iron metabolism
 Iron = 4-5g Per person
 Hb 65 % of total iron
 Reticuloendothelial system + liver = 15-30 %
 Myoglobin = 4%
 Intracellular oxidating heme compounds = 1% Ferritin
 Transferrin = 0.1 %
 Absorption of Iron:
• Mianly from Duodenum.
• Heme-Fe+2 from Meat (Myoglobin, hemoglobin)
• Fe+2 from small intestine (Fe+3 reduced by Vit C &
ferrireductase(FR) to Fe+2 for absorption)
 Transport of Iron:
• Iron + Apotransferrin [protein from liver]  Transferrin (Bound)
 is taken up by endocytosis into erythroblasts and cells of the
liver, placenta, etc. with the aid of transferrin receptors.
 Storage & Recycling:
• Ferritin  one of the chief forms in which iron is stored in the
body, storage occurs mainly in the intestinal mucosa, liver,
bone marrow, red blood cells, and plasma. (4500 Fe+3 ions i.e.
600mg as readily available store).
• Hemosidrin  In marcophages of liver and bone marrow
(250mg) slow release.
60
• 97 % recycled by phagocytes of liver, spleen and bone marrow
FR=ferrireductase

Daily Iron Loss

Male: 1mg/day
Females: 2mg/day

Daily Iron
Requirement
Male: 1mg/day
Females: 2mg/day

61
 Blood Transfusion
 Whole blood transfusions are routine when blood loss is
rapid and substantial.
 In all other cases, infusions of packed red cells (whole
blood from which most of the plasma has been removed)
are preferred for restoring oxygen-carrying capacity.
 The usual blood bank procedure involves collecting blood
from a donor and then mixing it with an anticoagulant,
such as certain citrate or oxalate salts, which prevents
clotting by binding with calcium ions.
 The shelf life of the collected blood at 4°C is about 35
days.
 Because blood is such a valuable commodity, it is most
often separated into its component parts so that each
component can be used when and where it is needed.

62
 ABO Blood Group
 BLOOD TYPES
 The membranes of human red cells contain
a variety of blood group antigens, which are
also called agglutinogens.

Antibodies against red cell antigens are
called agglutinins.
• When the plasma of a type A individual
(containing Anti-B antibodies) is mixed with type
B red cells, the anti-B antibodies cause the type
B red cells to clump (agglutinate).
 The most important and best known of
these are the A and B antigens, but there
are many more. eg
• MNSs, Lutheran, Kell, Kidd,
63
64
 ABO Blood Group
 The individuals are divided into four major blood types
on this basis of presence of these antigens.

Type A individuals have the A antigen,

Type B have the B,

Type AB have both, and

Type O have neither.
• These antigens are found in many tissues in addition to blood:
• E.g.. salivary glands, saliva, pancreas, kidney, liver, lungs, testes,
semen, and amniotic fluid.
 Chemsitry of Anitgens:
• The A and B antigens are complex oligosaccharides that differ in
their terminal sugar.
• On red cells they are mostly glycosphingolipids,
• whereas in other tissues they are glycoproteins.
• An H gene codes for a fucose transferase that puts a fucose1
(hexose dexoy sugar) on the end of these glycolipids or
glycoproteins, forming the H antigen
• H-antigen is usually present in individuals of all blood types.

65
 ABO Blood Group

Individuals who are type A have a gene which codes for a
transferase that catalyzes placement of a terminal N-
acetylgalactosamine on the H antigen,
 Individuals who are type B have a gene which codes for a
transferase that places a terminal galactose.
 Individuals who are type AB have both transferases.

Individuals who are type O have neither, so the H antigen
persists.

66
 ABO Blood Group
 Subgroups of blood types A and B

Most important being A1 and A2.
• A1 cell has about 1,000,000 copies of the A antigen on its
surface,
• A2 cell has about 250,000 copies of the A antigen on its
surface
 Antibody Development:
• Antigens very similar to A and B are common in intestinal
bacteria and possibly in foods to which newborn individuals
are exposed.
• Therefore, infants rapidly develop antibodies against the
antigens not present in their own cells.
Thus,
• type A individuals develop anti-B antibodies,
• type B individuals develop anti-A antibodies,
• type O individuals develop both,
• and type AB individuals develop neither.
 Blood Typing Test:
Blood typing is performed by mixing an individual's red
blood cells with antisera containing the various
agglutinins on a slide and seeing whether agglutination
occurs. 67
 Bombay phenotype
Missing H-gene so no fucose tranferase so no fucose and no H-antigen that
Forms the base for A and B Antigen.

No fucose

68
 Bombay Phenotype
 This blood phenotype was first discovered in Bombay, now
known as Mumbai, in, by Dr. Y.M. Bhende.
 hh is a rare blood group also called Bombay Blood group.
Individuals with the rare Bombay phenotype (hh) do not
express H antigen (the antigen which is present in blood group
O).
 So whatever alleles they may have of the A and B blood-group
genes, they cannot make A-anitgen or B-antigen on their
red blood cells,because A antigen and B antigen are made
from H antigen.
 As a result, people who have Bombay phenotype can donate to
any member of the ABO blood group system (unless some
other gene, such as Rhesus, is checked for compatibility), but
they cannot receive any member of the
ABO blood group system's blood (which always contains one or
more of A and B and H antigens), but only from other people
who have Bombay phenotype.
 The usual tests for ABO blood group system would show them
as group O, unless the hospital worker involved has the means
and the thought to test for Bombay group.
69
 Rh Blood Groups
 45 different types of Rh agglutinogens, each called an Rh
factor.
 Three, the C, D, and E antigens, are fairly common.
 Rh antigen  first identified in rhesus monkeys.
 As a rule, ABO and Rh blood groups reported together eg,
O+, A–, and so on.
 If an Rh– person receives Rh+ blood, the immune system
becomes sensitized and begins producing anti-Rh
antibodies against the foreign antigen soon after the
transfusion.
 Hemolysis does not occur after the first such transfusion
because it takes time for the body to react and start
making antibodies.
 But the second time, and every time thereafter, a typical
transfusion reaction occurs in which the recipient’s
antibodies attack and rupture the donor RBCs. eg
Erythorblastosis fetalis1
 Prevention:
70
• Anit-Rh antibodies given after every Rh+ birth. [RhoGAM]
Rh Factor

71
 Blood Transfusion Reactions
 When mismatched blood is infused, a transfusion reaction occurs
 Donor’s red blood cells  attacked by the recipient’s plasma
agglutinins.
 Donor’s plasma antibodies may also agglutinate the host’s RBCs,
but they are so diluted that this does not usually present a serious
problem.
 Initially, agglutination clogs small blood vessels throughout the
body.
 During the next few hours, the clumped red blood cells begin to
rupture or are destroyed by phagocytes, and their hemoglobin is
released into the bloodstream.
 These events lead to two easily recognized problems:
• The oxygen-carrying capability of the transfused blood cells is disrupted
• The clumping of red blood cells in small vessels hinders blood flow to tissues
beyond those points.
 Less apparent, but more devastating, is the consequence of
hemoglobin escaping into the bloodstream.
 Circulating hemoglobin passes freely into the kidney tubules,
causing cell death and renal shutdown. If shutdown is complete
(acute renal failure), the person may die.
72
 Blood Transfusion Reactions
 Transfusion reactions can also cause
• fever,
• chills,
• low blood pressure,
• rapid heartbeat,
• nausea,
• vomiting, and general toxicity;
but in the absence of renal shutdown, these reactions are rarely lethal.
 Treatment of transfusion reactions is directed toward preventing
kidney damage by administering fluid and diuretics to increase urine
output, diluting and washing out the hemoglobin.
 Some laboratories are developing methods to enzymatically convert
other blood types to type O by clipping off the extra (A- or B-specific)
sugar residue.
 Autologous (auto = self) transfusions.
 The patient predonates his or her own blood, and it is stored and
immediately available if needed during or after the operation. .
 Iron supplements are given, and as long as the patient’s preoperative
hematocrit is at least 30%, one unit (400–500 ml) of blood can be
collected every 4 days, with the last unit taken 72 hours prior to
surgery.
73
 Hemostatis
 Hemostasis or stoppage of bleeding (stasis = halting).
 No hemostasis  No sealing  bleed to death from minor
wounds
 The hemostasis response is
• fast
• localized and
• carefully controlled
 Involves many blood coagulation factors normally present
in plasma as well as some substances that are released
by platelets and injured tissue cells.
 During hemostasis, following steps occur:

Vascular spasms,

Platelet plug formation,
 Coagulation, or blood clotting.

Growth of fibrous tissue in clot to close the hole in vessel.
 Blood loss at the site is permanently prevented when
fibrous tissue grows into the clot and seals the hole in
the blood vessel.
74
75
76
 1-Vascular Spasms
– The immediate response to blood vessel injury is
constriction of the damaged blood vessel
(vasoconstriction).
 Factors that trigger this vascular spasm include
• Direct injury to vascular smooth muscle,
• Chemicals released by endothelial cells and platelets,
• Reflexes initiated by local pain receptors.
spasm mechanism becomes more and more efficient
as the amount of tissue damage increases, and is most
effective in the smaller blood vessels.
 Advantage:
• A strongly constricted artery can significantly reduce blood
loss for 20–30 minutes, allowing time for platelet plug
formation and blood clotting to occur.
• It is claimed that for a time after being divided transversely,
arteries as large as the radial artery constrict and may stop
bleeding.
• But arterial walls cut longitudinally or irregularly do not
constrict in such a way that the lumen of the artery is
occluded, and bleeding continues. 77
 2 - Platelet Plug Formation
Platelets play a key role in hemostasis by forming
a plug that temporarily seals the break in the
vessel wall.
– They also help to initiate subsequent events that lead
to blood clot formation.
– As a rule, platelets do not stick to each other or to the smooth
endothelial linings of blood vessels.
– But, when the endothelium is damaged and
underlying collagen fibers are exposed, platelets, with
the help of a large plasma protein called von
Willebrand factor (VWF) synthesized by endothelial
cells, adhere to the collagen fibers and undergo some
remarkable changes.
Swell,
Form spiked processes or pseudipodia,
78
Become sticky.
 2 - Platelet Plug Formation

Once attached, the platelets are activated and their granules
begin to break down and release several chemicals.
serotonin, enhance the vascular spasm.
Adenosine diphosphate (ADP), (potent aggregating agents that attract
more platelets to the area and cause them to release their contents).
Thromboxane A2 , a short-lived prostaglandin derivative, stimulates
both events (Vasoconstriction & Activation).
 So a positive feedback cycle begins that activates and attracts
greater and greater numbers of platelets to the area

within one minute, a platelet plug is built up, which further
reduces blood loss.

Limiting the platelet plug to the immediate area where it is
needed is the task of prostacyclin (also called PG I2), a
prostaglandin produced by intact endothelial cells that is a strong
inhibitor of platelet aggregation.

Platelet plugs are loosely knit, but when helped by fibrin threads
they are quite effective in sealing the small tears in a blood vessel
that occur with normal activity.
 Once the platelet plug is formed, the next stage, coagulation,
comes into play.

79
80
 3-Coagulation
 Coagulation or blood clotting
 Complicated process, Liquid Blood  becomes gel,
 Over 50 Substances are involved
 Factors that enhance clot formation are called clotting
factors or procoagulants.
 Factors that inhibit clotting are called anticoagulants.
 Balance between these two groups of factors.
Normally, anticoagulants dominate and clotting is
prevented; but when a vessel is ruptured,
procoagulant activity in that area increases
dramatically and clot formation begins.

The procoagulants are numbered I to XIII according to
the order of their discovery; hence the numerical order
does not reflect the reaction sequence.
 Most of these factors are plasma proteins made by the
liver that circulate in an inactive form in blood until
mobilized.
81
82
 3-Coagulation
 Three Phases of Coagulation:
• A complex substance called prothrombin activator
is formed.
• Prothrombin activator converts prothrombin (a
plasma protein) into thrombin, (an enzyme).
• Thrombin catalyzes the joining of fibrinogen
molecules present in plasma to a fibrin mesh.
 Role of Vitamin K in coagulation.
Vitamin K not directly involved in
coagulation, this fat-soluble vitamin is
required for the synthesis of four of the
procoagulants made by the liver i.e (II,
VII, IX and X). 83
84
Phase 1- Formation of prothrombin Activator
 Clotting may be initiated by either the
• Intrinsic Pathway
• Extrinsic pathway
 Both pathways are usually triggered by the
tissue-damaging events. Clotting of blood outside
the body (such as in a test tube) is initiated only
by the intrinsic mechanism.    
– Critical components in both mechanisms are

negatively charged membranes, particularly

those on platelets that contain
phosphatidylserine (platelets phospholipids),
also known as PF3 (platelet factor 3).
– Many intermediates of both pathways can be
activated only in the presence of PF3. 85
Phase 1- Formation of prothrombin Activator
 Intrinsic Pathway
In the slower intrinsic pathway, all factors needed for clotting are
present in (intrinsic to) the blood.
 Extrinsic Pathway
By contrast, when blood is exposed to an additional factor in
tissues underneath the damaged endothelium called tissue factor
(TF), factor III, or tissue thromboplastin, the “shortcut” extrinsic
mechanism, which bypasses several steps of the intrinsic
pathway, is triggered.
 Role of calcium

Each pathway requires ionic calcium and involves the activation
of a series of procoagulants, each functioning as an enzyme to
activate the next procoagulant in the sequence.
 The intermediate steps of each pathway cascade toward a
common intermediate, factor X.
 Activated factor X complexes with calcium ions, PF3, and factor V
to form prothrombin activator.
 Slowest step of the blood clotting process, but once formed, the
clot forms in 10 to 15 seconds. 86
 Blood Trauma or
Intrinsic Pathway
contact with collagen

(1) XII Activated XII (XIIa)

(Hageman)
HMW Kininogen, Prekellikerein

(2) XI Activated XI (XIa)

(PTA)
Ca++

(3) IX Activated IX (IXa)

(PTC) VIII (AHF-A)
Thrombin
VIIIa
Ca++
(4)
X Activated X (Xa)
(SPF) Ca++
Thrombin
V
(5)
Va
or PF3 Prothrombin
Activator
Ca++

Prothrombin Thrombin
87
 Extrinsic Pathway
Tissue trauma

(1)

(2) VII Activated VII (VIIa)

(Proconvertin)

Ca++
(3) X Activated X (Xa)
(SPF) Ca++
Thrombin
V

Va
Prothrombin
Activator
Ca++

or PF3
Prothrombin Thrombin

88
 Phase 2: Common Pathway to
Thrombin
 Prothrombin activator catalyzes the
transformation of the plasma protein
prothrombin to the active enzyme
thrombin.
Intrinsic Extrinsic
pathway pathway

Prothrombin
Activator complex

Prothrombin Thrombin
89
 Phase 3: Common Pathway to the Fibrin
Mesh
 Thrombin catalyzes the polymerization of
fibrinogen (another plasma protein made by the
liver).

 Thrombin is a protein enzyme with weak proteolytic

capabilities. It acts on fibrinogen to remove four
low-molecular weight peptides from each molecule
of fibrinogen, forming one molecule of fibrin
monomer.

 Fibrin monomers has the automatic capability to

polymerize with other fibrin monomer molecules to
form fibrin fibers.

 Many fibrin monomer molecules polymerize within

seconds into long fibrin fibers.
90
 Lets watch it
 During early polymerization,
fibrin fibers are held together
by weak non covalent
hydrogen bonding, No cross-
linkage with one another.

 fibrin-stabilizing factor causes

the cross linkage of fibrin
fibers (Released from
platelets entrapped in the
clot).

 Activated by thrombin

 This activated substance

operates as an enzyme to
form covalent bonds between
fibrin monomer molecules, as
well as multiple cross
linkages between adjacent
fibrin fibers.

91
 4-Clot Retraction and Repair
1. Within 30 to 60 minutes, the clot is stabilized further by
a platelet-induced process called clot retraction.
2. Platelets contain contractile proteins (actin and myosin),
and they contract in much the same manner as muscle
cells.
3. As the platelets contract, they pull on the surrounding
fibrin strands, squeezing serum (plasma minus the
clotting proteins) from the mass, compacting the clot
and drawing the ruptured edges of the blood vessel more
closely together.
4. Even as clot retraction is occurring, vessel healing is
taking place.
5. Platelet-derived growth factor (PDGF) released by
platelet degranulation stimulates smooth muscle cells
and fibroblasts to divide and rebuild the wall.
6. As fibroblasts form a connective tissue patch in the
injured area, endothelial cells, stimulated by vascular
endothelial growth factor (VEGF), multiply and restore
the endothelial lining. 92
 Fibrinolysis
 A process called fibrinolysis removes unneeded clots when healing
has occurred.
 Because small clots are formed continually in vessels, this cleanup
is important. Without fibrinolysis, blood vessels would gradually
become completely blocked.
 The critical natural “clot buster” is a fibrin-digesting enzyme called
plasmin, which is produced when the plasma protein plasminogen
is activated.
 Large amounts of plasminogen are incorporated into a forming clot,
where it remains inactive until appropriate signals reach it.
The presence of a clot in and around the blood vessel causes the endothelial
cells to secrete
 tissue plasminogen activator (tPA).
Along with that
 Activated factor XII and

thrombin
released during clotting also serve as plasminogen activators. As a result,
most plasmin activity is confined to the clot, and any plasmin that strays into
the plasma is quickly destroyed by circulating enzymes.
 Fibrinolysis begins within two days and continues slowly over
several days until the clot is finally dissolved.

93
 Factors Limiting Normal Clot Growth
 Normally, two homeostatic mechanisms prevent clots from becoming
unnecessarily large:
• swift removal of clotting factors, and
• inhibition of activated clotting factors.
 Limiting the Activity of Thrombin
 As a clot forms, almost all of the thrombin produced is bound onto the
fibrin threads.
 This is an important safeguard because thrombin also exerts positive
feedback effects on the coagulation process prior to the common
pathway.
• It speed up the production of prothrombin activator by acting through factor
V,
• It also accelerates the earliest steps of the intrinsic pathway by activating
platelets.
 Thus, fibrin effectively acts as an anticoagulant to prevent enlargement
of the clot and prevents thrombin from acting elsewhere.
 Thrombin not bound to fibrin is quickly inactivated by antithrombin III,
a protein present in plasma. It inactivates the protease activity of
thrombin and factors IXa, Xa, XIa and XIIa by forming complexes with
them.
 Heparin, the natural anticoagulant contained in basophil and mast cell
granules, inhibits thrombin by enhancing the activity of antithrombin III.

94
 Disorders of Hemostasis
1. Thromboembolic disorders
• Resulting from conditions that cause undesirable clot formation.
2. Disseminated intravascular coagulation (DIC)
• Involving both wide spread clotting and severe bleeding.
3. Bleeding disorders
• Arising from abnormalities that prevent normal clot formation.
 -Thromboembolic Conditions

A clot that develops and persists in an unbroken blood vessel is
called a thrombus. It may block circulation to the cells beyond
the occlusion and lead to death of those tissues.
• eg coronary thrombosis.

Free Floating thrombus in the bloodstream is called an embolus
(plural: emboli). Casue embolism by obstructing the vessel.
• For example, emboli that become trapped in the lungs (pulmonary
embolisms).
• A cerebral embolism may cause a stroke.

Conditions that roughen the vessel endothelium, like
atherosclerosis or inflammation, cause thromboembolic disease
by allowing platelets to clump.

Slowly flowing blood or blood stasis is another risk factor, eg in
bedridden patients (No quick washing away of clotting factors).95
 1. Disorders of Hemostasis
 Disseminated Intravascular Coagulation
 DIC is a situation in which widespread
clotting occurs in intact blood vessels and
the residual blood becomes unable to clot.
• Blockage of blood flow
• Severe bleeding follows
 DIC is most commonly encountered as a
complication of pregnancy or a result of
septicemia or cancers.

96
 1. Bleeding Disorders
 The most common causes are
 Platelet deficiency (thrombocytopenia)
 Deficiencts of some procoagulants, (which can result from impaired liver
function)
 Hemophilias (certain genetic conditions.)
 Thrombocytopenia  
– A condition in which the number of circulating platelets is deficient,
evidenced by many small purplish blotches, called petechiae (pe-te′ke-e),
on the skin.
– Cause:
• Condition that suppresses or destroys the bone marrow, such as bone marrow
malignancy,
• exposure to ionizing radiation, or certain drugs.
 A platelet count of under 50,000/µl of blood is usually diagnostic for
this condition.
 Impaired Liver Function  
– When the liver is unable to synthesize its usual supply of procoagulants,
abnormal, and often severe, bleeding occurs.
– Cause:
• Vitamin K deficiency (common in newborns or after taking systemic
Antibiotics)
 Destruction of Intestinal flora

Intestinal malabsorption
• Impairment of liver function (as in hepatitis or cirrhosis). 97
 Bleeding Disorders
 Hemophilias   
 The term hemophilia refers to several different hereditary bleeding disorders
that have similar signs and symptoms.
 Hemophilia A, or classical hemophilia,
• Results from a deficiency of factor VIII (antihemophilic factor).
• It accounts for 77% of cases.
 Hemophilia B
• Results from a deficiency of factor IX.
 Both types are X-linked conditions occurring primarily in males.
 Hemophilia C,
 A less severe form of hemophilia seen in both sexes, is due to a lack of factor
XI. The relative mildness of this form, as compared to the A and B forms,
reflects the fact that the procoagulant (factor IX) that factor XI activates may
also be activated by factor VII
 Symptoms:
• Symptoms of hemophilia begin early in life;
• even minor tissue trauma causes prolonged bleeding into tissues that can be life
threatening.
• Commonly, the person’s joints become seriously disabled and painful because of
repeated bleeding into the joint cavities after exercise or trauma.
 Treatment:
• Transfusions of fresh plasma or injections of the appropriate purified clotting factor.
These therapies provide relief for several days but are expensive and inconvenient.

98
 Blood Testing
 Blood Sample

Complete Blood Count (CBC)
• WBC’s (4000-10800 cells/mm3 )
• Plateltes (150,000-400,000 cells/mm3)
• RBC’s (Male: 4.6–5.9, Female: 4.2–5.4 million
cells/mm3 )

Direct measurements
• RCC (Red cells Count)
• Hb Concentration (g/dl)
• Hematocrit (Hct) (Vol of RBC’s / Vol of whole blood)

Calculated from direct measurements
• MCH (Mean Corpuscular Hb Mass / RBC)
• MCV (Mean Corpuscular volume/RBC)
• MCHC (Mean Corpsucular Hb Conc. per Liter (RBC)
99
Blood Testing

100
 Anemias
 Diagnostic Classification
1. Kinetic Approach
• Production vs. destruction or loss
 Reticulocyte Production Index (RPI)

2. Morphological Approach
• Red blood cell size
 Microcytic (Cells Smaller than normal size i.e. MCV< 80 fl)
 Normocytic (Cells Normal sized i.e. MCV = 80-00 fl)
 Macrocytic (Cells bigger than normal size i.e. > 100 fl)
• Concentration of Hb

Hyperchromic (Increased Hb Concentration)

Normochromic (Normal Hb Concentration)

Hypochromic (Decreased Hb Concentration- cells paler
than normal) 101
 Anemias
 Anemia means deficiency of hemoglobin in the blood

Cause
• Too few red blood cells or
• Too little hemoglobin in the cells.
2. Aplastic Anemia
– Anemia due to lack of functioning of Bone Marrow or
bone marrow aplasia. Aplastic anemia patients have
lower counts of all three blood cell types: termed
pancytopenia.
– Causes
• Hereditary
 Congenital hypoplastic anemia (or constitutional aplastic
anemia) refers to a type of aplastic anemia which is
primarily due to a congenital disorder (defects or damage
to a developing fetus).

Examples include:
• Fanconi anemia (Caused by short Stature, Skeletal Abnormalities)
• Diamond-Blackfan anemia (Congenital Erythroid Aplasia-
Characterized by anemia with decreased erythroid progenitors in
bone marrow)
102
 Anemias
• Acquired

Pure Red cell Aplasia (PRCA)

Sideroblastic anemia (Sideroachrestic anemia)1 The body
has iron available, but cannot incorporate it into
hemoglobin
 Myelophthisic anemia2 (Normal marrow space is replaced
by nonhematopoietic or abnormal cells). Cause e.g.
tumors

2. Nutritional Anemia

 Hemolytic Anemia

103