Composition and function of

BLOOD

.

Introduction

A. The internal environment is regulated by the fluids which bathe the cells: 1. Interstitial fluid (IF) is found outside cells and in between the cells 2. Lymph inside lymph vessels 3. Blood within blood vessels B. Hematology is the science which studies blood and blood disorders

Blood Components: Plasma Transports Solutes 
Water, ions, trace elements  Gasses: O2 & CO2  Organic Molecules
Glucose N wastes Proteins Antibodies Hormones

Blood Components: Plasma Transports Solutes

Figure 16-1: Composition of blood

Blood Components: Cells 
Erythrocytes
Red Blood Cells (RBC) O2 & CO2 transport 

White Blood Cells (WBC)
Immune defense Phagocytosis 

Platelets: clotting

Blood Cells and Platelets

Blood 
Fluid connective tissue  Functions include
Transporting dissolved gases, nutrients, hormones, and metabolic wastes Regulating pH and ion composition of interstitial fluids Restricting fluid loss at injury sites Defending the body against toxins and pathogens Regulating body temperature by absorbing and redistributing heat

Figure 19.1 The Composition of Whole Blood

Figure 19.1a

.

Physical Characteristics of Blood

A. Greater viscosity than water (Water s viscosity = 1; Blood s viscosity = 3.0 -4.5) B. Temperature of blood: 38°C (100.4oF) C. pH of 7.35 to 7.45 (Below 7.35 = Acidosis; Above 7.45 = Alkalosis) D. Blood constitutes approximately 8% of body weight E. Blood volume ranges between 4 to 6 liters (for females: 4-5 L; for males: 5-6 L)

Composition of Blood 

Total Blood Volume : 5-6 liters (8% of body weight or 80ml/kg body weight)  Specific Gravity : 1050-1060  Viscosity : 4-5 times that of water  Ph : 7.4 (.05); it is alkaline in nature  In acidosis Ph of blood falls below 7.38 and in alkalosis Ph is more than 7.42

Composition of Blood 

Blood contains plasma & formed elements(cells)  Cells represent 45% of blood volume  Hematocrit represents the percentage of red blood cells in blood (called Packed Cell Volume (PCV)  1. A lower than normal hematocrit is
representative of a condition known as anemia  2. An abnormally high hematocrit is representative of polycythemia 

Hematocrit for males: 40%-54% (47%);

Females: 38%-46% (42%)

Plasma is a clear ,straw colored fluid portion of blood & represents 55% of the total blood volume. It contains 91% water and 9% solids( 1% inorganic molecules ,8% organic molecules) 

The major inorganic 

Of 8% organic

molecules are Na+ ,Ca++, Hco3-, K+ , Mg++, Cu++, Po4-3

molecules  7% are Plasma Proteins  (6.4-8.3 gm%) 

1% are Non Protein

Nitrogenous substances(urea,Uric
acid,Creatinine,Xanthin)

. Blood plasma composition 1. 91.5% water 2. 8.5% solutes a. Proteins (albumins; globulins (alpha, beta, gamma); fibrinogen) b. Nutrients c. Enzymes d. Hormones e. Respiratory gases (oxygen, carbon dioxide) f. Electrolytes (sodium, chloride, potassium) g. Waste products (urea, uric acid, creatinine, H+, etc)

Figure 10.1

Figure 19.1 The Composition of Whole Blood

Figure 19.1b

Figure 19.1 The Composition of Whole Blood

Figure 19.1c

Plasma 

Accounts for 46-63% of blood volume
92% of plasma is water Higher concentration of dissolved oxygen and dissolved proteins than interstitial fluid

Plasma 
Water  Plasma proteins
Albumin (60%)
‚ Osmotic Gradient

Globulins (Ab) Fibrinogen 

Molecules
Electrolytes Nutrients Trace Elements Dissolved gases

Serum = Plasma Clotting factors

‡ Albumin ‡ Globulin‡ Fibrinogen

,

,

,

,

Origin: Liver- Albumin & Fibrinogen Plasma cells, lymphocytes, tissue macrophages- Globulin

Plasma proteins 

more than 90% are synthesized in the liver  Albumins
55% of plasma proteins Responsible for viscosity and osmotic pressure of blood 3-5 gm% (average 4.8gm%)

Plasma Proteins 

Globulin is 38%of plasma Proteins  2-3 gm%(average 2.3gm%)  13%

globulin: 0.79-0.84 gm%  14% globulin : 0.78-0.81 gm%  11% globulin: 0.66-0.70 gm%  A/G ratio :1.7-1 
7% Fibrinogen: 0.3 gm%  Prothrombin: 40 mg % 

Globulins
~38% of plasma proteins Include immunoglobins which attack foreign proteins and pathogens Include transport globulins which bind ions, hormones and other compounds 

Fibrinogen
Converted to fibrin during clotting Removal of fibrinogen leaves serum

Other proteins 
Antithrombin-III  Haptoglobulin  Transferrin  Angiotensinogen  Apolipoprotein B  Clotting factors  Antithrombin C

Plasma proteins 

concentration 65 80 g f l (6.4-8.3gm%)  simple or conjugated (glycoproteins, lipoproteins)  separation:

a) salting-out methods p albumin, globulins, fibrinogen b) electrophoresis p albumin, globulin E1, E2, F, K fractions: K F E2 E1

+

albumin

Elfo fractions of plasma proteins
Fraction
Albumins: albumin pre-albumin (transthyretin)
E1-globulins: thyroxin-binding globulin, transcortin, E1-acid glycoprotein, E1-antitrypsin, E1-lipoprotein (HDL), E1-fetoprotein E2-globulins: haptoglobin, macroglobulin, ceruloplasmin F-globulins: transferrin, hemopexin, lipoprotein (LDL), fibrinogen, C-reactive protein, C3 and C4 components of the complement system K-globulins: IgG, IgM, IgA, IgD, IgE

Rel. amount (%)
52 ± 58 2,4 ± 4,4

c (g)
34 ± 50 2-4

6,1 ± 10,1 8,5 ± 14,5

5±9 6 ± 11

10 ± 21

8 ± 15

Plasma proteins participate in:
1. 2. 3. 4. blood coagulation maintenance of homeostasis (pH, osmotic pressure) defence against infection transport of nutrients metabolites hormones drugs metabolic waste

General properties of plasma proteins 

Most are synthesized in the liver Exception: K-globulins synthesized in plasma cells  Synthesized as pre-proteins on membrane-bound polyribosomes;

then they are subjected to posttranslational modifications in ER and Golgi apparatus 

Almost all of them are glycoproteins

Exception: albumin 
They have characteristic half-life in the circulation (albumin 20

days) 

 Many of them exhibit polymorphism (immunoglobulins,

transferrin )

Functions of Plasma Proteins 
Exert Osmotic Pressure :  Contribution to Blood Viscosity  Role in Coagulation of Blood  Role in Defense Mechanism of Body  Role in maintaining acid base balance of body  Transport Function

Exert osmotic pressure  Plasma proteins can t cross capillary membrane so exert colloidal osmotic pressure of about 25mmHg on capillary membrane. 80% of COP is contributed by Albumin  COP plays an important role in exchange of water between blood & tissue fluid  At arterial end of capillaries, due to high Hydrostatic Pressure than COP there occurs filtration of fluid out in tissues  At venous end of capillaries due to lower HP than COP there occur absorption of fluid from tissues in to vessels

Contribution to Blood Viscosity  Shape of Plasma Proteins Greatly contribute

to Viscosity of Blood.  Fibrinogen & globulins are main contributors due to their asymetrical shape  The Blood Viscosity plays an important role in the maintenance of Blood Pressure by providing resistance to flow of blood .

Role in Coagulation of Blood  The Fibrinogen ,Prothrombin & other

coagulation proteins present in plasma play important role in the coagulation of Blood. 
Whenever ,there is injury to blood vessels, the fibrinogen is converted in to fibrin which form blood clot

Role in Defense 
The Gamma globulins are anti bodies which plays an important role in the immune system meant for defense of the body against the micro-organisms

Acid-Base Balance  Plasma Proteins acts as Buffers  Contribute for 15% buffering capacity of

Blood  PP are amphhoteric in nature means can combine with acids and bases.  In Acidic Ph the NH2 group of the proteins acts as base & accept proton & is converted to NH4  In Alkaline Ph the COOH group of the protein act as acid & can donate a proton & thus become COO-

Transport Function 

PP can combine easily with many substances & play an essential role in their transport  CO2 is transported by PP in the form of carbamino compounds  Thyroxine- is transported by an alpha globuline called TBP  Cortisol is transported by transcortin(mucoprotein)

Transport Function  Vitamins A,D & E are transported by the high and low density lipoproteins.  Bilirubin is transported with Albumin & also with fractions of alpha globulin.  Drugs are transported with Albumin  Ca++ of plasma is 50% bound to proteins for transport.  Cu is bound to ceruloplasmin(alpha globulin) for transport  Free HB in the vessels is bound to haptoglobin & is carried to reticulo-endothelial system

cute phase reactants (APRs) 
Their levels change during acute inflammatory response  APRs concentration changes in: infection surgery injury cancer

Acute phase reactant response

Types of APRs:

Positive
E1-antitrypsin

C-reactive protein (CRP): ~1000fold increase! fibrinogen haptoglobin (HP) C3, C4

Negative
albumin transferrin

ALBUMIN 
Concentration in plasma: 45 gl  b 55% of the total plasma protein  Functions:

maintenance of the osmotic pressure of plasma transport of: ‚ steroid hormones ‚ free fatty acids ‚ bilirubin ‚ drugs (sulfonamides, aspirin) ‚ Ca2+ ‚ Cu2+

Causes of Albumin Deficiency 

Liver diseases (cirrhosis)

decrease in the ratio of

albumin to globulins 
Protein malnutrition  Excessive excretion by kidneys (renal disease)  Mutation causing analbuminemia (affects splicing)

Variations in plasma protein levels
Reduced albumin: (i) Infancy (ii) Pregnancy (iii)Hepatitis (iv) Cirrhosis (v) Nephrosis (vi) burns

Increased globulin: (i) Cirrhosis of liver (ii) Tuberculosis (iii)Lymphatic leukemia Decreased Fibrinogen: (i) Congenital (ii) DIC Increased Fibrinogen: (i) Pregnancy (ii) malaria

TRANSFERRIN 



Concentration in plasma: 3 gl Functions: transport of iron: from catabolism of heme and from food (gut) to the sites where iron is required, i.e. to the bone marrow and other organs 2 moles of Fe3+ per 1 mol of transferrin

Receptor-mediated transferrin endocytosis 

Ferro-transferrin binds to the receptors on the cell surface the complex is internalized into an endosome  In endosomes, iron dissociates from transferrin (enabled by low pH & Fe3+ p Fe2+ reduction) and enters cytoplasm  Iron is delivered to intracellular sites or bound to ferritin (Fe2+ p Fe3+ oxidation and Fe3+ storage)  Apotransferrin, associated with the receptor, returns to the membrane, dissociates from the receptor and reenters plasma.

Causes of transferrin deficiency: 
  

Burns Infections Malignancies Liver and kidney diseases

Cause of relative transferrin excess: excess: 

Iron-deficiency anaemia

FERRITIN 
Intracellular protein; only small portion in plasma  24 subunits surround 3000 - 4500 ions of Fe3+  Function: stores iron that can be called upon for use when needed 

Primary hemochromatosis genetic disorder characterized by

increased absorption of iron from the intestine   accumulated iron damages organs such as the liver, skin, heart, and pancreas. Concentration of ferritin is elevated.

CERULOPLASMIN 

Conc. in plasma: 300 mgl  Functions:

carries 90% of copper in plasma (copper cofactor for a variety of enzymes); 1 molecule binds 6 atoms of copper; binds copper more tightly than albumin that carries other 10% of copper   albumin may be more important in copper transport (donates copper to tissues more readily)

Causes of ceruloplasmin decrease: 
Liver diseases, in particular Wilson´s disease:

genetic disease in which copper fails to be excreted into the bile and accumulates in liver, brain, kidney, and red blood cells cause: mutations in the gene encoding for copper-binding ATPase consequences: ‚ accumulation of copper in liver, brain, kidneys   liver disease, neurologic symptoms ‚ coupling of copper to apoceruloplasmin   low plasma levels of ceruloplasmin

Causes of ceruloplasmin increase: 
Inflammatory states  Carcinomas, leukaemia  Rheumatoid arthritis

HAPTOGLOBIN 
E2- globulin, tetrameric  Exists in 3 polymorphic forms  Functions:

binds free hemoglobin and delivers it to the reticuloendothelial cells complex Hb-Hp is too large to pass through glomerulus   prevention of loss of free Hb
X

free Hb passes through glomeruli, enters tubules and precipitates therein   kidney damage

Causes of Hp increase 
Hp belongs to APRs  
inflammation, infection injury malignancies

Causes of Hp decrease 
Haemolytic anaemia: half-life of Hp = 5 days x of complex Hp-Hb = 90 min (the complex is being rapidly removed from plasma)   Hp levels fall when Hb is constantly being released from red blood cells (as in haemolytic anaemia)

transferrin ferritin ceruloplasmin haptoglobin hemopexin (binds heme and transfers it to the liver)
- act as antioxidants:

remove Fe 2+ and thus prevent the Fenton reaction:

H2O2 + Fe 2+ p Fe 3+ + OH· + OH-

E1- ANTITRYPSIN (E1-antiproteinase) 
Synthesized by hepatocytes and macrophages  Major component ("90 %) of the E1-fraction  Glycoprotein, highly polymorphic  Function: principal plasma inhibitor of serine protease (inhibits

trypsin, elastase) deficiency has a role in emphysema proteolytic damage of the lung methionine involved in AT binding to proteases is oxidized by smoking   AT no longer inhibits proteases   increased proteolytic damage of the lung, particularly devastating in patients with AT-deficiency

Blood Components: Cells 
Erythrocytes
Red Blood Cells (RBC) O2 & CO2 transport 

White Blood Cells (WBC)
Immune defense Phagocytosis 

Platelets: clotting

FORMED ELEMENTS 
Red Blood cell
Are disk-shaped and biconcave; no nucleus; contains hemoglobin Live for about 120 days in males and 110 days for females. Main component is the hemoglobin which is responsible for 98.5% of the oxygen transported in the blood 6.9 7.5 µm in diameter(average=7.2) Function: transports oxygen and carbon dioxide

Red blood cells (RBCs) 
transport oxygen  specialised to do

this Also carry some CO2

Abundance of RBCs 
Erythrocytes account for slightly less than half the blood volume, and 99.9% of the formed elements  Hematocrit measures the percentage of whole blood occupied by formed elements
Commonly referred to as the volume of packed red cells

Structure of RBCs 

  

Biconcave disc, providing a large surface to volume ration Shape allows RBCs to stack, bend and flex RBCs lack organelles Typically degenerate in about 120 days.

Red blood cells specialisations
1) biconcave shape 2) no nucleus p extra space inside 3) contain haemoglobin p the oxygen carrying molecule p 250million molecules / cell

increases the surface area so more oxygen can be carried

Figure 19.2 The Anatomy of Red Blood Cells

Figure 19.2

Red Blood Cell 
Diameter of each RBC is 7.2 µm(range6.97.4µm)  Thickness-in periphery is 2µm and in center is 1µm.  Surface area of each RBC is 120-140µm2  Volume is about 80µm3

Advantages of Biconcave Shape 
It renders the cells quite flexible so that they can pass through capillaries(3.5µm) 

Biconcave shape increases surface area

20-30%. 
Thus it allows easy exchange of gases.  Biconcavity allows considerable alteration in cell volume .So RBCs can withstand considerable changes of Osmotic Pressure & resist hemolysis.

Normal Counts 
At birth RBC count-6-7million/cubic m.m.  In Adult male =5-6.5million/cubic m.m.of blood(average=5.5million/cubic m.m.)  In females =4.5-5.5 million/cubic mm (average=4.8 million/cubic mm )  Clinically a count of 5million/cubic mm is considered as 100%

Variation in size, shape & counts of RBCs 

Variation in size is called anisocytosis:  Microcytosis i.e. occurs in

In size of red blood cells

- iron deficiency anaemia, thalassaemia (MCV<80) Macrocytosis i.e. In size of RBCs seen in Megaloblastic Aneamia (MCV>95)

Hypochromic Microcytic RBC

CWM-20353-Meg.An

Variation in shape is called Poikilocytosis 
Abnormal shapes of RBCs are Crenation or shrinkage of RBCs is seen when they are supended in hypertonic solution.  Spherocyte is a globular shape RBC. It is seen in hereditary disease called spherocytosis. These cells are more fragile.

Her. Spherocytosis: 

Elliptocytes i.e. elliptical shape of RBCs seen in some aneamia

Sickle cell,i.e. crecent shape of RBCs due to presence of HB S

Sickle Cell Disease:

Variations in counts 
Physiological factors  Age- at birth 6-7 million/cmm of blood  Sex- adult females =4.8million/cmm

adult males =5.5million/cmm High Altitude- persons residing at mountains (>10,000feet) have high RBC counts 7million/cmm because of hypoxic stimulation of erythropoisis

Physiological 
After sleep

in RBC count 

In pregnancy- due to haemodilution  At high barometric pressure

Polycythaemia-pathological in RBC count above 7million/cmm 
Primary polycythaemia or polycythaemia vera(PV) malignancy of bone marrow  Secondary polycythaemia-occurs due to state of chronic hypoxia in the body such as: -congenital heart diseases  Chronic respiratory disorder like emphysema  Phosphorus & arsenic poisoning

Polycythemia 
Description:
an increase in the number of circulating erythrocytes and the concentration of hemoglobin in the blood; also known as polycythemia vera, PV, or myeloproliferative red cell disorder, polycythemia can be primary or secondary

Polycythemia 
Etiology and Pathophysiology
a. Primary  Neoplastic stem cell disorder characterized by increased production of RBCs, granulocytes, and platelets  With the over production of erythrocytes, increased blood viscosity results in congestion of blood in tissues, the liver, and spleen  Thrombi form, acidosis develops, and tissue infarction occurs as a result of the diminished circulatory flow of blood caused by the increased viscosity

Polycythemia 
b. Secondary

 Most common form of polycythemia vera  The disturbance is not in the development of red blood cells but in the abnormal increase of erythropoietin, causing excessive erythropoiesis  The increase in red blood cell production caused by increased erythropoietin release is a physiologic response to hypoxia; hypoxia stimulates the release of erythropoietin in the kidney

Polycythemia
 Chronic hypoxic states may be produced by prolonged exposure to high altitudes, pulmonary diseases, hypoventilation, and smoking  The results of an increased RBC production include the increased viscosity of blood, which alters circulatory flow

Hematocrit 
Percent of formed elements

Normal Hematocrit is around 45%, depending on gender

Hematocrit

Packed Cell Volume (PCV) 
Hematocrit represents the percentage of red 

blood cells in blood (called Packed Cell Volume (PCV)

1. A lower than normal hematocrit is representative of a condition known as anemia  2. An abnormally high hematocrit is representative of polycythemia 

Hematocrit for males: 40%-54%

(47%); Females: 38%-46% (42%) 

True Haematocrit = is calculated by multiplying observed haematocrit by 0.98. it is calculated because 2% of plasma is trapped in between the cells.  Body Haematocrit = is calculated by multiplying the observed haematocrit with 0.87. it is calculated because of the fact that haematocrit estimated from venous blood whose haematocrit is greater than the whole body.

MCV (Mean Corpuscular Volume) 
It refers to average volume of single red blood cell.  Normal value=80-100µm3  one cubic micro meter is equal to one
famto letre(fl) 

When MCV is normal=RBCs are termed Normocytes.  When MCV =Microcytes seen in IDA (<80µm3 )  When MCV =Macrocytes seen in Megaloblastic Anaemia (>100µm3 )

MCH(Mean Corpuscular Haemoglobin)  It means average amount of Hb present in

each red blood cell  In IDA - low  HS and Megaloblastic Anaemia -high

MCHC(Mean Corpuscular Haemoglobin Concentration) 
It refers to the amount of Hb expressed as percentage of the volume of a RBC  Normal value= 33.3%(range 3034%)  RBCs with normal value of MCHC are called Normochromic.  MCHC<30% = Hypochromic RBCs seen in IDA,Thalassemia  MCHC can t be more than36% 

So RBCs can t be Hyperchromic  since RBC s can t hold the HB beyond the saturation point.  Whole enzymatic machinery of RBC s after full working can t form HB more than 36% of the volume of a RBC  MCHC has greater clinical importance as it is independent of RBC count & RBC s size  It is simply ratio of MCH/MCV ×100

C.B.C 
Haemoglobin - 15±2.5, 14 ±2.5 - g/dl  PCV - 0.47 ±0.07, 0.42 ±0.05 - l/l (%)
Haematocrit, effective RBC volume 

RBC count - 5.5 ±1, 4.8 ± 1 x1012/l  MCHC - Hb/PCV - 30-36 - g/dl
Hb synthesis within RBC 

MCH - Hb/RBC - 29.5 ± 2.5 pg/l
Average Hb in RBC 

MCV - PCV/RBC- 85 ± 8 - fl

Roulaeux Formation 
Is the tendency of RBC s to pile one over the another like a pile of coins.  Albumin decreases the rouleaux formation, while fibrinogen and globulin & other products of tissue destruction increases it.  This is a reversible phenomina ,but it promotes sedimentation of RBC;s  It does not occur in normal circulation,however within a blood vessel in absence of significant flow & when the blood is taken out the red cells tend to form roulaeux.

ESR(Erythrocyte Sedimentation Rate) 
Is the rate at which the red blood cells sediment(settle-down) when the blood containing an anti coagulant is allowed to stand in a vertically placed tube. It is expressed to m.m. at the end of 1st hour.  Westergren s Method normal value 0-15 mm 1st hour(M) 0-20mm1st hour (F)  Wintrobe s Method: 0-9mm 1st hr(M), 0-20(increase with age)

Measurement of ESR
Put anti-coagulated blood in vertical tube, then RBC will sink slowly for its larger density. ESR is expressed by RBC sinking distance during the first hour. Normal value by Westergreen method: male,0~15mm/h; female,0~20mm/h

ESR 
Has no specific diagnostic value.  However ,raised level of ESR do suggest presence of some chronic inflammatory condition in body  Estimation of ESR is more useful as a prognostic test i.e. to judge the progress of the disease in patients under treatment.

Factors affecting ESR 
Rouleaux formation- = ESR ,fibrinogen & proteins which enters in plasma in inflammatory and neoplastic diseaes favours RF  In size of RBC s=  ESR increases when the viscosity of blood is & vice versa.  Males =ESR is higher then females  Pregnancy=ESR is High  New Born=ESR is low

ESR  When no of RBC s increased the ESR is decreased & when the no of RBC s dereased(as in aneamia) the ESR is increased

Pathological variation of ESR
ESR 
Tuberculosis  Malignancy  Chronic infections  All anaemias except Sickel cell  Collegen diseases.

ESR 
Polycythemia  Decreased fibrinogen level  Sickle Cell Anemia  Allergic Conditions

Cell shrinkage or swelling Isotonic: cell neither shrinks nor swells Hypertonic: cell shrinks (crenation) Hypotonic: cell swells (lysis)

Properties of RBC
3. Osmotic Fragility
The resistance of RBC to hypotonic solution.
0. % 0.8% 

0.46% 0.34%

Glucose Metabolism in RBCs 1- Glycolysis 2- Hexose mono-phosphate shunt (HMPS)

Glycolysis in RBCs
Glucose 2 NAD 1,3bisphosphoglycerate 2 NADH+H 2 ATP 2,3bisphosphoglycerate 2 Lactate Glucose

Importance of glycolysis in red cells: a) Energy production: it is the only pathway that supplies the red cells with ATP. haemolytic anaemia may occur due to an inherited deficiency of glycolytic enzymes mainly pyruvate kinase deficiency. b) Reduction of methaemoglobin: glycolysis provides NADH for reduction of met Hb by NADHCyto.b5 reductase c) In red cells 1,3 bisphosphoglycerate is converted to 2,3 bisphosphoglycerate which binds to oxy Hb and helps release of O2 to tissues.

Importance of HMPS in Red cells:  Red cells are liable for oxidative damage by H2O2 due to their role in O2 transport.  In RBCs, H2O2 can cause both oxidation of iron in haemoglobin (to form methaemoglobin) and lipid peroxidation (increases the cell membrane fragility).  The major role of HMS in red cells is the production of NADPH, which protect these cells from oxidative damage by reduction of glutathione that helps removal of H2O2.

Oxidative Phase Irreversible Regulatory 6 moles of G-6-P
6 H2O 12 NADP

Non- Oxidative Phase Reversible Non-Regulatory 6 moles of Pentose-P

6 CO2

12 NADPH +12 H

6 moles of Pentose-P

5 moles of G-6-P

Role of NADPH+H in reduction of glutathione
Glutathione Reductase
NADPH+H NADP

G-S-S-G

2-GSH

2 GSH +H2O2

Glutathione peroxidase

G-S-S-G + 2 H2O

HAEMOGLOBIN 
     

INTRODUCTION STRUCTURE TYPES SYNTHESIS FATE DERIVATIVES CLINICAL- ANAEMIAS - HAEMOGLOBINOPATHIES - PORPHYRIAS 

Pigment present inside RBCs  Carries O2 and CO2  Responsible for red color of blood  Deficiency is called anaemia

Disadvantages of Free Hb 
increase in blood viscosity, causing a rise in blood pressure.

osmotic pressure of plasma to @ 100mm Hg which interferes with fluid exchange between capillaries & tissue spaces.  Loss in urine (Haemoglobinuria) kidney damage by forming acid haematin.  Taken up & rapidly destroyed by the tissue Macrophase system. 

Normal values
´ In foetus- just before birth the Hb concentration of blood from umbilical cord is =16.5 to 18.5 gm/dl. ´ After birth=23gm% this occurs due to transfusion of cells from placenta to infant & haemoconcentration by reduction of plasma volume ´ At the end of 3 months = 10.5 gm/dl ´ At the end of 1 year

= 12.5gm/dl ´ Adult male =14-18 gm/dl ´ Adult female =12-15.5 gm/dl ´ Clinically 14.8gm% Hb is considered 100% Hb

Hemoglobin
´ Human hemoglobin A, present in adults, consists of four

subunits: two -subunits and two ß-subunits.
´ The - and ß-subunits are homologous and have similar three-

dimensional structures.
´ The capacity of hemoglobin to bind oxygen depends on the

presence of a bound prosthetic group (heme).
´ The heme group is responsible for the distinctive red color of

blood. 

The heme group consists of a protoporphyrin, and

a central iron atom. 
Protoporphyrin is made up of four pyrrole rings

linked by methene bridges to form a tetrapyrrole ring. 
Four methyl groups, two vinyl groups, and two

propionate side chains are attached.

STRUCTURE OF HEMOGLOBIN 
Iron-protoporhyrin-globin forms a subunit  4 subunits join to form a molecule of Hb  Iron is in ferrous ( Fe2+) form  Fe2+ binds 4 pyrrole rings, polypeptide chain and a molecule of O2 

4 pyrroles join to form a ring

called porphyrins  4 polypeptide chains constitute globin  , , and are four important polypeptide chains  chain has 141 a.a  and chain have 146 a.a

GLOBIN (protein part or apoprotein): 

It is a simple protein (histone) which is characterized by its high content of histidine and lysine.  It is composed of four polypeptide chains 2 and 2 chains. 

The -chain contains 141 amino acids and -chain contains 146 amino acids. 
Each -polypeptide chain is folded into 8 right handed helices termed A-H starting from NH2-terminal, 

-subunit is folded into 7 -helices. 

The ratio of haem to globin is 4:1. So each haem moiety is linked to one peptide chain.

Attachment of haem to globin 
One Hb molecule contain 4 units of Haem each attached to one of the 4 polypeptide chains, constituting globin.  Thus one Hb mol. Can carry 4 molecules (8 atoms) of oxygen.  Oxygenation of 1st haem mol. In Hb increases the affinity of second mol. for O2

Attachment of haem to globin 
and    

affinity for O2 in 3rd haem mol. Therefore 4th haem-gr of Hb has highest affinity for O2. This shifting affinity of Hb for O2 result in sigmoid shape of O2-Hb curve. Oxygenation of Hb is very rapid <.01 seconds. Hb at it s normal con. Increases the O2 carying capacity of blood 70 folds

TYPES OF HEMOGLOBIN 
HbA- Adult Type HbF- Fetal Type2 2 2 2 

HbA2- 2% of adult Hb2 2 

Hb gower1 Hb gower2-

2 2 2 2 

HbA- Adult Type- 2 2  It is the main form of normal adult Hb.  It is spheroidal molecule with a

molecular weight of 68000  HbA2- 2% of adult Hb- 2 2 
In Hb A2 10 individual Amino Acids differ from those in beta chain of HbA

III. Fetal haemoglobin = HbF ( 2 2):  It is present normally in newborn and early fetal life and at age of 7 months 90 % of fetal haemoglobin is replaced by adult haemoglobin ( HbA1) - It consists of 2 alpha chains and 2 gamma chains. - In gamma chain there is more than one amino acid different from those in -chain - HbF has a great affinity for O2 under physiological conditions, because -chains do not bind 2,3 DPG well. DPG is responsible for lowering the O2 affinity of Hb and allowing Hb to release O2 at the typical PO2 of tissues.

Hemoglobin Genes and Gene Products

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 ( 2 2 ) ‚ Hb A2 = 2 alpha (141 AA)+ 2 delta (146 AA) chains (2.5%) 1 (
2 2)

(10 AA differ)
133

Fetal Hb
‚ Hb F = 2 alpha (141 AA)+ 2 gamma (146 AA) chains 2 ( ‚ 99% replaced with adult Hb with in a year of birth.
2 2)

(37 AA differ)

Myoglobin and Hemoglobin Structure
oxyMb (MbO2) deoxyMb deoxyHb
E F

O2

F

E

DERIVATIVES OF Hb
1) Oxyhaemoglobin (HbO2) oxygenation reaction combination with Fe 2+ 2) Carbaminohaemoglobin ( CO2Hb) carbon dioxide combines with globin part 3) Carboxyhaemoglobin (COHb) carbon monoxide binds with Fe 2+ { 250 times more affinity than oxygen}

4) Methhaemoglobin ( HHb) oxidized haemoglobin 5) Sulfhaemoglobin 6) Glycated haemoglobin (HbA1c) Glucose attached to terminal Valine in chain. integrated index of Diabetic control over 4 to 6 weeks

Methemoglobinemias:  This is oxidized Hb,  The Fe2+ normally present in heme being replaced by Fe3+,the ability to react as an O2 carrier is lost.  The normal erythrocyte contains small amount of met Hb, formed by spontaneous oxidation of Hb.  Met Hb is normally reconverted to Hb by reducing systems in the RBC, the most important of which is NADH-methemoglobin reductase.

Congenital methemoglobinemias A. Hemoglobin M (Hb-M): It is a congenital condition due to mutation in globin biosynthesis in which distal or proximal histidine is replaced by tyrosine. B. Deficiency of NADH cytochrome b5 methemoglobin reductase system Acquired (toxic) methemoglobinemea Usually arises following the ingestion of large amounts of drugs e.g. phenacetin or the sulphonamides, excess of nitrites or certain oxidizing agents present in the diet.

II. Glycosylated haemoglobin (Hb A1c): - It is modified form of haemoglobin similar to haemoglobin A1 but it contains glucose linked to amino group present on lysyl residues and at the NH2 - terminal ends. The reaction is non enzymatic and its rate depends on the concentration of glucose .It is present in normal value 5% of the total haemoglobin. - This percentage is increased in prediabetic and diabetic patients up to 8-14%. This glycohaemoglobin gives an idea about the blood glucose level during the last three months and is useful in the assessment of diabetic control

FUNCTIONS OF HEMOGLOBIN 
1. Transports O2 from lungs to tissues in the form of oxy-hemoglobin  2. Transports CO2 from tissues to lungs in the form of carbamino-hemoglobin:- 30% of total CO2 transport  3. Acts as a buffer- important in acid-base balance- 6 times more than plasma proteins

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 -ketoglutaric Acid Succinyl Co A (Krebs Cycle) ‚ Globin (polypeptide chain) is synthesized by Ribosomes

141

SYNTHESIS
SUCCINYL Co-A + GLYCINE
ALA synthase

-AMINOLEVUNIC ACID PORPHOBILINOGEN PROTOPORPHYRIN IX

Fe2+
GLOBIN

haem synthase

HAEM

HAEMOGLOBIN

FATE OF HAEMOGLOBIN 
RBCs are destroyed in RES ( mainly spleen and bone marrow) after 120 days of life.  Macrophages phagocytose hemolyse degrade haemoglobin form Bilirubin transport to liver with albumin conjugated and detoxified secreted in bile.

H e o bin a m glo

He am

G bin lo

P to rphy ro po rin

Iro n

C O

A inoa po l m cid o

B e iliv rdin

iro po l n o

B ilirubin

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.

146

HAEMOGLOBINOPATHIES
Sickle cell anaemia: Valine replaces glutamate in the 6th position of chain. Is common in African blacks Confers resistance against malaria Hb crystallizes & takes sickle shape under hypoxic conditions. Increased RBC sequestration

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

Sickle Cell Disease

Hemoglobin S (HbS) 50% Hb present.
Homozygotic HbSS (sickle cell anemia) - HbS = 100% Hb present, Giving Sickle cell disease

HbSA disease - Double heterozygote for HbS and HbA, with intermediate clinical severity. It is called Sickle cell trait

Molecular and cellular changes of hemoglobin S 
Basic abnormality - glutamic acid is replaced

by valine at the sixth position of the F-globin chain.  2 normal E-globin and 2 abnormal F-globin chains forms HbS.  HbS carries O2 normally but begins to form semisolid aggregate structures once O2 is unloaded to the tissues. These HbS aggregates distort RBCs and cause them to lose their normal elasticity.

Anemia - reduced O2 carrying capacity of the blood 
Abnormal hemoglobin in RBCs:
Sickle Cell - one amino acid in the 287 forming the beta chains is wrong.

In low O2 conditions the beta chains form stiff rods which cause RBCs to sickle blocking small vessels.

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.

Thalassaemias: 
The name is derived from the Greek word

thalassa,which means sea.Greek identified this disease present around Mediterranean sea.  They are hereditary hemolytic diseases in which the synthesis of either - or - globin chain is defective.  This decreased rate of synthesis of the globin chains is due to mutation affecting the regulatory gene rather than the structural gene.

A)

- thalassaemias: there are decreased or absent synthesis of -chains of haemoglobin with compensatory increase in the synthesis of other chains. a.Homozygous -chain thalassaemia (thalassaemia major): Incompatible with life, and present as hydrops foetalis usually die in utero , due to complete absence of -chains which are required for synthesis of HbF. b.Heterozygous -chain thalassaemia thalassaemia minor,(trait):

B) - thalassaemias: Synthesis of of -chains is decreased or absent whereas synthesis of -chains is normal and will combine with -chains giving excess of HbA2 ( 2 2) or it may combine with -chains producing excess of HbF ( 2 2). The abnormal haemoglobin do not function as normal haemoglobin - Homozygous (Thalassaemia - major = Cooley's anaemia = Mediterranian sea anaemia): There is complete absence of -globin chain and there is marked increase of HbF. - Heterozygous thalassaemia (Thalassaemia - minor): ´ There is slow rate of synthesis of -globin chain.

Heterozygous thalassaemia (Thalassaemia minor): 

There is slow rate of synthesis of -globin

chain.

Other molecules containing Haem:i) Myoglobin present in muscles - combination with single polypeptide chain. ii) Neuroglobin present in CNS iii) Cytochrome enzymes present in mitochondria iv) Peroxidases

PORPHYRIAS
´ DEF: Inherited disorders involving specific enzymes in heme biosynthetic pathway ´ TYPES: ´ a) Hepatic porphyrias i) ALA dehydratase deficient porphyrias ii) Acute intermittent porphyrias iii) Porphyria cutanea tarda b) Erythropoetic porphyrias i) X-linked sideroblastic anemias ii) Congenital erythropoetic porphyrias

ERYTHROPOIESIS
Dr. Raghuveer Choudhary Assistant Prof of Physiology Dr S.N.M.C, Jodhpur

HEMOPOIESIS: INTRO 
Hemo: Referring to blood cells  Poiesis: ´The development or production

ofµ  The word Hemopoiesis refers to the production & development of all the blood cells:
Erythrocytes: Erythropoiesis Leucocytes: Leucopoiesis Thrombocytes: Thrombopoiesis. 

Begins in the 20th week of life in the

fetal liver & spleen, continues in the bone marrow till young adulthood & beyond! 

It is the process of development, differentiation and maturation of RBCs from primitive stem cells  Monophyletic and polyphyletic theory  Things to learn:- Site, Stages, Duration, Regulating factors, Clinical abnormalities

Sites of erythropoiesis
‡ Mesoblastic stage-

in the yolk sac Starts at 2 weeks of intrauterine life intravascular ‡ Hepatic stage2-7 months Both liver and spleen ‡ Myeloid stage

Myeloid stage
‡ Occurs in bone marrow ‡ Starts at 5 months of fetal life and takes over completely at birth ‡ Red bone marrow of all bones ‡ Late adult life, red marrow of flat bones

SITES OF HEMOPOIESIS 
Active Hemopoietic

marrow is found, in children throughout the:
Axial skeleton:
‚ ‚ ‚ ‚ ‚ Cranium Ribs. Sternum Vertebrae Pelvis

Appendicular skeleton:
‚ Bones of the Upper & Lower limbs 

In Adults active

hemopoietic marrow is found only in:
The axial skeleton The proximal ends of the appendicular skeleton.

In the adult, red blood cells, many white blood cells, and platelets are formed in the bone marrow. In the fetus, blood cells are also formed in the liver and spleen, and in adults such extramedullary hematopoiesis may occur in diseases in which the bone marrow becomes destroyed or fibrosed

In children, blood cells are actively produced in the marrow cavities of all the bones. By age 20, the marrow in the cavities of the long bones, except for the upper humerus and femur, has become inactive . Active cellular marrow is called red marrow; inactive marrow that is infiltrated with fat is called yellow marrow.

The bone marrow is actually one of the largest organs in the body, approaching the size and weight of the liver. It is also one of the most active. Normally, 75% of the cells in the marrow belong to the white blood cell-producing myeloid series and only 25% are maturing red cells, even though there are over 500 times as many red cells in the circulation as there are white cells. This difference in the marrow reflects the fact that the average life span of white cells is short, whereas that of red cells is long.

STEM CELLS 
These cells have extensive proliferative

capacity and also the:

Ability to give rise to new stem cells (Self Renewal) Ability to differentiate into any blood cells lines (Pluripotency) 

They grow and develop in the bone

marrow.  The bone marrow & spleen form a supporting system, called the  ´hemopoietic microenvironmentµ

CLONAL HEMOPOIESIS
PLURIPOTENT STEM CELL MULTIPLICATION COMMITTMENT COMMITTED STEM CELL MULTIPLICATION COMMITTED STEM CELL PROGENITOR CELL

STEM CELL

CFU: COLONY FORMING UNIT

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. There are separate pools of progenitor cells for megakaryocytes, lymphocytes, erythrocytes, eosinophils, and basophils; neutrophils and monocytes arise from a common precursor.

Stem cells
‡ Totipotential stem cells- convert into any tissue type ‡ Pluripotent stem cell- Pluripotent hematopoeitic stem cell ‡ Committed stem cells- CFU E, CFU G, CFU M, etc

PROGENITOR CELLS 
Committed stem cells lose their capacity

for self-renewal.  They become irreversibly committed.  These cells are termed as ´Progenitor cellsµ  They are regulated by certain hormones or substances so that they can:
Proliferate Undergo Maturation.

ERYTHROID PROGENITOR CELLS 
BFU-E: Burst Forming Unit ±

Erythrocyte:
Give rise each to thousands of nucleated erythroid precursor cells, in vitro. Undergo some changes to become the Colony Forming Units-Erythrocyte (CFU-E) Regulator: Burst Promoting Activity (BPA)

ERYTHROID PROGENITOR CELLS 
CFU-E: Colony Forming Unit-

Erythrocyte:
Well differentiated erythroid progenitor cell. Present only in the Red Bone Marrow. Can form upto 64 nucleated erythroid precursor cells. Regulator: Erythropoietin. 

Both these Progenitor cells cannot be

distinguished except by in vitro culture methods.

The HSCs are few in number but are capable of completely replacing the bone marrow when injected into a host whose own bone marrow has been completely destroyed. The HSCs are derived from uncommitted, totipotent stem cells that can be stimulated to form any cell in the body. Adults have a few of these, but they are more readily obtained from the blastocysts of embryos. There is not surprisingly immense interest in stem cell research due to its potential to regenerate diseased tissues

Microenvironment

Stages of erythropoiesis
Pronormoblast Early normoblast Intermediate normoblast Late normoblast Reticulocyte Erythrocyte

Figure 19.6 Stages of RBC Maturation

Figure 19.6

ERYTHROPOIESIS 15-20µm- basophilic cytoplasm, nucleus with nucleoli. 14-17µm-mitosis, basophilic cytoplasm, nucleoli disappears. 10-15µm- POLYCHROMASIA Hb appears, nucleus condenses. 7-10µm- PYKNOTIC Nucleus. Extrusion, Hb is maximum. 7.3µm- Reticulum of basophilic material in the cytoplasm. 7.2µm- Mature red cell with Hb.

Pronormoblast
‡15-20 microns ‡Mitosis present ‡Nucleus with multiple nucleoli ‡Basophilic cytoplasm with polyribosomes ‡No hemoglobin

Basophilic normoblast 
Large nucleus  Basophilic cytoplasm  Active mitosis  Slight reduction in size

Polychromatophilic normoblast 
Chromatin lumps  Hb starts appearing  Reduced mitoses

Orthochromatic normoblast 
Small and pyknotic nucleus  Eosinophilic cytoplasm  Mitoses absent

Reticulocyte 
Reticular nuclear fragments  Nucleus extruded  Slightly larger than RBCs

1.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

Erythrocytes Production (Erythropoiesis)
PHSC
Myeloid Stem cells Hemocytoblasts:
‡Cell size large 20-25 mircon ‡Nucelus large ‡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

199

Erythrocytes Production (Erythropoiesis)
1. 2. 3. 4. 5. 6. 7.

PHSC Myeloid stem cells Proerythroblast Basophilic Erythoroblasts (Early erythroblasts) (early Normoblast) Polychromatophil Erythroblasts (Intermediate erythroblast or Normoblast) Orhochromatic Erythroblasts (Late Erythroblast or Normoblasts) 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
200

8. Erythrocytes

Duration
Differentiation phase: from pronormoblast to reticulocyte phase- 5 days Maturation phase: from reticulocyte to RBC2 days

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
202

Hematopoiesis
Factors affecting erythropoiesis:C) Hormonal factors: i-Androgens: increase erythropoiesis by stimulating the production of erythropoietin from kidney. ii-Thyroid hormones:  Stimulate the metabolism of all body cells including the bone marrow cells, thus, increasing erythropoiesis.  Hypothyroidism is associated with anemia while hyperthyroidism is associated with polycythaemia.

Hematopoiesis
Factors affecting erythropoiesis:C) Hormonal factors: iii-Glucocorticoids: 

Stimulate the general metabolism and also stimulate the bone
marrow to produce more RBCs. 

In Addison¶s disease (hypofunction of adrenal cortex) anemia
present, while in Cushing¶s disease (hyperfunction of adrenal cortex) polycythaemia present.

Hematopoiesis
Factors affecting erythropoiesis:C) Hormonal factors: iv-Pituitary gland: Affects erythropoiesis both directly and indirectly through the action of several hormones. v- Haematopoietic growth factors: Are secreted by lymphocytes, monocytes & macrophages to regulate the proliferation and differentiation of proginator stem cells to produce blood cells.

Hematopoiesis
Factors affecting erythropoiesis:D)-State of liver & bone marrow: i-Liver: Healthy liver is essential for normal erythropoiesis because the liver is the main site for storage of vitamin B12 , folic acid, iron & copper. In chronic liver disease anemia occurs. ii-Bone marrow: When bone marrow is destroyed by ionizing irradiation or drugs, aplastic anemia occurs.

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.
208

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.

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
²

Drop in normal blood oxygen levels may result due to
‡ Reduced number of RBCs
Hemorrhage Excess RBC Destruction High Altitude Lung Diseases Aerobic Exercises
         

‡ Reduced Availability of Oxygen

‡ Increase Tissue demands of Oxygen

210 

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

Regulation of erythropoiesis
1. Tissue Oxygenation

Regulation of Erythropoiesis
Stimulates

CFU ² E

Proerythroblasts ERYTHROPOIETIN Mature Erythrocytes
Decreases

Tissue Oxygenation Factors decreasing:
‚ Hypovolemia
x Anemia

An example of a Negative feed back mechanism

‚ Poor blood flow ‚ Pulmonary Disease

Erythropoietin 
Glycoprotein with 165 amino acids, 4 oligosaccharide chains and molecular weight of 34,000  Production- 85% by peritubular capillary bed interstitial cells(Kidney) and 15% by perivenous hepatocytes( Liver)  Also seen in brain, salivary glands, uterus, oviducts

Factors increasing erythropoietin secretion: (i) Hypoxia (ii) Androgens (iii)Growth Hormone (iv) Catecholamines (v) Prostaglandins Factors inhibiting erythropoietin secretion: (i) Estrogen (ii) Theophylline

Action of Erythropoietin: 1. Formation of Pronormoblast from stem cell 2. Speeds up the differentiation through various stages of erythropoiesis Mechanism of Action: ‡ Formation of ALA synthetase ‡ Activation of Adenylyl Cyclase ‡ Synthesis of transferrin receptors

Erythropoietin receptors: Member of cytokine superfamily Receptor has Tyrosine kinase activity

Maturation factors
Vitamin B12 and Folic acid:  Essential for DNA synthesis (Thymidine triphosphate)  Abnormal and diminished DNA  Failure of division and maturation  Macrocytic / Megaloblastic anemia

Pernicious Anemia
Intrinsic factor of Castle- secreted by oxyntic cells of gastric mucosa Essential for absorption of Vitamin B12 by enteric route

Other Factors 
Cobalt  Copper  Proteins  Vitamin C

Clinical Aspects
Anemias: Reduced RBC count / reduced Hb concentration Polycythemia: Increased RBC count  Polycythemia vera  Secondary polycythemia- due to hypoxia

Anemia 
Anemia means a decrease in hemoglobin content,  or RBCs count,  or both of them below the normal range.  Anemia leads to a decrease in blood ability to transport oxygen to tissue cells.

Anemia 
Types & causes of anemia: I-Blood loss anemia: A-Acute blood loss anemia:  Due to severe hemorrhage.  Plasma volume is replaced rapidly by the fluids present in tissue spaces.  This leads to marked dilution of the blood.  RBCs are replaced within 2-3 weeks.  Sufficient iron gives normocytic cells but insufficient iron will produce microcytic RBCs.

Anemia 
Types & causes of anemia: I-Blood loss anemia: B-Chronic blood loss anemia:  Due to repeated loss of small amounts of blood over a long period e.g.: -Gastrointestinal bleeding (peptic ulcer) -Excessive menstruation. -Hemorrhagic diseases.  Due to depletion in iron stores the newly formed RBCS are microcytic.

Anemia
Types & causes of anemia:
II-Aplastic anemia: It results from destructione of bone marrow.  It may result from: 1-Excessive exposure to x-rays or gamma rays. 2-Chemical toxins e.g. cancer therapy & prolonged exposure to insecticides or benzene. 3-Invasion of bone marrow by cancer cells. 4-Following infection by hepatitis.  Damaged bone marrow don¶t produce any RBCs, so in aplastic anemia RBCS are normocytic.  It is associated with decrease in WBCs & platelets.

Anemia
Types & causes of anemia: III-Hemolytic anemia: It results from increased rate of destruction of RBCs inside the cardiovascular system.  Causes of hemolytic anemia: A-Hereditary: 1-Membrane abnormalities. 2-Enzyme deficiency e.g. G-6-P Dehydrogenase. 3-Hemoglobin abnormalities. B-Acquired: 1-Incompatible blood transfusion. 2-Parasitic infection e.g. malaria. 3-Toxic agents e.g. snake venom & insect poisons. 4-Thermal e.g. several burns.

Anemia
Types & causes of anemia: IV-Dyshemopoietic anemia: Which may be due to: 1-Iron deficiency anemia. 2-Maturation failure (megaloblastic) anemia:a-Vitamin B12 deficiency. b-Folic acid deficiency. 3-Anemia of endocrine disorders. 4-Nutritional anemia. 5-Anemia of renal failure.

Table 16-3: Causes of Anemia

RBC
Biconcave shape increases surface area 20-30%. Readily deform and pass through capillaries. Cytoskeleton is unique. Spectrin is major protein. Hemoglobin. Energy from anaerobic respiration of glucose. (No mitochondria present.) Lifespan is 120 days. Removed by spleen and liver.

Figure 19.4 Sickling in Red Blood Cells

Figure 19.4

Haemoglobin 
gives red blood

cells their colour  can carry up to 4 molecules of O2 
associates and

dissociates with O2 
contains iron

RBC life span and circulation 
Replaced at a rate of approximately 3 million new

blood cells entering the circulation per second.  Replaced before they hemolyze  Components of hemoglobin individually recycled
Heme stripped of iron and converted to biliverdin, then bilirubin 

Iron is recycled by being stored in phagocytes, or

transported throughout the blood stream bound to transferrin

Figure 19.5 Red Blood Cell Turnover

Figure 19.5

C.B.C 
Haemoglobin - 15±2.5, 14 ±2.5 - g/dl  PCV - 0.47 ±0.07, 0.42 ±0.05 - l/l (%)
Haematocrit, effective RBC volume - better 

RBC count - 5.5 ±1, 4.8 ± 1 x1012/l  MCHC - Hb/PCV - 30-36 - g/dl
Hb synthesis within RBC 

MCH - Hb/RBC - 29.5 ± 2.5 pg/l
Average Hb in RBC 

MCV - PCV/RBC 85 ± 8 - fl

Her. Spherocytosis:

Hereditary Elliptocytosis:

Sickle Cell Disease:

Hypochromic Microcytic RBC

CWM-20353-Meg.An

Polycythemia 
Description:
an increase in the number of circulating erythrocytes and the concentration of hemoglobin in the blood; also known as polycythemia vera, PV, or myeloproliferative red cell disorder, polycythemia can be primary or secondary

Polycythemia 
Etiology and Pathophysiology
a. Primary  Neoplastic stem cell disorder characterized by increased production of RBCs, granulocytes, and platelets  With the over production of erythrocytes, increased blood viscosity results in congestion of blood in tissues, the liver, and spleen  Thrombi form, acidosis develops, and tissue infarction occurs as a result of the diminished circulatory flow of blood caused by the increased viscosity

Polycythemia 
b. Secondary

 Most common form of polycythemia vera  The disturbance is not in the development of red blood cells but in the abnormal increase of erythropoietin, causing excessive erythropoiesis  The increase in red blood cell production caused by increased erythropoietin release is a physiologic response to hypoxia; hypoxia stimulates the release of erythropoietin in the kidney

Polycythemia
 Chronic hypoxic states may be produced by prolonged exposure to high altitudes, pulmonary diseases, hypoventilation, and smoking  The results of an increased RBC production include the increased viscosity of blood, which alters circulatory flow

Polycythemia 
Diagnostic and laboratory tests
Elevated hemoglobin and erythrocyte count Decreased MCHC Increased WBC and basophilia Increased platelets Elevated leukocyte alkaline phosphatase Elevated uric acid Elevated cobalamin levels Increased histamine levels Bone marrow examination shows hypercellularity

Polycythemia 
Therapeutic management
Management of the underlying condition (such as COPD) causing the chronic hypoxia Repeated phlebotomy to decrease blood volume; the goal is to keep the hematocrit less than 45 to 48 percent Hydration to decrease blood viscosity

Anemia - reduced O2 carrying capacity of the blood 
Insufficient number of RBCs:
Hemorrhagic - due to blood loss associated with an injury, undiagnosed bleeding ulcer, etc.

Hemolytic - due to blood loss due to transfusion reactions & certain bacterial and parasite infections.

Aplastic - due to destruction or inhibition of red marrow by drugs, ionizing radiation or certain bacterial toxins.

Anemia - reduced O2 carrying capacity of the blood 
Insufficient hemoglobin content in RBCs:
Iron Deficiency - inadequate intake or absorption of iron.

Pernicious - dietary deficiency of Vitamin B12 or inadequate production of intrinsic factor for absorption of Vitamin B12.

Anemia - reduced O2 carrying capacity of the blood 
Abnormal hemoglobin in RBCs:
Sickle Cell - one amino acid in the 287 forming the beta chains is wrong.

In low O2 conditions the beta chains form stiff rods which cause RBCs to sickle blocking small vessels.

Focus on RBCs:

Figure 16-5c: Bone marrow

Focus on RBCs:

Figure 16-7a, b: Bone marrow

Iron Metabolism: Key to Hemoglobin O2 Transport

Figure 16-8: Iron metabolism

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