Blood

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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

Pigments (Biluribin)

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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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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

Functions of blood

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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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Albumin StructureR

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Causes of decreased plasma albumin:

Decreased synthesisA. malnutrtionB. malabsorptionC. advanced chronic liver disease

Abnormal distribution or dilutionA. overhydrationB. increased capillary permeability like in septicemia

Abnormal excretion or degradationA. nephrotic syndromeB. burnsC. hemorrhageD. 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.

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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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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

Erythrocytes

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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

Leuckocytes

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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)

ChemotaxisThe 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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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

Production of Leukocytes

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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

Production of Granulocytes

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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 µm3

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

Why count is different? 1

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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.

Erythrocytes (RBCs)

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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

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

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Erythrocytes Production (Erythropoiesis)Erythrocytes Production (Erythropoiesis)

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1. PHSC

2. Myeloid stem cells

3. Proerythroblast (Megaloblasts)

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

5. Polychromatophil Erythroblasts (Intermediate erythroblast or Normoblast)

6. Orhochromatic Erythroblasts (Late Erythroblast or Normoblasts)

7. 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

8. Erythrocytes

Erythrocytes Production (Erythropoiesis)

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Proerythroblast or

pronormoblast

Basophilic erythroblast

or Early

Normoblast

Polychromatophilic (or intermediate)Erythroblast or

Normoblast

DividingPolychromatophilic

Erythroblast orNormoblast

Orthochromatic(Acidophilic) erythroblast

OrLate

Erythroblast

Orthochromatic erythroblast

ExtrudingNucleus

Reticulocyte

Reticulocyte(brilliant cresyl

blue dye) 1

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Factor needed of Erythropoiesis1. 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 synthesis7. Iron & copper Heme synthesis8. Intrinsic factor Absorption of Vit B 129. Hormones

Physiological Variations in RBC count1. Diurnal Variation (During 24 hours)

• 5 % • Lowest - Sleep and early morning hours• Highest - Evening

2. Temperature3. High Altitude4. Hypoxia5. Radiations

• X-rays

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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.

Fate and destruction of RBCs 1

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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 productionRegulation of RBC production

A negative Feed back mechanismA 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)• 99% replaced with adult Hb with in a year of birth.

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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++)

Hemoglobin (Hb)

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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.

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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.

Hb Abnormalities

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Hb AbnormalitiesHb 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% 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.

• 97 % recycled by phagocytes of liver, spleen and bone marrow

FerritinFerritin

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FR=ferrireductase

Daily Iron LossMale: 1mg/dayFemales: 2mg/day

Daily Iron RequirementMale: 1mg/dayFemales: 2mg/day

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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.

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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,

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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.

ABO Blood Group

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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.

ABO Blood Group

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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.

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Missing H-gene so no fucose tranferase so no fucose and no H-antigen thatForms the base for A and B Antigen.

Bombay phenotypeBombay phenotype

No fucose

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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.

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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:• Anit-Rh antibodies given after every Rh+ birth. [RhoGAM]

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Rh Factor

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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.

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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.

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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.

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– 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.

1-Vascular Spasms

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2 - Platelet Plug FormationPlatelets 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, Become sticky.

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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.

2 - Platelet Plug Formation

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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.

3-Coagulation

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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).

3-Coagulation

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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.

Phase 1- Formation of prothrombin Activator

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Intrinsic PathwayIn the slower intrinsic pathway, all factors needed for clotting are present in (intrinsic to) the blood.

Extrinsic PathwayBy 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.

Phase 1- Formation of prothrombin Activator

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Intrinsic PathwayBlood Trauma or

contact with collagen

XII(Hageman)

Activated XII (XIIa)

HMW Kininogen, Prekellikerein

XI(PTA)

Activated XI (XIa)

IX(PTC)

Activated IX (IXa)

Ca++

X(SPF)

Activated X (Xa)

Ca++

VIII (AHF-A)

VIIIaThrombin

Ca++

ThrombinV

Prothrombin Activator

Va

Prothrombin Thrombin

or PF3

Ca++

(1)

(2)

(3)

(4)

(5)

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Extrinsic Pathway

Tissue trauma

VII(Proconvertin)

Activated VII (VIIa)

X(SPF)

Activated X (Xa)

Ca++

Ca++

ThrombinV

Prothrombin Activator

Va


or PF3

Ca++

(1)

(2)

(3)

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Phase 2: Common Pathway to Thrombin

Prothrombin activator catalyzes the transformation of the plasma protein prothrombin to the active enzyme thrombin.

Prothrombin Activator complex


Extrinsic pathway

Intrinsic pathway

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Phase 3: Common Pathway to the Fibrin Mesh

1. Thrombin catalyzes the polymerization of fibrinogen (another plasma protein made by the liver).

2. 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.

3. Fibrin monomers has the automatic capability to polymerize with other fibrin monomer molecules to form fibrin fibers.

4. Many fibrin monomer molecules polymerize within seconds into long fibrin fibers.

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Lets watch it

5. During early polymerization, fibrin fibers are held together by weak non covalent hydrogen bonding, No cross-linkage with one another.

6. fibrin-stabilizing factor causes the cross linkage of fibrin fibers (Released from platelets entrapped in the clot).

7. Activated by thrombin

8. 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.

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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.

4-Clot Retraction and Repair

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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.

Fibrinolysis

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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.

Factors Limiting Normal Clot Growth

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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.

1)-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).

Disorders of Hemostasis

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2. 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.

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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).

3. Bleeding Disorders

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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.

Bleeding Disorders

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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)

Blood Testing

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Blood Testing

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Diagnostic Classification1. 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)

Anemias

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Anemia means deficiency of hemoglobin in the blood Cause

• Too few red blood cells or • Too little hemoglobin in the cells.

1. 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)

Anemias

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• 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

1. Nutritional Anemia

2. Hemolytic Anemia

Anemias

Blood

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