Physiology of Haemostasis

Physiology of Haemostasis Lecture by:

Usi Sukorini

Department of Clinical Pathology

Faculty of Medicine GMU

Yogyakarta

2008

Vessel walls

The vascular intima

1. Innermost vascular lining
   
2. Endothelial cells (endothelium)
   
3. Supporting the endothelial cells
   
4. Basement membrane composed of collagen surrounds endothelium
   
5. Subendothelial connective tissue
   

• Collagen, fibroblast in veins
  
• Collagen, fibroblast, and smooth muscle cells in arteries
  

The vascular intima in haemostasis

Vascular intima of vessel wall is lined by a layer called endothelial cell

Endothelial cells have a smooth, unbroken surface that promotes the fluid passage of blood and prevents turbulence that may otherwise activate platelet and plasma enzymes

Any harmful local stimulus, be it mechanical or chemical, induces vasoconstriction in arterial or arterioles and initiate hemostasis mechanism

Vessel walls

Thrombogenic surface or Procoagulant properties

Procoagulant properties of vascular intima

First, any harmful local stimulus induces vasocontriction in arteries and arterioles

(endothelin = vasoconstrictor))

The SMCs contract, the vascular lumen narrows or closes, and blood flow to the injured site is minimized

Second, the BM and subendothelial connective tissues of arteries & veins are rich in collagen that binds and activates platelets

Third, ECs secrete von Willebrand factor (vWF), a glycoprotein that is necessary for platelets to adhere to exposed subendothelial collagen

Fourth, upon activation, ECs secrete and coat themselves with P-selectin, an adhesion molecules that promotes platelet and leucocyte binding

ECs also secrete immunoglobulin-like adhesion molecules called ICAMs (intercellular adhesion molecules) and PECAM (platelet endothelial cell adhesion molecules) that promote leucocytes binding

Finally, subendothelial cells, that is, smooth muscle cells and fibroblast, support a constitutive surface protein called tissue factor (TF)

Exposed TF activates the plasma coagulation system through factor VII

While damaged vessels have procoagulant properties, the intact vascular intimal layers prevent intravascular thrombosis by several mechanism

Fibrinolytic properties of vascular intima

ECs support fibrinolysis with 2 secretions: TPA (Tissue Plasminogen Activator) and PAI-1 (Plasminogen Activator Inhibitor-1)

Platelet

The blood platelets are fragments of the cytoplasm of the megakaryocyte, hence they are non-nucleated and formed chiefly in the bone marrow

Platelet activation

Platelet adhesion

Release reaction

Platelet aggregation

Platelet secretion/release reaction

The series of platelet events

Platelet adhere to exposed cellular matrix (collagen) at sites of endothelial injury and become activated

Upon activated, platelets secrete granules (ADP) and synthezise TXA2 (Thromboxane A2)

Platelets also exposed phospholipid complexes important in the intrinsic coagulation pathway

Injured or activated endothelial cells release TF to activate the extrinsic coagulation cascade

Released ADP stimulates formation of primary haemostatic plug, which is eventually converted into a larger definitive secondary plug

Fibrin deposition stabilizes and anchors the aggregated platelets

Screening Test in Hematology

Tourniquet Test/ Rumpel Leede

• Determines capillary fragility, measures the endothelial support function of platelet and the strength of capillary wall
  
• By impeding venous flow to increase capillary pressure by a blood pressure cuff wrapped around the upper arm for a period of time
  

Platelet Count

• Determines the amount of platelet in blood volume
  
• Could be manually or using blood analyzer
  
• Reference range : 150,000-450,000 / uL
  

Platelet morphology

• When examining peripheral blood smears, platelet size and morphology should be observed
  
• Normal platelets are seen to contain a variable number of azurophilic granules concentrated in the center and surrounded by a clear area
  

Bleeding Time

• A sharp skin puncture is made and bleeding time is noted
  
• It measures interaction between platelets and injured vascular endothelium
  
• Reference value : 1-7 minutes
  
• Prolonged values are found in some vascular defects, qualitative and quantitative platelet abnormalities
  
• Purpose:
  
  • to screen patients with platelet dysfunction
    
• Principle:
  
  • The bleeding time is time (in minutes) that it takes for bleeding to cease from a small, superficial wound made under standardized condition
    
  • The bleeding time is mainly affected by primary hemostatic mechanism (platelet number & function), but is also affected by a variety other conditions
    
• Methods of bleeding time
  
• Duke method
  
  • using an ear lobe
    
  • normal range: 1-3 minutes
    
  • the reproducibility is poor
    
  • Ivy method
    
  • the skin of hand surface of forearm is incised
    
  • vein pressure is standardized by pressuring constantly to the upper arm
    
  • normal range: 1-7 minutes
    
  • Template Ivy method
    
  • a template and fixation plate for blade are used instead of a blade make a constant incision
    
  • normal range: 2-7 minutes (with 9 mm incision)
    

Prothrombin Time (PT)

• Purpose:
  
  • The PT assay is used to screen for inherited and acquired abnormalities in the extrinsic (factor VII) and common (factors V, X, protrombin and fibrinogen) pathways
    
  • The PT is also used to monitor the effect of oral anticoagulant therapy
    
  • Principle:
    
  • Clotting is initiated by a commercial tissue factor reagent, called thromboplastin, and calcium are mixed and the clotting time determined.
    
• Normal range (depend on reagent used)
  
  • 10-12 seconds
    
  • 12-14 seconds
    
• When patients are receiving oral anticoagulants, the ratio of the patient's PT to that of the normal control is useful in monitoring this therapy
  

Prothrombin Time (PT)

• Interpretation:
  
  • The common causes of prolonged PT are:
    
    • The administration of oral anticoagulant drugs (vit K antagonist/Warfarin)
      
    • Liver disease
      
    • Vitamin K deficiency
      
    • Disseminated intravascular coagulation
      
    • Deficiency of extrinsic coagulation factors
      
    • Inhibitor to F. VII
      

INR

• Control of oral anticoagulant
  
• Variation in reagent of PT
  
• Standardization of thromboplastin: ISI (International Sensitivity Index)
  

  
   

Activated Partial Thromboplastin Time (APTT)

Purpose:

• The APTT assay is used to detect inherited and acquired coagulation factor deficiency and quality of the intrinsic pathway, to screen for lupus anticoagulant and to monitor heparin therapy
  

Principle:

• The APTT is an assay of the intrinsic and common pathway. A platelet substitute (crude phospholipid), and a surface-activating agent such as micronized silica (to activate factor XII) are added to plasma. This achieves optimal contact activation
  
• Calcium is then added and the clotting time is recorded
  
• The APTT assay measures all factors except factors VII and XIII
  
• Activated Partial Thromboplastin Time (APTT)
  
• Normal range: depending on the reagent used
  
  • 25-45 seconds
    
  • 23-35 seconds
    

 

• The common causes of prolonged APTT are:
  
  • Disseminated intravascular coagulation
    
  • Liver disease
    
  • Massive transfusion with stored blood
    
  • Administration of heparin or contamination with heparin
    
  • A circulating anticoagulant
    
  • Deficiency of a coagulant factor other than factor VII
    
  • Inhibitor to intrinsic factors (e.g. antibody to f. VIII in hemophiliac)
    

Thrombin Time (TT)

• Purpose:
  
  • The TT is used to screen for abnormality in the conversion of fibrinogen to fibrin
    
  • Principle:
    
  • Thrombin is added to plasma and the clotting time measured
    
  • The TT is affected by the concentration & reaction of fibrinogen and the presence of inhibitory substances including fibrinogen/fibrin degradation products (FDP) and heparin
    
• Interpretation: normal 15-19 seconds
  
  • The common causes of prolonged thrombin time are:
    
    • Hypofibrinogenemia
      
    • Increased of FDP
      
    • Presence of heparin
      
    • Dysfibrinogenemia
      

Fibrinogen

• Quantity : fibrinogen concentration
  
• Quality : Thrombin time (TT)
  

Methods of PT, APTT , fibrinogen and TT test

• Manually: tilt tube
  
• Semiautomatic: coagulometer
  
• Fully automatic: coagulometer
  

 

PHARMACOLOGY of DRUGS in HEMOSTASIS DISORDER

Major Drugs

1. ANTICOAGULANTS
   
2. ANTITHROMBOTICS
   
3. THROMBOLYTICS
   
4. HEMOSTATICS
   

• Coagulation is Complex
  
• BLOOD COAGULATION
  
• Disorders of Hemostasis
  
• Vascular disorders
  
  • Scurvy, easy bruising,
    
• Platelet disorders
  
  • Low Number or abnormal function
    
• Coagulation disorders
  
  • Factor deficiency.
    
• Mixed/Consumption: DIC (Disseminated intravascular coagulation)
  
• ANTICOAGULANTS
  
• Parenteral Anticoagulant
  

    * Heparin

• Oral Anticoagulant
  

    * Warfarin

• Heparin
  
• Abundance in liver
  
• Water soluble mucopollysaccharide
  
• Strongly acidic ( because of its content of covalently linked sulphate and carboxylic acid groups)
  
• Prepared from bovine lung and porcine intestinal mucosa or from sheep and whales
  
• Anti Factor-Xa activity and plasma lipolytic activity of porcine intest. muc. heparin is more potent than is that of bovine lung. But, all heparins are biologically equivalent. However, the incidence of thrombocytopenia is lower with porcine intestinal mucosa heparin
  
• Heparinoid
  
• Heparin from mammalian tissue sources is in limited supply
  
• Semisynthetic sulphated polymers that have been prepared from disaccharides composed of D-glucosamine and D-glucuronic acid
  
• Have high anticoagulant and lipolytic activities
  
• Parmacological Properties
  

Action on Blood Coagulation and Antithrombin III :

     * The anticoagulant effect is essentially         immediate

    * Heparin acts indirectly by means of a             plasma cofactor (antithrombin III)

    * Antithrombin III neutralizes several             activated clotting factors

• cont.
  
• Activated clotting factors :
  

    * XII a

    * Kallikrein (activated Fletcher factor)

    * XI a

    * IX a

    * X a

    * II a

    * XIII a

• Heparin Absorption
  
• Crosses membranes poorly because of its polarity and large molecule size
  
• Not absorbed from GI and sublingual sites
  
• Can't passage across placenta and hindered into maternal milk
  
• The deep subcutaneous or intrafat injection site is used when therapy with low doses of heparin is chosen and for treatment of ambulatory patients
  
• Intramuscular injection of heparin should be avoided because large hematomas can form at the site of injection
  
• Miscellaneous Action of Heparin
  
• When added to blood, it doesn't alter routine chemical determination
  
• But, it distorts the morphology of red and white blood cells
  

It has been reported

• to suppress the secretory rate of aldosterone
  
• increase the concentration of free thyroxine in plasma
  
• Inhibit fibrinolytic activators
  
• Retard wound healing
  
• Depress cell-mediated immunity
  
• Accelerate the healing of thermal burns
  
• II. ANTITHROMBOTICS
  
• Aspirin
  
• Dipyridamol
  
• Dextran
  
• Ticlopidine
  
• Clopidogrel
  

    The action of Antithrombotics

• = Platelet aggregation inhibitors
  
• = Suppress platelet function
  
• = Prevent platelet aggregation
  
• III. THROMBOLYTICS
  
• = Fibrinolytics
  
• = Promote the dissolution of thrombi by stimulating the activation of endogenous plasminogen to plasmin (fibrinolysin = a proteolytic enzyme that hydrolyzes fibrin)
  
• STREPTOKINASE
  
• = Interact with and activates plasminogen
  
• = The complex of Streptokinase and plasminogen
  

     has protease activity and catalyzes the conver-     sion of plasminogen to plasmin

• = Following the administration of Strkase. there is a high incidence of bleeding from sites of per- cutaneous trauma and in wounds (because plasmin lyses fibrin in hemostatic plugs and degrades fibrin
  

ogen and factors V and VII)

• ADVERSE REACTION
  
• Fever
  
• Allergic reaction and even anaphylaxis
  

    

ADMINISTRATION

• Intravenously loading dose of 250.000 I.U. over a 30 min. period
  
• Followed by 100.000 I.U. /hour, adjusted according to the thrombin time
  
• Therapy is continued for 24 to 72 hours and must be monitored by the thrombin time
  
• UROKINASE
  
• = Originally isolated from human urine
  
• = Prepared from cultures of human renal cell
  
• = Contraindicated in children or in patients with any kind of healing wound, recent trauma visceral or intracranial malignancy, pregnancy or recent cerebrovascular accident
  
• = IV loading dose 4400 IU/kg over a period of 10 minutes. Followed by a continous infusion of 4400 IU/kg/hour for 12 hours and then by heparin or oral anticoagulants.
  

    Thrombin time should be evaluated before heparin is given

• IV. HEMOSTATICS
  
• 1. Absorbable hemostatics
  

    = Arrest bleeding when applied directly to denuded     or bleeding surfaces:

         * by formation of an artificial clot

         * by providing a mechanical matrix that facili-     tates clotting

    = Absorbed from the site of application after periods     of time

    = Used to control oozing from minute vessels

    = Will not effective combat bleeding from arteries     or veins

• 2. Orally and parenterally hemostatics
  
• Vitamin K
  

    = Coagulation vitamin

    = A dietary principle essential for the normal bio –

        synthesis of several factors required for clotting     of blood

    = Fat soluble substance

    = Synthezised by the bacteria in the intestinal tract

    = In plants : is concentrated in the chloroplast of     plant leaves and in many vegetable oils

• Hemostasis
  
• Clot retraction: platelets contract and pull the torn edges of the damaged vessel closer together.
  
• Fibrinolysis: here the clot gradually dissolves through the action of plasmin, the activated form of circulating plasminogen.
  
• Manipulation of hemostasis: clotting may be prevented by administering drugs that depress the clotting response or dissolve existing clots. Important anticoagulant drugs include heparin, dicumarol, coumadin, streptokinase, urokinase and aspirin.
  
• Disorders of hemostasis
  
• Thromoboembolytic disorders: undesirable clot formation
  
• Thrombocytopenia-a deficit of platelets, causes spontaneous bleeding. Hemophilia. Liver disease can cause this because the liver manufactures many of the coagulation proteins.
  
• ABSORBABLE HEMOSTATICS
  
• Absorbable gelatin sponge
  

    = Sterile, absorbable, water insoluble, gelatin base     sponge

    = Used for the control of capillary oozing and frank     hemorrhage, particularly from highly vascular areas that     are difficult to suture

    = Before use, it is frequently moistened with sterile     isotonic sodium chloride solution or with thrombin     solution

    = When implanted in tissues it is absorbed completely     in 4     to 6 weeks without inducing excessive formation of scar     tissue

 

• Thrombin
  
• = Applied topically to control capillary oozing in     operation procedures
  
• = To shorten the duration of bleeding from     punctured sites in heparinized patients (e.g. after     hemodialysis)
  
• = Thrombin should never be injected, because of the     possibility of thrombosis and death
  
• = Thrombin is dusted as a powder , applied as a     solution or combined with a suitable sponge     matrix (e. g. , absorbable gelatin sponge)
  
• VITAMIN K
  
• Physiological function in animals and man:
  

    *To promote the hepatic biosynthesis of     #prothrombin (factor II)

        #proconvertin (factor VII)

        #plasma thromboplastin component (PTC,             Christmas factor, factor IX)

        #Stuart factor (factor X)

• Human requirement : 0.5 - 1µg/kg of BW/day
  
• PREPARATIONS of VITAMIN K
  
• Phytonadione (phylloquinone, vitamin K1)
  

    Toxicity:

        Rapid intravenous administration has produced     flushing, dyspnea, chest pains, cardiovascular     colapse and rarely death

• Menadione (vitamin K3)
  

    Toxicity:

        *Large doses of menadione and its derivatives     irritating to the skin and the respiratory tract.     *In new born (especially premature infants), pro-

        duce hemolytic anaemia, hyperbilirubinemia,     kern icterus.

• Menadione also can induce hemolysis in individuals who are genetically deficient in glucose-6-phosphate dehydrogenase (G6PD)
  
• ! In patients who have severe hepatic disease, the administration of large doses of menadione or phytonadione may further depress function of the liver
  

 

Hematology Lecture from Department of pharmacology Faculty of Medicine Gadjah Mada University

 

The Clinical Aspect Of Anemia

• THE CLINICAL ASPECT OF ANEMIA
  
• DEFINITION of ANEMIA
  

Normally defined as haemoglobin concent less than :

13.5 g/dl in adult male

11.5 g/dl in adult female

Children : Newborn Hb 15-21 g/dl

        3 month Hb 9.5-12.5 g/dl

        1 year-puberty Hb 11.0-13.5 g/dl

• INTRODUCTION — Although anemia can be defined as a reduced number of circulating red blood cells (ie, a reduced red blood cell volume), such studies are not practical, cost-effective, or generally available.
  
• As a result, anemia has been defined as a reduction in one or more of the major red blood cell (RBC) measurements: hemoglobin concentration, hematocrit, or RBC count
  
• Hb concentration (HGB) measures the concentration of the major oxygen-carrying pigment in whole blood. Values may be expressed as grams of hemoglobin per 100 mL of whole blood (g/dL) or per liter of blood (g/L)
  
• Hematocrit (HCT) is the % of a sample of whole blood occupied by intact RBC
  
• RBC count is the number of red blood cells contained in a specified volume of whole blood, usually expressed as millions of red blood cells per microL of whole blood.
  
• Normal range — One set of "normal ranges" (95 percent confidence limits) for HGB, HCT, and RBC count is shown in the table .
  
• If anemia is defined as values which are more than two standard deviations (SD) below the mean, then, by using these ranges, a HGB <13.5 g/dL or a HCT <41.0 percent represents anemia in men and a value <12.0 g/dL or <36.0 percent, respectively, represents anemia in women.
  
• Normal ranges other than the above have been proposed: Other authors have proposed diffrent lower limits of normal, ranging from 13.0 - 14.2 g/dL for men & 11.6 to 12.3 g/dL for women [1].
  
• WHO criteria for anemia in men and women are <13 and <12 g/dL, respectively [2].
  
• Other lower limits according to sex, age, and race, based on data from NHANES III and Scripps-Kaiser studies, have been proposed [1]. These values are as low as 12.7 g/dL for black men >60 years of age and 11.5 g/dL for black women >20 years of age.
  
• HEMOGLOBIN
  
• The Hb protein and its nonprotein iron-containing porphyrin cofactor, heme, are so central to the evolution of our understanding of blood diseases that the field of hematology takes name from the molecules.
  
• The processes of the globin proteins and heme biosynthetic pathway occurred throughout evolution.
  
• Understanding of the Hb molecule underlies the pathophysiology of many of the red cell diseases.
  
• INTRODUCTION- HEMATOPOIESIS
  
• Hematopoiesis is difined as the process by which pluripotent hematopoietic stem cells both self-renew and differentiate into all of th specialized circulating blood cells, including white blood cells, red blood cells, and platelets.
  
• Some factors such as a humoral factor was originally favored as containing a critical stimulatory factor.
  
• KEY POINTS- HEMATOPOIESIS
  
• There is ordered progression of hematopoietic development during ontogeny: blood elements are first produced by precursor cells in the yolk sac, then in the fetal liver, and finnally in the bone marrow.
  
• A wide variety of informative experimental assays exits for hematopoietic stem and progenitor cells. Each assay has limitations. The only true assay for long-term repopulating stem cells is reconstitution of hematopoiesis in vivo.
  
• Hematopoietic stem cells can be defined by the expression pattern of spesific cells surface proteins, cell cycle quiescence, and telomerase activity.
  
• Hematopoiesis occurs in a specialized bone marrow microenvironment, composed of cellular and cellular elements critical to localization and control of blood cell production.
  
• Hematopoietic … count
  
• The processes of stem cell mobilization and homing are governed by modulation of interactions between primitive hematopoietic cells and their microenvironment.
  
• Populations of nonhematopoietic stem cells in the bone marrow, including mesenchymal stem cells, are capable of generating bone, cartilage, and other tissues, along with more undifferentiated cell populations that potentially contribute to a wide variety of adult tissues.
  
• THE RBC LIFE CYCLE
  
• Overview — Erythropoiesis in the adult takes place within the bone marrow under the influence of the stromal framework, cytokines, and the erythroid specific growth factor, erythropoietin (EPO).
  
• EPO is a true endocrine hormone produced in the kidney by cells that sense the adequacy of tissue oxygenation relative to the individual's metabolic activity (show figure 2).
  
• EPO enhances the growth and differentiation of the two erythroid progenitors: burst forming units-erythroid (BFU-E) and colony forming units-erythroid (CFU-E) into normoblasts of increasing maturity.
  
• When the normoblast extrudes its nucleus to form a red blood cell, it still has a ribosomal network which, when stained supravitally, identifies it as a reticulocyte, a cell still capable of a limited amount of hemoglobin and protein synthesis
  
• Subtances needed for erythropoiesis
  
• 1. Metal : Fe, Mg, Co
  
• 2. Vitamins : B1, B6, B12, riboflavin, panthothenic acid, Folate, Vit C, Vit E
  
• Amino acids
  
• Hormones: erythropoietin, androgens, thyroxine
  
• Main function Hb :
  
• Carry oxygen® the tissue
  
• Return carbon dioxide (CO₂) from the tissue to the lung.
  
• PATHOPHYSIOLOGY — Most patients with breathing discomfort can be categorized into one of two groups: respiratory system dyspnea or cardiovascular system dyspnea.
  
• Respiratory system dyspnea includes discomfort related to disorders of the central controller, the ventilatory pump, and the gas exchanger.
  
• While cardiovascular system dyspnea includes cardiac diseases (eg, acute ischemia, systolic dysfunction, valvular disorders, pericardial diseases), anemia, and deconditioning.
  
• Anemia — Anemia can severely impair oxygen delivery because the bulk of oxygen carried in the blood is hemoglobin-bound. Nevertheless, the exact mechanism by which anemia produces dyspnea is unknown.
  
• To the extent that the local pH of metabolically active cells decreases due to the inability to sustain aerobic metabolism, there may be stimulation of "ergoreceptors" which are believed to be located in the muscles and which respond to such changes in the microenvironment of the cell
  
• Anemia also leads to increased cardiac output, which may necessitate elevated left ventricular volume and pulmonary vascular pressures. However, the quality of dyspnea is usually quite different in these two clinical situations.
  
• IV. Marrow examination
  A. Aspirate
  1. E/G ratioa
  2. Cell morphology
  3. Iron stain
  B. Biopsy
  1. Cellularity
  2. Morphology
  

E. Cell morphology

1. Cell size

2. Hemoglobin content

3. Anisocytosis

4. Poikilocytosis

5. Polychromasia

II. Reticulocyte count

III. Iron supply studies

A. Serum iron

B. Total iron-binding capacity

C. Serum ferritin, marrow iron stain

• CAUSES OF ANEMIA — There are two general approaches one can use to help identify the cause of anemia:
  
• A kinetic approach, addressing the mechanism(s) responsible for the fall in hemoglobin concentration
  
• A morphologic approach categorizing anemias via alterations in RBC size (ie, mean corpuscular volume) and the reticulocyte response
  
• Kinetic approach — Anemia can be caused by one or more of three independent mechanisms:
  
• Decreased RBC production,
  
• Increased RBC destruction,
  
• Blood loss
  
• Decreased RBC production —The more common causes for reduced (effective) RBC production include:
  
• Lack of nutrients, such as iron, B12, or folate. This can be due to dietary lack, malabsorption (eg, pernicious anemia, sprue), or blood loss (iron deficiency)
  
• Bone marrow disorders (eg, aplastic anemia, pure RBC aplasia, myelodysplasia, tumor infiltration)
  
• Bone marrow suppression (eg, drugs, chemotherapy, irradiation).
  
• Low levels of trophic hormones which stimulate RBC production, such as EPO (eg, chronic renal failure), thyroid hormone (eg, hypothyroidism), and androgens (eg, hypogonadism).
  
• The anemia of chronic disease/inflammation, associated with infectious, inflammatory, or malignant disorders, is characterized by reduced availability of iron due to decreased absorption from the gastrointestinal tract and decreased release from macrophages, a relative reduction in erythropoietin levels.
  
• Increased RBC destruction — A RBC life span below 100 days is the operational definition of hemolysis
  
• Hemolytic anemia will ensue when the bone marrow is unable to keep up with the need to replace more than about 5 percent of the RBC mass per day, corresponding to a RBC survival of about 20 days.
  
• Examples include Inherited hemolytic anemias (eg, hereditary spherocytosis, sickle cell disease, thalassemia major) Acquired hemolytic anemias (eg, Coombs'-positive autoimmune hemolytic anemia, thrombotic thrombocytopenic purpura-hemolytic uremic syndrome, malaria)
  
• Morphologic approach — The causes of anemia can also be classified according to measurement of RBC size, as seen on the blood smear and as reported by automatic cell counter . The normal RBC has a volume of 80 to 96 femtoliters (fL, 10)
  
• The anemia is first classified via the mean corpuscular volume (MCV), which is part of the CBC (show algorithm 1): Microcytic anemias are associated with an MCV below 80 fL. The most commonly seen causes are iron deficiency thalassemia, and the anemia of chronic disease (see "Microcytic anemia" above and see "Evaluation for iron deficiency" above).
  
• Macrocytic anemias are characterized by an MCV above 100 fL The most common causes include alcoholism, liver disease, folic acid and vitamin B12 deficiency, and myelodysplasia. (See "Macrocytosis", section on Evaluation).
  
• The MCV is between 80 and 100 fL in patients with normocytic anemia or would raise suspicion of an acute or chronic hemolytic state (eg, spherocytes, sickle forms, ovalocytes).
  
• Therapy
  

Mild to moderate ® Tx underlying Disease

( Hb levels greader than 9 -10 g/dl)

Severe :

Transfusion support

Erythropoetin Therapy

• OTHER HYPOPROLIFERATIVE ANEMIAS IN SYSTEMIC DISEASE
  

By Ibnu Purwanto

• Approach Anemia
  
• Introduction
  
• Anemia is common in patient :
  
• Acute & chronic inflammatory disease, Renal inssufficiency
  
• Hypothyroidism
  
• There is an apparent failure erythropoetin stimulation of the marrow
  
• Needed skill in evaluating patient with a hyproliferative
  
• The of erythropoetin production
  
• Anemia of chronic inflamatory states
  (The Anemia of chronic Disease)
  
• When the inflamatory states is present®for a long period® manifestasion of iron Deffisien
  
• The smear because mild microcytic (MCV: 75-85%)
  
• Hypochromic
  
• Hb falls to levels below 10 gr%
  
• Reticulosit ¯
  
• Low serum iron
  
• Ion TIBC
  
• Ferritin normal/ elevated
  
• Active Rheumatoid Artritis
  
• Hb level range 8 -12 g/dL
  
• Hematocrit range 25 -35%
  
• The severity of the anemia »IL1 level
  
• AIDS
  
• The Anemia of renal disease
  
• Characterized :
  
• Normocytic normocromic morphology
  
• MCV normal
  
• Reticolusit ¯
  
• The severity anemia » with severity of the renal failure
  
• Acute loss of renal functions as acute tubuler necrosis ® as associated with severe anemia (Hb level below 7 gr/dL)
  
• The serum iron , TIBC, Ferritin® level normal.
  
• Hypomethabolic states
  
• Protein Deprivation
  
• Endocrine deficiency Status
  
• Protein Deprivation
  
• The hypoproliferative anemi of protein deprivation is mild ® 1 to 3 g/dl reduction in Hb level.
  
• Poor protein nutritions as a possible cause of hypoproliferative anemi in elderly.
  
• Endocrine deficiency Status
  
• Diagnosis of Hypoproliferative Anemia
  

STAGES OF IRON DEFICIENCY

These can be divided into three stages.

1. Negative iron balance
   
2. Blood loss,
   
3. Pregnancy
   
4. Rapid growth spurts in the adolescent
   
5. Inadequate dietary iron intake.
   

Lecture by:

dr.Ibnu Purwanto,SpPD-KHOM

Sub division of Haematology & Medical Oncology

Department of Internal Medicine

Medical Faculty of Gadjah Mada University /

Sardjito Hospital

Jogjakarta

 

 

Erythrocytes Metabolism

• Erythrocytes metabolism
  

1. Introduction

2. Cytoskeletal Proteins

3. Metabolism and clinical correlation

4 Synthesis of hemoglobin

5. Porphyrias

6. Catabolism of hemoglobin

 

• Introduction
  

Although devoid of nucleus and intracellular

organelles, the red cell:

- squeezed through small capillary beds, buffeted by high shear forces in heart,

- subjected to sequestration,

- static

- substrate deprivation in spleen,

- has a life-span approximating four month

- maintains its shape, pH, ionic equilibrium, protects hemoglobin integrity and function by virtue of its membrane- cytoskeletal structure and its metabolic activity

• Cytoskeletal proteins membrane
  

- Inner surface of the membrane consist 5 principal proteins:

1. Spectrins 2. Actin 3. Ankyrin 4. Bands 4.1

5. Bands 4.2

- Other membrane proteins serve transport functions

1. Non-polar substances enter RBCs by diffusion

2. Polar solutes enter at special transport sites

• Glucose
  
  

- enters the cell by facilitated diffusion mediated by the glucose transporter (bands 4.5) because the glucose transporter can supply much more glucose than is required by the glycolytic pathway

- glucose is not rate limiting in normal red cell

metabolism

Na⁺ and K⁺

- The concentrations Na⁺ and K⁺ within the red cell

are regulted by the Na⁺/K⁺ ATPase pump

- The intracellular conc. of these cations approximate

130 mM of K⁺ and 8mM of Na⁺ and in the plasma about 140 mM of Na⁺ and 4 mM of K⁺

• continued
  

- Passive diffusion of the Na⁺ and K⁺ by the active transport mechanism, the cell must continuously move Na⁺ out and K⁺ in by way of the Na⁺/K⁺ ATPase powered pump

Ca²⁺ and Mg²⁺

- Ca²⁺ and Mg²⁺ con. are regulated by the Ca²⁺/Mg²⁺ ATPase pump.

- Band 3 both links the lipid bilayer to the cytoskeleton and functions as an anion exchanger for chloride-bicarbonate exchange

Glycosylphosphatidylinositol (GPI)

- embedded in the outer leaflet of the bilayer is a phospholipid anchor exposed hydrophilic protein

 

• continued
  

- There are two complement - regulatory proteins

1. Decay accelerating factor (DAF): to protects the cell against complement- induced lysis

2. Membrane inhibitor of reactive lysis (MIRL = CD59): to protects against complement-mediated by binding to C8

- Defect in GPI synthesis, the red cell becomes vulnerable to complement-induced lysis

- Defect in GPI synthesis can be caused by somatic mutation in a totipotent hematopoietic stem cell

• Erythrocytes Metabolism
  

- red cell is dependent on a continuous of glucose for it energy requirement

- is supplied by glycolysis, the pathway begins with facilitated insulin-dependent, uptake glucose and the production of lactate

- the role glycolysis in supporting the energy-requiring housekeeping duties of membrane maintenance and osmotic stability, methemoglobin reduction, regulation of hemoglobin function,

- In normal red cell, 90% of glucose follow the an aerobic pathway to lactate and 10% flows through the aerobic HMPS.

• The function energy in red cell
  

- is required to maintain

* cationic balance. shape and membrane integrity of cells

* prevent oxidative damage

* maintain hemoglobin in its un oxidized functional deoxy-form

* provide 2,3 BPG - a facilitator of hemoglobin function

- The function are made possible through:

1. Generation of energy in the form of ATP

2. Generation of NADPH and reduced glutathione (GSH) in

HMPS

3. The methemoglobin reduction NADH system

4. The Rapoport-Luebering shunt that produces 2,3

BPG

 

 

• Anaerobic glycolysis
  

- The final product is lactate because the latter reaction, by the enzyme lactic dehydrogenase (LDH) converts pyruvat to lactate

- In this reaction only two molecules ATP are produced per mol of glucose consumed

The Hexose Monophosphate Shunt

(aerobic glycolysis)

- The role of the reactions to product NADPH and reduced glutathione (GSH) under condition stress, to protect the cell membrane and cell contents against oxidative damage

- The key enzyme of HMPS is glucose 6-PO4 dehydrogenase (G6PD) in the production of NADPH

- NADPH is use to reduction of oxidized

glutathione (GSSG) to be reduced form (GSH)

- GSH is an essential intracellular reducing agent

that protects cell against oxidant injury

 

Rapoport-Luebering Shunt

- The role of the shunt reaction

* produces 2,3-BPG which is a regulator of hemoglobin oxygenation-deoxygenation and modulates ATP synthesis

 

- The BPG mutase converts 1,3-BPG to 2,3-BPG, while BPG phosphatase removes the phosphate at the 2 position of 2,3-BPG, and return 3-PG to glycolytic pathway has two primary roles:

* when 1,3-BPG converted directly to 3-PG

the high energy phosphate from the 1

position generates ATP

* In the convertion of 1.3-BPG to the lower

energy 2.3-BPG through the action of

BPG mutase, no ATP is form.

• Methemoglobin reduction
  

- is formed under condition the oxidation of heme Fe2⁺ to Fe3⁺

- can not transport O2

- deoxyhemoglobin is essential for transport in O2

- methemoglobin is converted to deoxyhemoglobin

is mediated through the transfer of an electron

from NADH to the iron atom of heme via cyt. B5 and cyt. B5 reductase

- NADH is generated in convertion of G-3P to 1,3-BPG by G 3-P dehydrogenase

• Clinical correlation
  

Defect in red cell metabolism

- changes in lipid content or cholesterol/phospholipid ratios with expansion of the lipid bilayer to cell volume is reflected in changes of red cell morphology frequently in patients with liver diseases

- hereditary defects, manifest by structurally altered or unstable glycolytic enzymes, occur in both the anaerobic and aerobic pathways.

- produce varying degrees of shortened red cell life-span - congenital jaundice, splenomegaly and cholithiasis. The most common hereditary enzyme defect of red cell are PK and G6PD deficiencies

• G6PD deficiency
  

- result s in decreased NADPH synthesis with inability to maintain GSH levels the oxidants damage the red cell membrane, oxidize heme to methemoglobin, and denature globin which precipitates on the membrane as Heinz bodies

- the membrane changes reduce deformability, increase cation leakage, and result in a fragile cell

hemolysis with hemoglobinuria

- Methemoglobinemia

- due to a deficiency of NADH-cytochrome b5

reductase (methemoglobin reductase)

• Hemoglobin
  

- hemoglobin is protein constitutes 90% of the dry weigh of erythrocytes serves as oxygen transporting

- also plays a vital role in the transport of carbon dioxide and hydrogen ion

- the capacity of hemoglobin to bind oxygen depend on the presence of non polypeptide unit a heme group

- is composed by heme and globin

- globin is polypeptides synthesized in erythropoitic

cells

- heme, a cyclic tetrapyrrole that imparts a red color

• Synthesis of heme
  

- is synthesized in living cell , most mammalia cell with the exception of nature erythrocytes, from from succinyl-CoA & glicin

- succinyl-CoA is dirived from reaction:

1. The citric acid cycle in mitochondria

2. Methyl malonyl to succinyl-CoA by coenzyme deoxyadenosylcobalamin (active form vit. B12)

- in the reaction to "active" glicin, is pyridoxal phosphate necessary

- there are 7 enzymes for heme synthesis of heme

1. ALA synthase (d-amino levulinic acid synthethase)

- is key regulatory enzyme in heme biosynthesis, act as a negative regulator of the synthesis of ALA synthase

- to be the role-controlling enzyme in porphyrin biosynthesis in mammalia liver

- the reaction by this enzyme occur in the mitichondria

- many xenobiotics, when administrated to humans

hepatic ALA synthase (xenobiotics is metabolized by a system in liver that utilizes a specific hemoprotein: cytochrome P-450)

- during the metabolism of xenobiotics utilization of heme in turn diminishes the intracellular heme concentration ALA-synthase heme synyhesis

- the factor affect the induction ALA-synthase in the liver:

a. Glucose: prevent the induction of ALA-sinthase

b. Iron : in chelated form exerts a synergistic

effect on induction of hepatic ALA-synthase

c. Steroid : permissive role in the drug-mediated

derepression of ALA-synthase in vivo

d. Hematin: prevent the drug-mediated derepression

of ALA-synthase in vivo

2. ALA-dehytratase (ALA-dehydrase)

- is a Zn -containing enzymes and is senzitive to inhibition by lead, because lead combine with SH groups in enzyme ALA-dehydrase

- the reaction occurs in the cytosol

3. Uroporohyrinogen I synthase (porphobilinogen deaminase

- the reaction occurs in the cytosol

4. Uroporphyrinogen III cosynthase

- the reaction occurs in cytosol

5. Uroporphirinogen decarboxylase

- the reaction occurs in cytosol

6. Coproporphirinogen oxidase

- the reaction occurs in mitochondria

7. Protoporphyrinogen oxidase

- the reaction occurs in mitochondria

8. Ferrochelatase = heme synthase

- the reaction occurs in mitochondria, can be inhibited by lead, because lead combine with SH-group in ferrochelatase

- regulation of heme synthesis occurs at ALA-synthase by a repression-dereprission mechanism mediated by heme and its hypothetical aporepressor

- lesions of enzymes 2 - 8 cause the porphyrias

• Pophyrias:
  - group of inborn errors of metabolism due to
  mutation in genes direcly the synthesis of the
  enzymes involved in the biosynthesis of heme
  - in general, the porphyrias describe are inherited in
  an autosomal manner
  - can be classified on the basis the organs or cells
  that are most affected
  - these are generally organs or cells in synthesis of
  heme is particularly active:
  * the bone marrow: synthesis hemoglobin
  * liver is active in the synthesis of another hemo-
  protein: cytochrome P-450 classification
  of the porphyrias is to designate as erythropoietic,
  hepatic, and erythrohepatic
  
• Globin synthesis
  - globin polipeptides are synthesized in erythropoietic cells, defects in globin chain synthesis in thalassemias
  

Catabolismm of hemoglobin

- under physiologic condition in human adult, 1-2 X 10 degree 8 erythrocyte are detroyed per hour

in 1 day turns over approximately 6 gram of hemoglobin

- globin, may be reutilized oer in the form of its constituent amino acids

- iron of heme enters the iron pool, the iron-free porphyrin is degraded mainly in the reticuloendothelial cells of the liver, spleen etc.

• + NADPH + NADPH
  Heme (Fe2⁺) hemin (Fe³⁺) + O2
  - NADP - NADP
  Fe3+ (reutilized) + CO(exhaled) + biliverdin IX-a
  
  
  + NADPH
  - NADP
  yellow pigment bilirubin IX-a
  

- the further metabolism of bilirubin occurs in primarily in liver and then divided into 3 process

1. Uptake of bilirubin by liver parenchymal cells

2. Conjugation of bilirubin in the smooth e. r.

3. Secretion of conjugated bilirubin into bile

• 1. Uptake bilirubin by liver parenchymal cell.
  

- bilirubin sparingly soluble in plasma, in plasma it is protein-bound (albumin)

- Each mol. albumin have one high-affinity site and one low-affinity site for bilirubin

- such antibiotic and othe drugs compete with bilirubin for the high-affinity binding site on albumin these compounds can displace bilirubin from albumin significant clinicalseffect

2 In the liver

- bilirubin are removed from the albumin and take up at the sinusoidal surface of the hepatocytes

• 3. Conjugation in the liver
  

- bilirubin is converted to a water soluble form that can be secreted into the bile

- in the smooth endoplasmic reticulum, bilirubin is excreted in the bile in the form of a bilirubin diglucuronide which is catalysis by UDP-glucuronyltransferase

- UDP-glucuronyltransferase can be induced by phenobarbital

- Hyperbilirubinemia causes jaudice

Reference: lecture from:

Wiryatun Lestariana

Biochemistry Department

Fac. of Medicine GMU

Yogyakarta

 

HEMOPOIESIS LECTURE

Hemopoiesis = Hematopoiesis = the formation and the development of blood cells

All the cells that circulate in the     peripheral blood are derived from primitive mesenchymal cells i.e. pluripotential hematopoietic stem cells

Embryology

Pluripotential hematopoietic stem cells =tutipotent haemal stem cells (1 per 10⁴-10⁵ cells in the bone marrow)

Origin: mesenchymal tissue in the yolk sac during the first 6 week of gestation.

In the 2nd month a number of intraembryonic sites appear and replace the yolk sac.

These sites succeed but overlap each other in time, each site gradually increasing in importance then waning: liver & spleen (extramedullary hemopoiesis) --> bone marrow (myeloid tissues) (all blood cells) (medullary hemopoiesis) --> peripheral lymphoid tissues (only lymphocytes)

Sites of hematopoiesis after birth

Birth: mostly bone marrow; spleen and liver when needed

Beginning at 4 years: hemopoietic activity move to axial skeleton (flat bones, skull, ribs, sternum, clavicle, vertebrae, pelvic bones) and proximal ends of long bones (humerus, femur) --> complete at 18 years

Adult: remaining marrow cavities are fill with fat (medulla osseum flavum). By age 40 marrow in sternum, ribs, vertebrae and pelvis is composed of equal amount of hemopoietic tissues and fat.

Sites of hematopoiesis after birth

In the adults, in times of great demand the marrow in the long bones shafts may become hemopoietic again.

Extramedullary hemopoiesis may occur under certain conditions:

- if the bone marrow is no longer functional

- when bone marrow is not able to keep up with the demand for blood.

--> liver & spleen will become enlarged

Characteristics of stem cells

1. The parent cells to all of the cells that circulate in the peripheral blood

2. Morphologically indistinguishable from small to medium sized lymphocytes

3. While reside primarily in the bone marrow, they migrate freely in the peripheral blood

4. They may:

a. "rest" for long interval without dividing (prolonged intermitotic interval),

b. divide without differentiating further,

c. differentiate down any one of the several myeloid or lymphoid pathways of development.

Hemopoietic differentiation

1. Commitment of the stem cell to either one of two larger pathways, myeloid or lymphoid     (committed progenitor cells)¹ (tutipotent ---> pluripotent)²

2. Progeny of cells committed to each of these     2 pathways --> obligated to specific sublines of development

3. A. Three branches of myelod pathway:

    erythroid, megakaryocytic, and phagocytic

B. lymphoid pathway subdivides into

    B cell, T cell and "null" cell pathway

Stages of blood cells development

To generate a complete set of blood cells from a single tutipotent cells a definite sequence of major stages can be recognized:

1. Final commitment to a particular line of differentiation

2. Early cell proliferation to form a large pool of dividing cells

3. Differentiation as specific protein characterizing the particular line are synthesized

4. Final maturation: close of protein synthesis

5. Release of the cells from bone marrow

Stem cell à late stemm cell à proerythroblast à basophilic erythroblast à polychromatophilic erythroblast à normoblast à extrosion of nucleus à reticulocyte

Normally it takes 7 - 10 days for erythrocytes to develop from their precursor

It takes 10 - 14 days for blood neutrophils to develop from precursors

Structure & Function of Bone Marrow

Bone marrow has a vascular compartment and an extravascular compartment. 

Hematopoiesis takes place in the extravascular compartment. 

The extravascular compartment consists of a stroma of reticular connective tissue and a parenchyma of developing blood cells, plasma cell, macrophages and fat cells. 

The high activity of the bone marrow is demonstrated by its daily output of mature blood cells: 2.5 billion erythrocytes, 2.5 billion platelets, 50-100 billion granulocytes. The numbers of lymphocytes and monocytes is also very large.

Structure & Function of Bone Marrow (cont.)

• Bone marrow also function as the site of the removal of aged and defective erythrocytes and the differentiation of B lymphocytes. 
  
• It is also the site of numerous plasma cells.
  

The process of hematopoiesis

- Involves a complex interplay between the intrinsic genetic processes of blood cells and their environment.

- The interplay determines whether HSCs, progenitors, and mature blood cells remain quiescent, proliferate, differentiate, self-renew, or undergo apoptosis.

- All of the genetic and environmental mechanisms that govern blood production operate by affecting the relative balance of these fundamental cellular processes.

- In normal conditions, most HSCs and many progenitors are quiescent in the G0 phase; many progenitors are proliferating --> producing mature offspring. In the absence of any stresses, this is balanced by the rate of apoptosis in progenitors and mature cells.

- In the event of a stress e.g. bleeding / infection, several processes occur.

- Stored pools of cells in the marrow are quickly released into the circulation in order to localize to the site of injury.

- Fewer progenitors and mature cells undergo apoptosis.

- Quiescent progenitors and HSCs are stimulated by a variety of growth factors to proliferate and differentiate into mature white cells, red blood cells, and platelets.

Control of hematopoiesis

Probably the best characterized environmental regulators of hematopoiesis are cytokines (Hemopoietic Growth Factors) (Hemopoietic inductive microenvironment)

Cytokines are a broad family of proteins that mediate positive and negative affects on cellular quiescence, apoptosis, proliferation, and differentiation. In general, cytokines function by engaging a specific receptor and activating a variety of signaling pathways.

Hemopoietic cytokines

The response to these cytokines develops only when specific receptors for each of the cytokines are acquired by the cells.

Hematopoietic cytokines: produced through both autocrine and paracrine mechanisms and in many cases are produced by nonhematopoietic cells including bone marrow stroma and endothelium.

- Erythropoietin is produced primarily by     cells in the kidney

- thrombopoietin is produced by the liver

- IL-1 & TNFa are produced by     macrophages eating bacteria.     Stimulated by these cytokines --> Tcells, endothelial cells or fibroblast -->    produce G-CSF, GM-CSF

Hematopoietic inductive microenvironment

Microenvironment of the bone marrow is essential for hematopoiesis

1. Endothelial cells, fibroblast, and macrophage in the stroma are sources of hemapoietic cytokines that act on stem cells and early committed progenitor cells

2. It provides sorting mechanism that permits only the most immature cells (stem cells,     committed progenitor cells) and the most mature cells (RBC, neutrophils, platelets)     to exit the marrow

Microenvironment, cytokines, & cytokines receptors

Dynamics / kinetics in the blood

- Blood half-live of neutrophil: 6 - 8 hr

--> new blood populations of neutrophil is formed every 24 hours

--> neutropenia is the most frequent hematologic consequence when bone marrow is damaged

 

- Erythrocyte life span 100 -120 days

--> transfusion of erythrocytes and platelets is feasible

Reference: Lecture from Djoko Prakosa