by: anna rose d. magtalas€¦ · megakaryopoiesis megakaryopoiesis is a process wherein there will...

By: Anna Rose D. Magtalas

Compiled notes on Megakaryopoiesis, Primary and Secondary Hemostasis, Laboratory evaluation, and Mixing studies

Magtalas ‖ Hematology 2

Rodak’s Hematology: Clinical Principles and Applications

Clinical Hematology: Principles, Procedures, Correlations by Steininger

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HEMATOPOIESIS / HEMOPOIESIS

Hemopoiesis is a complex process of blood formation of bone-forming organs like the long bones of sternum and femur(most important bone-forming organ). The lungs may also form blood cells such as megakaryocytes.

Multipotential stem cell - the most stem cell line of blood located and produced in the bone marrow. It is capable of becoming several types of blood cells.

Cytokines - in the presence of these, the maturation process of the blood cells are greatly affected.

MEGAKARYOPOIESIS

Megakaryopoiesis is a process wherein there will be megakaryocyte maturation and eventually the production of platelets. It is a part of hemopoiesis.

Megakaryocyte Primary cell involved in megakaryopoiesis and the bone

marrow cell responsible for the production of platelets. Largest cell in the body the accounts for 1% of total

nucleated cells in the bone marrow. Considered as polyploid cell -possess multiple chromosomes

Characteristics: a. The only cell line where cell become smaller in size as

they mature, because it has to prepare itself for platelet shedding phase (a process wherein meg. fragments to produce platelets).

b. Do not go mitosis. It undergo ENDOMITOSIS c. Do not increase in number as they mature d. Can be harvested in the lungs and BM e. On blood films (Wright stained smear), 2-4

megakaryocyte per LPF f. A single megakaryocyte may shed 2000-4000 platelets

where 1/3 of it is being sequestered by the spleen. FACTORS THAT AFFECT MEGAKARYOPOIESIS:

1. Hormone: Thrombopoietin (TPO) It is a glycoprotein hormone produced mainly by liver,

smooth muscle cells, and kidney that regulates the production of platelets by the bone marrow.

Stimulates production and differentiation of meg. It means that from a primitive cell line, TPO affects the growth of megakaryocyte until it becomes a mature one. Has 23% homology with erythropoietin Concentration of TPO is inversely proportional with

megakaryocyte and platelet mass.

2. Cellular Derived Stimulators: Cytokines Cytokines are cellular secretions that gives signal in order for

the cell to proliferate and differentiate. Among which are: IL3 - acts synergistically with TPO to induce early

differentiation of stem cells. Results in the progenitor lineage.

IL6 with IL11 - acts in the presence of TPO to enhance the later phenomenon of the following processes: Endomitosis, Megakaryocyte maturation, and Platelet release/shedding

Different cells produce different cytokines depending on what cell it is: Monocyte produce monokines and lymphocyte produce lymphokines.

3. Growth Factors Growth factors are necessary for the growth, maturation

and proliferation of megakaryocytes. Among which are: Stem cell factors/Kit Ligand/Mast cell growth factor GM-CSF G-CSF EPO - atleast 20% of circulating epo is needed for

meg and platelet maturation

4. Inhibitors of Megakaryopoiesis Inhibitors control and regulate production of platelet in the

circulation because accumulation of platelet in the blood may lead to formation of thrombus that cuts the oxygen supply in the body causing tissue necrosis and eventually death.

Platelet factor 4 β-thromboglobulin NAP2 (Neutrophil activating peptide 2) IL8 - the only cytokine that inhibits megakaryopoiesis

HORMONES AND CYTOKINES THAT CONTROLS MEGAKARYOPOIESIS




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Platelets Come from mature megakaryocytes Anucleated Triggers primary hemostasis especially in exposed in

endothelial lining, subendothelium, and presence of plasma procoagulants in the blood vessels.

Characteristics: DIAMETER: 2-5 um and commonly mistaken as debris MEAN PLATELET VOLUME (MPV): 8-10 fL SHAPE: disc-shaped or circular that appears lavander

and granular under wright’s stain. Normal values:

(Under OIO): 7-21 platelets/10fields

(Metric): 150-400 x109/L

(SI): 150-400 x103/ul

STRUCTURE OF PLATELET:

I. PERIPHERAL ZONE: Outermost layer Consist of 2 major portions:

A. Plasma Membrane - made up of a phospholipid bilayer where each and every phospholipid especially in the inner layer has their own functions especially during primary hemostasis.

1. Outer layer is composed of: a. Phosphatidylcholine b. Sphingomyelin

2. Inner layer consists of: a. Phosphatidylserine b. Phosphatidylethanolamine

- capable of flipping out during platelet activation. It is a phospholipid layer where coagulation enzymes can assemble.

These enzymes that will adhere to phosphatidylethanolamine are called PROTHROMBINASE COMPLEX consisting of Factors Va, Xa, IV-Calcium.

Prothrombinase complex is essential for the formation of a stable fibrin clot. c. Phosphatidylinositol

- supports platelet activation by supplying arachidonic acid which is needed to produce thromboxane A2, an enzyme that is strictly needed for platelet activation.

B. Glycocalyx - an adhesive layer that responds

readily to hemostatic requirement. It provides a distinct connection between the inside part of the platelet to the outside environment.

* It is made up of different glycoproteins (V, VIII, and fibrinogen) that are necessary for the 2 major process of primary hemostasis: 1. Platelet Adhesion- attachment of the platelet to the site of injury. 2. Platelet Aggregation- adhesion of platelets with one another to form a temporary platelet clot.

Classification of Plasma membrane receptors that provide for Cell Adhesion Molecule (CAM):

CAM Integrin Family - clinically significant CAM. This is made up of Glycoprotein IIb-IIIa, a calcium-dependent membrane protein complex for fibrinogen receptor, necessary for platelet aggregation.

CAM Leucine Rich Family - clinically significant CAM because this is the CAM that makes up the Glycoprotein Ib which serves as binding site for vWF, necessary for platelet adhesion process. Another members of CAM Leucine rich family: GPV and GPIX

CAM Ig gene Rich Family CAM Selectin Family CAM Seven Transmembrane Receptor(STR) Family

II. SOL-GEL ZONE It lies beneath the platelet membrane. Sol-gel zone provides support to entire platelet especially during changing of shape and during activation process. Composed primarily of 2 different proteins:

A. Microtubule contains protein tubulin that maintains the disc shape of the platelet

B. Microfilaments this contains the actin and myosin which upon the stimulation of platelet will interact to form actomyosin (thrombosthenin) for clot retraction. It enables the platelet to change in shape.

*Clot retraction Formed because of the aggregated platelets in the red blood cells due to presence of microfilaments.

III. ORGANELLE ZONE: Centromere The part where the secretory products of platelet come from. These secretory products are components of the platelet itself, needed for platelet activation:

a. alpha granules 1. Platelet Factor 4 2. β-thromboglobulin 3. Platelet-derived growth factor 4. Fibrinogen 5. Factor V 6. Von Willebrand Factor (vWF)- platelet can also

produce vWF aside from liver. vWF acts as a ligand with GPIb especially in platelet adhesion.

7. Thrombospondin 8. Fibronectin

b. Dense granules - provides energy for the platelets during platelet activation. 1. ADP 2. ATP 3. Calcium and Magnesium 4. Serotonin

c. Mitochondria - for ATP synthesis used in platelet metabolism

d. Lysosomal granules 1. Acid phosphatase 2. Hydrolytic enzyme - enables the platelet to fight

bacterial infection




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IV. PLATELET MEMBRANE SYSTEM Consist of 2 parts:

1. Dense Tubular System (DTS) control center for platelet activation derived from smooth endoplasmic reticulum and

sequesters calcium for platelet activation process. it is also the site of platelet cyclooxygenase and of

prostaglandin synthesis Contains series of enzymes necessary for platelet

activation: A. PHOSPHOLIPASE A- enzyme that activates phosphatidylinositol to release arachidonic acid. B. CYCLOOXYGENASE- acts on arachidonic acid to synthesize Prostaglandin 2. C. PEROXIDASE- cleaves PG2 to synthesize PG3 D. Thromboxane synthetase- takes action on PG3 to release TXA2 which is needed for platelet activation. It is the last enzyme secreted by DTS to make vasoconstriction happen. E. PHOSPHOLIPASE C- cleaves phosphatidylinositol to release Inositoltriphosphate and diacylglycerol (DAG) which is another pathway of platelet activation.

2. Open Canalicular System (SCCS) surface connecting canalicular system an invagination of the plasma membrane acts as a canal for the release of the granule

constituents and cytoplasm to the exterior of the platelet

PLATELET FUNCTION: Participates in a sequence of events that lead to the formation of a

platelet plug and ultimately to the formation of a stable fibrin clot at the site of vessel interruption.

a.Primary hemostasis by adhesion, secretion and aggregation with the end view of hemostatic plug. (Platelet plug formation)

b.Blood coagulation by releasing platelet factor 3 that plays a big role in forming fibrin clot.

c.Clot retraction by its actomysin d.Helps in localization of bacteria and other small objects and

producing aggregates too large to pass through capillaries.

Composition of a Temporary Platelet Plug: RBCs WBCs Platelet Trapped bacteria/microbes

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HEMOSTASIS - process wherein upon tissue injury the body responds to promote stoppage of blood - a complex process wherein there will be an interaction of blood vessels, cellular components (red cells, platelet, wbc), coagulation factors and fibrinolytic factors that are being secreted by the liver, fibrinolysis and tissue repair.

Why the body has to perform hemostasis: 1. To produce clot formation to stop the bleeding 2. To confine the clot that has been formed by the platelets & different coag factors 3. If the wound is already stable within 48 hours the body should respond in order to lyse the clot that has been formed as the wound heals.

Components of hemostasis: 1. Extravascular tissue factor/ TF factor - It is the tissue surrounding

the vessel. The subendothelium and the endothelial lining that is being exposed upon vessel injury.

2. Vascular intima - The blood vessel through which the different cell components (platelets) would flow

3. Intravascular component - made up of different plasma proteins (coagulation factors I-XII including pre kallikrein & high molecular weight kininogen, inhibitors of fibrinolysis, factors for fibrinolysis, VWF, platelets)

Concept of normal blood coagulation The body has its vessels through which the blood flow and it should be maintained in balance because upon excessive production of coagulation factors, the patient is susceptible in having thrombotic episode and upon excessive production of fibrinolytic factor, there will be increased lysis of plasma protein on the clot formed which will lead to bleeding tendencies Hypocoagulation Increased prod of fibrinolytic factor – these are the plasma proteins that will lyse the clot that has been formed inside the body which will make the body susceptible to bleeding tendencies. There is poor clot formation and excessive fibrinolysis. Hypercoagulation There will be uncontrolled thrombi formation – if your body’s platelet is capable of adhesion and aggregation, this will further be reinforced by the different coagulation factors (prothrombinase complex), if there is continuous activity of coagulation factors, the body will be susceptible in developing thrombotic episodes and hypercoagulable disease.




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PRIMARY HEMOSTASIS

Primary hemostasis is based on: 1. Subendothelium/ endothelial lining 2. Platelets

2 most important phases in primary hemostasis:

1. platelet adhesion phase 2. platelet aggregation phase

Sequence of events in primary hemostasis:

EVENT COMMENT

1 Vasoconstriction Controlled by vessel muscle Happens in exposed smooth muscles and enhanced by chemicals secreted by platelets as well as signals secreted by the brain

2 Platelet Adhesion Adheres to exposed subendothelial connective tissue

3 Platelet Aggregation Interaction of platelets to one another to create a temporary platelet plug at the site of injury.

4 Fibrin Platelet Plug Formation

Coagulation factors interact on platelet surface to produce fibrin.

5 Fibrin Stabilization Fibrin clot must be stabilized by coagulation factor XIII

*Steps 4-5 are included in secondary hemostasis

STEP 1: VASOCONSTRICTION

Upon vessel injury your brain is capable of secreting a stimulus which is known as the serotonin (other source: dense granules of platelet). It is capable of causing the very first stage of hemostasis which is vasoconstriction.

Vasoconstriction occurs as a neurogenic response and causes

the site of injury to swell so platelets could easily adhere to the actual site. It is capable of exposing the different types of collagen that would serve as a ligand for platelet adhesion and aggregation. Collagen promotes thrombus formation by causing the adherence of platelets to the area of injury.

2 kinds of collagen that are exposed upon vasoconstriction: 1. for platelet adhesion phase – collagen types 4 & 5 2. for aggregation – collagen 1 & 3

* upon adhesion and aggregation, there will now be a temporary platelet plug. The body’s response would be reinforcement to produce a stable fibrin clot and then there will be secondary hemostasis activity. * vWF – the most important ligand

STEP 2: PLATELET ADHESION Factors that should be present in for platelet adhesion:

1. VWF 2. Glycoprotein1B –always exposed in the plasma membrane

Process of cell adhesion: 1. Upon injury, there is an exposed subendothelial tissue, causing

bleeding. 2. The brain secretes serotonin in order to give signal to the

subendothelial tissue to expose collagen 4&5 and 1&3 at the same time.

3. Once it’s already exposed, circulating platelet should be able to bind in the site of injury only with the help of vWF which is produced by the liver (together with factor VIII) and alpha granules of active platelets.

4. VWF will bound to GP1B and this will promote adhesion of platelet to the damaged subendothelial tissue.

5. The platelet that adheres to the site of injury will undergo a change in shape – the Pseudopod extrusion – which is needed for the next phase of hemostasis which is the platelet secretion phase.

STEP 3: PLATELET AGGREGATION It is the recruitment of additional platelets, which interact with

each other to form clumps. Requires fibrinogen and calcium; Involves binding of fibrinogen to specific platelet receptor complex GpIIb-IIIa

Platelet stimulating agents (collagen, epinephrine , thrombin, ADP) binds to platelets, causing them to adhere to one another. Platelet to platelet interaction is necessary to produce a temporary platelet plug. 3 Important Mediators of Platelet Aggregation:

ADP –causes secondary, irreversible platelet aggregation by inducing a change in the conformation of the IIb-IIIacomplex allowing the binding of fibrinogen Thrombin –stimulates the platelets to release ADP and activates the platelet membrane phospholipases TXA2–promotes platelet aggregation directly or in synergy with ADP; acts as a potent vasoconstrictor

Summary of Primary Hemostasis

Initiated by the exposure of platelets to the subendothelium connective tissue components of blood vessels (Collagen, microfilaments, basement membrane)

If acute injury occurs, the small vessels constrict and platelets immediately adhere to the exposed surfaces and release ADP and ATP.

Thromboxane A2 is also released to promote further vasoconstriction.

PLATELET SECRETION / RELEASE MECHANISM Platelet will extend its pseudopod so that the open canalicular

system will be open resulting the granules to localize at the center for it to readily be secreted by the platelets. Contents of the granules that are released outside:

1. Inhibitors of megakaryopoiesis (factor 4 and β-thromboglobulin) 2. ADP – for aggregation and energy 3. Serotonin – for vasoconstriction 4. VWF – ligand for adhesion 5. Platelet derived growth factor – will support megakaryopoiesis

Causes of Release mechanism:

1. Exposed collagen 1 & 3 2. Secretion of thrombin – an activated factor II 3. Thromboxane A2 – for platelet activation and vasoconstriction 4. Secretion of epinephrine – for aggregation




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A reversible primary platelet aggregation takes place during which

platelet adhere to one another. Platelets also change in shape and then organelles become centralized.

At this point, platelets may disaggregate in the absence of further stimulation.

Important substances released during platelet aggregation includes:

ADP – promotes secondary platelet aggregation and recruits additional platelets to the site of injury.

ATP Serotonin – promotes further vasoconstriction.

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Laboratory Evaluation of Primary Hemostasis

I. Test of the Vascular and Platelet Phases A. BLEEDING TIME - principally a measure of platelet plug formation.

Factors which affect bleeding time: 1. Elasticity of the cut tissue 2. Ability of the blood vessels to constrict and retract. 3. Mechanical and chemical action of platelets in the formation of hemostatic plug.

Methods of Bleeding Time Determination

A. Duke’s Method Principle: Lobe of the ear is the site of choice of puncture. It should be warmed before the test or before incision is made. Then take bleeding time. Normal Values: 2-5minutes B. Modified Ivy’s Method

Best method to assess platelets. It is considered to be the best screening test available for platelet’s role in hemostasis. Principle: Capillary pressure is standardized and the average of bleeding time from three separate incisions in the forearm is noted. This provides a very accurate technique if the incisions are identical. Sphygmomanometer cuff: Pressure raised to 40mmHg Puncture of wound = 3mm deep NormalValues: 2.5-7minutes

C. Coply Lalitch Method

Procedure involves: (1)Puncture of finger to a depth of 6mm (2)Immersion of punctured finger in sterile physiologic saline solution warmed at 37degC

Normal Values: >3minutes

D. Adelson-Crosby Method – same with Copley-lalitch E. Macfarlane’s method- same principle with Adelson-Crosby method but it only uses ear lobe as the site of puncture. F. Aspirin tolerance Test- assesses the effect of a standard dose of aspirin on the Duke’s Bleeding Time. BLEEDING TIME IS PROLONGED IN THE FF. CONDITIONS:

1.When the blood platelets are greatly reduced. a. Thrombocytopenic purpura- dec. In platelets b. Acute Leukemia c. Aplastic anemia

2. Injury of capillary wall a. Scurvy b. Toxins (infection, chemical, snake venom) c. Allergy

3. PT deficiency a. Destructive disease of the liver b. HDN

4. Slightly prolonged severe anemia B. PLATELET COUNT

Reasons why platelets are hard to count: 1. They adhere to foreign surfaces (skin and dried walls of pipets) 2. Platelets easily disintegrate 3. They are hard to differentiate from debris 4. Platelets are unevenly distributed in the blood because they tend to

clump

A. Indirect Method Platelets are counted in their relationship to red cells on a

fixed-stained smear. This method is NOT RELIABLE because the results depend upon the distribution of platelets and on the red cell count. 1.Fonio’s Method- 14% MgSO4 + 1ggt of blood + Wright Stain.

2.Dameshek Method- Brilliant Cresyl Blue, Sodium Citrate, sucrose and formalin; counter stain with Wright’s Stain.

Formula: Platelets/uL= Plts ctd X RBC count 1000 Reference range: 250 000 –500 000/uL or 250 –500 x 10^9/L

B. Direct Method

Platelets are counted in a hemocytometer as in RBC and WBCs

1. Guy and Leake- diluent is made of sodium oxalate, 40% formalin and crystal violet. Calculation: Platelets/uL= platelet counted X 5 X 10 X 100

2. Rees and Ecker- diluent is made up of sodium oxalate, brilliant cresyl blue, formalin and dist. Water. Calculation: Platelets/uL= Platelets counted X 10 X 200 4 3. Brecher-Cronkite- diluent is 1% ammonium oxalate. This is the most accurate method because there is no difficulty in distinguishing platelets from debris. Computation: Platelet/uL= platelets X 5 X 10 X 100




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Reference range: 150 000 –450 000/uL or 150 –450 x 10^9/L

Sources of Error (Falsely Elevated Ct.): Fragments of leukocyte cytoplasm Inclusions from lysed RBCs Debris

Sources of Error (Falsely Low Ct.): Platelet satellistism Platelet clumping d/t agglutinins Spontaneous aggregation Clotting d/t faulty blood collection & handling Delay in sampling (skin puncture)

C. Electronic Method- red cells must first be removed from whole blood, either by sedimentation or by controlled centrifugation.

1.Voltage-pulse counting 2.Electro-optical Counting.

D. Platelet Estimation

IMPORTANCE: (1)Determination of causes of artifactual low counts (2)Detection of abnormalities in platelet structure (3) as a cross-check of direct counts

NORMAL DISTRIBUTION: 1 platelet for every 10-40 RBCs at 1000x magnification or 5-20 platelets per oil immersion field in the area where RBCs are just touching each other ESTIMATED PLATELET COUNT:

Platelets/uL= ave.no. of plts per OIF X 20 000

C. TESTS FOR SPECIFIC PLATELET FUNCTION A. Test for Adhesion of Platelets or Platelet Adhesiveness Test Principle: The adhesiveness of platelets may be measured in vitro by their ability to adhere glass surfaces.

1. In vivo method of Borchgrevink- measures adhesion of platelets to the wound surface. 2. Salzmann Method- test of the retention of platelets within glass bead column 3. Test for adhesion of platelets to collagen fibers- platelet rich plasma containing EDTA is assessed for adhesion collagen in the absence of aggregation. The technique is based on enumeration of free and adherent platelets. Platelet retention (%)= 1 PC – Final PC X100

1 platelet count

B. Platelet Aggregation Test - the most commonly used quantitative method that uses various aggregometers. Platelet Aggregometer:

In this machine, a light beam is passed through the suspension. The platelet aggregation in response to an added chemical stimulus can be monitored by changes in light transmittance. Discoid to spheroid shape change is seen as an initial decrease in transmittance whereas the subsequent formation of platelet clumps allows more light to pass through the suspension to the photo detector and is recorded as an increase in light transmittance. C. Platelet Factor 3 Availability Test- platelets are incubated with kaolin and epinephrine, and are stimulated to provide PF3 activity.

D. DETERMINATION OF CLOT RETRACTION

When blood coagulation is complete, the clot normally undergoes retraction (serum is expressed from the clot and the clot becomes denser). Normal clot retraction requires normal number of functioning platelets, calcium, ATP, and a normal fibrinogen, and fibrin ABNORMAL with: •Thrombocytopenia •Low/Abnormal Fibrinogens •Paraproteinemias (Multiple myeloma, macroglobulinemia) that interfere with fibrin formation •Glanzmann’s Thrombasthenia (platelet is incapable of interacting with fibrin

INCREASED in: Anemic states METHODS: 1.Qualitative Test

a.Hirshboeck or Castor Oil Method Add one drop of blood into castor oil. Observe for formation

of dimpling/ droplet-like serum on the surface of the blood drop. NV: 15 –45 minutes b.Single Tube Method

Blood in a test tube used for clotting time determination is saved and left at room temperature in order to note retraction, red cell fall-out and clot lysis. Note the shape and character of the clot, serum expressed and volume of red cell fall out. NV: 30-60minutes; complete in 18-24hours

2. Quantitative Test a. Stefanini Method- similar with single tube method. b. Macfarlane Method

Blood is allowed to clot in a test tube containing a glass rod and retraction is observed after the incubation period. The clot attached to the rod is then removed and the serum extracted from the clot is measured.

Retraction is expressed in terms of the volume of serum obtained from the original volume of blood. CRT= Amt. of serum left in tube x100

Amt. of blood used NV: 44-67%




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E. CAPILLARY FRAGILITY TEST / RESISTANCE TEST

Tests the stability of the small blood vessels to retain the red cell in their lumen under conditions of stress and trauma.

Platelets and Vitamin C are important in the maintenance of normal capillary integrity or resistance.

Measures platelet’s ability to maintain capillary integrity

Correlates with the degree of thrombocytopenia, as with the bleeding time

Clinical Significance: Positive in Purpura, Dengue Fever, & Scurvy Reference Range: 1-10 petechiae

Known by various names: 1. GERMANY: Rumpel/ Leede 2. ENGLAND: Hess 3. FRANCE: Weil 4 .ITALY: Grocco-Frugoni

METHODS:

1.Tourniquet/ Rumpel-Leede/ Hess Test positive pressure technique. A tourniquet or blood pressure cuff (80 mmHg) is placed

above the elbow, preventing the blood in the veins from returning and creating conditions of increased hydrostatic pressure and hypoxia with veins and capillaries. A rupture of one or more blood vessels constitutes a positive test. Principle: by partially obstructing the venous blood, the capillary pressure is increased. This will give rise to intravasation of blood which will be manifested in the form of small hemorrhages called petechiae.

a.Quick’s Method b.Gothlin’s Method

2. Suction Cup/ Petechiometer Method/ Negative Pressure Technique

In the negative pressure method, trauma is created by means of suction cups applied to the skin. Principle: employs the use of a modified da Silva Melle Instrument. The cup is applied to the outer surface of the arm for a period of one minute at 200 mmHg and the resistance of the capillaries is expressed as the negative pressure required to produce a macroscopic petechiae. Normal value: less than 4 petechiae

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Additional Notes:

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SECONDARY HEMOSTASIS

Involves the enzymatic activation of series of plasma proteins (serine proteases) in the coagulation system to form a fibrin meshwork.

COAGULATION Is a process whereby, on vessel injury, plasma proteins, tissue factors, and calcium interact on the surface of platelets to form a stable fibrin clot. Platelets also interact with fibrin to form a stable platelet-fibrin clot. This is a mechanism consisting of a series of cascading reactions involving development of enzymes from their precursors (zymogen) which will further be converted to an activated state (serine protease). COAGULATION FACTORS

Roman Preferred Descriptive

Synonyms Biochemistry

I Fibrinogen Ia-Fibrin Glycoprotein

II Thrombin IIa-Thrombin

III Tissue factor Thromboplastin Lipoprotein

IV Calcium Metal ion

V Proaccelerin Labile factor Glycoprotein

VI Omitted

VII Proconvertin Stable factor Serum prothrombin

conversion accelerator

Glycoprotein

VIII Antihemophilic Factor (AHF)

Antihemophilic Globulin (AHG)

Antihemophilic factor A

IX Plasma Thromboplastin

Component (PTC)

Antihemophilic factor B/ Christmas factor

X Stuart-Prower Factor

Stuart factor Prower factor

XI Plasma Thromboplastin

Antecedent (PTA)

Antihemophilic factor C Beta/Gamma Globulin

XII Hageman Factor Contact factor Glass factor

Sialoglycoprotein

XIII Fibrin Stabilization

Factor

Laki-Lorand Factor Fibrinase

Beta/Gamma Globulin

- Prekallikrein Fletcher factor Plasma Protein - High Molecular

Weight Kininogen (HMWK)

Fitzgerald/ William/Flaujeac factor

COAGULATION PROTEINS The intrinsic and extrinsic coagulation pathways are a series of reactions involving coagulation factors known as: 1 – enzyme precursors (zymogens/proenzyme): inactive form 2 – non-enzymatic (cofactors): help serine proteases in activating proenzymes 3 – calcium (Ca++) 4 – phospholipids (PL) All coagulation factors normally are present in the plasma, with PL being provided by the platelets.

Classification of Coagulation Factors: I. According to Hemostatic function

Zymogen 2, 5 , 9-12, Prekallikrein

Cofactors 3, 5, 8, HMWK

Enzymes Serine protease 1a, 9-11a, Transaminase, 13a, Prekallikrein

II. According to Physical properties

Contact group 11, 12, P, H

Prothrombin/Vit K Dependent group - synthetized by liver in the presence of K+

2, 7, 9, 10

Fibrinogen group - labile group; readily utilized

1, 5, 8, 13

PATHWAYS OF COAGULATION I. EXTRINSIC PATHWAY

Activated by the release of Tissue factor from the injured vessel into the plasma. Tissue factor is secreted by the ff organs: lungs, liver, kidney, brain, placenta, spleen.

II. INTRINSIC PATHWAY Activated in vivo by the contact of certain coagulation proteins. Activation occurs when a vessel is injured, exposing the (-)subendothelial basement membrane and collagen. This will lead to the activation of the “Contact Factor”, Factor XII together with Factor XII, HMWK and prekallikrein.




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III. COMMON PATHWAY

This pathway starts with the activation of Factor X to Factor Xa by either intrinsic or extrinsic pathway.

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LABORATORY EVALUATION OF SECONDARY HEMOSTASIS Tests for Phase II of Coagulation

A. The Coagulation Test

Tests the composite action of all plasma factors acting simultaneously.

Clotting time is a measure of the ability of the blood to clot and is not influenced by the platelet functions other than PF3.

It also measures only the time required for the formation of the traces of thrombin sufficient to produce a visible clot.

a. Micro Methods

1. SLIDE OR DROP METHOD Puncture the ring/middle finger Start timer then transfer blood to slide every 30

seconds. Observe for fibrin string.

2. CAPILLARY or DALE AND LAIDLAW’S METHOD Collect non-heparinized blood Start timer and fill 3 capillets After 30 secs, break the capillet Observe for fibrin string.

b. Macro Methods- superior for there is less contamination of the plasma with tissue fluids when blood is drawn from a vein.

1. Lee-White Method or Whole Blood Clotting Time PRINCIPLE:

The whole blood clotting time is the time required for freshly collected blood to form a rm clot in standardized glass tubes at C. Thus, the whole blood clotting time is a measure of the integrity of the intrinsic system.

Procedure: o Wash the test tube using sterile NSS o Collect 3 ml of blood and transfer it to the test tube o Every 30 secs, tilt the tube and observe for clot. Three-tube Method of Lee-white: o Same with the 1-tube mtd but take the average from 3

tubes. Put blood first in Tube 3>2>. Record the result and take the average.

o Tube 1 should be the first one to clot: it has less/no tissue juice

NORMAL VALUES: 5-10 minutes

c. Activated Coagulation Time of Whole Blood PRINCIPLE: The ac vated coagula on me of whole blood is the me necessary for fresh blood to form a clot when incubated at C in the presence of “surface contact” activation. This assay, like to whole blood clotting, measures overall activity of the intrinsic clotting system. NORMAL VALUES: 1-2 minutes d. Plasma Recalcification Time

-More sensitive method than the coagulation time of whole blood because there is an activator added. -May reveal abnormality which is not detectable by the determination of the clotting time of venous blood. -The activated recalcification time makes use of 0.25 M CaCl2 as activator. NORMAL VALUE: Less than 50 seconds

B. Partial Thromboplastin Time

-Simple test for the INTRINSIC and COMMON pathways of coagulation.

C. Activated Partial Thromboplastin time (APTT)

-a test for the deficiencies of factors in the INTRINSIC system. (Factor VIII, IX, XI, XII) NORMAL VALUES: 25-35 seconds

D. Differential Tests of Activated Partial Thromboplastin Time (DAPTT)

-used to differentiate factor deficiency and disorder of circulating anticoagulants.

E. Differential Partial Thromboplastin Time (DPTT)

-Another modification of APTT which is done by mixing the patient’s plasma with commercially available correcting reagents, Factor VIII and IX reagents. -If prolonged:

If PTT is corrected with Factor VIII (hemophilia A) If PTT is corrected with Factor IX (hemophilia B)

F. Plasma Prothrombin Time (PROTIME)

Measures the EXTRINSIC and COMMON pathway of coagulation.

It is used to monitor oral anticoagulant therapy.

This can detect prothrombin, fibrinogen, Factors V, VII and X deficiencies.

Methods: 1. One Stage Method of Quick (Stanley Brown Method)

Principle: “Tissue thromboplastin and calcium added to plasma react to fibrinogen to form a clot. The thromboplastin added to the plasma takes the place of the tissue juice formation of extrinsic thromboplasitn. The prothrombin time is therefore prolonged if there is a deficiency of Factors V, VII or X or a very severe defieciency of Factor I and II.”

Normal Value: 10-14 seconds




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2. Two-stage Prothrombin and Proconvertin Test (Owren and Aas)

- It offers a combined estimation of the levels of prothrombin and proconvertin. ADVANTAGES:

a. It is more sensitive with Stanley Brown Method. b. Fresh specimens are not necessary, and the method can be used for mailed samples of blood. c. The method is not affected by heparin.

3. Owren’s Thrombotest Method - for the control of Coumarin anticoagulant therapy, this is considered as the most sensitive test.

4. Fibrometer Method - it is an electromechanical semi-automated instrument that has been used extensively in one-stage prothrombin method of Quick.

5. Micromethod (Pro time) - this is microtechnique employed for childer and the method uses micropipettes; the principle of the test is similar with one-stage prothrombin time or Stypven Time.

6. Related Method- Stypven Time (Rusell’s viper venom Time) - it is used to distinguish deficiencies of Factor X and those of Factor VII. It is also used to detect deficiencies in prothrombin, fibrinogen and factors V and X. It differs from prothrombin time in that deficiencies in factor VII are not detected.

7. Prothrombin Activity or Index - reported in percentage, with 100% as the maximum level. PT activity (%)= Pro time (in secs of ctrl) X 100 Pro time (in secs of px)

8. International Normalized Ratio - this method of reporting has been proposed to monitor patients on oral anticoagulant therapy. - it is defined as the prothrombin time ratio had the test been perfored using international standard thromboplastin reagent.

G. Subtitution Tests

This can be adopted if primary tests like PT or APTT are abnormally prolonged and the indicate a factor deficiency.

The patient’s deficient plasma is diluted 1:1 with a plasma or serum substitute and the APTT or PT is repeated.

A correction if the original prolonged APTT or PT indicates that the deficient factor has been added to the patient’s plasma by substitution solutions as follow:

1. Aged plasma- lacks labile factors V & VIII but retains normal activity of all coagulation factors.

2. Fresh adsorbed plasma- lacks vitamin K dependent factors (II, VII, IX, X) but retains activity of al other coagulation factors.

3. Aged serum- lack factors I, II, V, VIII but retains normal activity of all coagulation factors.

Run samples for APTT and PT

Sub solutions PRESENT ABSENT Fresh plasma Aged plasma

ALL All except

None ,,,,,,,,5,8

Fresh serum Aged serum

All except All except

1,,,,5,8 ,1,2,5,8

Adsorbed plasma -plasma treated with BaSO4

Labile group (1,5,8) + 11,12

Vit K dep Group (2,7,9,10)

*FACTORS 3,4,6 and 13 are always ABSENT in the reagents.

Pathway Coagulation factors present Extrinsic 7

Intrinsic 8,9,11,12

Common 1,2,5,10

SAMPLE PROBLEMS: INTRINSIC PATHWAY DEFICIENCIES

1. APTT – 39.7s (abnormal; problem with intrinsic pathway) PT – 12.2s Corrected by Fserum but Not corrected by Fplasma

Intrinsic has = 8,9,11,12

Fplasma NC has 1,5,7,11,12,8

Fserum C has 2,3,7,9,10,11,12

The px has Factor IX(9) deficiency

Intrinsic has = 8,9,11,12

Aserum NC has 7,9,10,11,12

Adso plasma C has 1,5,8,11,12

The patient has factor VIII(8) deficiency ______________________________________________________ COMMON PATHWAY DEFICIENCIES

2. APTT – 52.7s (both are abnormal; problem with common) PT – 18.3s C: Aged serum not by aged and adsorbed plasma.

Common has = 1,2,5,10

Aged plasma NC has Adso plasma NC has

1,2,7,9,10,11,12 1,5,8,11,12

Aserum C has 7,9,10,11,12 The px does not have a factor deficiency therefore, circulating anticoagulants could be present.

Common has = 1,2,5,10

Adso plasma NC has 1,5,8,11,12

Aged plasma C has 1,2,7,9,10,11,12 The patient has Factors II(2) and X(10) deficiencies.




12

H. Serum Prothrombin Time or Prothrombin Consumption Test (PCT)

Best considered as a test of platelet phospholipid activity.

If the prothrombin time and the PTT are normal, a short PCT indicated a deficiency of PF3 due to thrombocytopenia or thrombopathia.

I. Thromboplastin Generation Test

1. Bigg’s and Macfarlane Method 2. Hick’s-Pitney Kaolin Modification Method

Tests for Phase III of Coagulation 1. Thrombin Time- test for the deficiency or inhibition of fibrinogen.

Principle: Commercially prepared thrombin reagent is added to citrated plasma, and the time required for clot formation is measured.

Normal Values: 10-20 seconds

2. Fibrindex Test- Commercially available test wherein upon addition of plasma containing fibrinogen, thrombin produces clotting.

3. Fit-test (Immunological Test) - a rapid slide test based on the

agglutination of fibrinogen-coated red blood cells by the latex anti-human fibrinogen reagent. Normally, presence of fibrinogen is indicated by agglutination.

4. Fibrinogen Titer Method - serial dilutions of plasma are diluted

with thrombin. The titers is the highest dilution in which a fibrin clot can be seen, and is related to the fibrinogen concentration and indirectly to the presence of circulating anticoagulants.

5. Assay of Plasma Fibrinogen - several accurate methods are now

available for the quantitative assay of plasma fibrinogen. Fibrinogen is usually converted into fibrin which is quantified by gravimetric, nephelometric, chemical, immunologic and precipitation methods.

Methods: a. Ellis and Stransky Method b. Stirland’s Method c. Turbidimetric Method of Partfantjev et.al d. Ratnoff and Menzie Method e. Fibrin Clot Method

Additional Notes:

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by: anna rose d. magtalas€¦ · megakaryopoiesis megakaryopoiesis is a process wherein there will...

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