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HEMATOLOGY 2
PLATELET PRODUCTION, FUNCTION, AND STRUCTURE
MEGAKARYOPOIESIS / THROMBOPOIESIS / THROMBOCYTOPOIESIS
¾ The maturation of the cells of the megakaryocytic system has been divided into four stages. (1) Megakaryoblast , (2) Promegakaryocyte, (3) Megakaryocyte , (4) Metamegakaryocyte ¾ Maturation sequence of megakaryoblast takes about 5 days. Platelets are produced directly from the megakaryocyte cytoplasm. ¾ A specific Hormone , Thrombopoietin (70,000daltons) , is responsible for megakaryopoieisis ¾ Megakaryocytic cells are unusual in that their nuclei are able to undergo multiple mitotic divisions without cytoplasmic division , generating giant multinucleated or polyploidy cells. This is referred to as Endomitosis. ¾ Endomitosis= a form of mitosis that lacks telophase and cytokinesis (Maturation series From Barbara Brown)
- Megakaryoblast x 20 - 50 um x Blue cytoplasm x Multiple nucleoli x N;C ratio about 10: x Fine chromatin x First recognizable stage
- Promegakaryocyte x 20 - 60 um x Less basophilic cytoplasm x Chromatin becomes coarse x Irregulary shaped nucleus , may show slight lobulation x N:C ratio of4:1 to 7:
- Granular megakaryocyte x 30 to 90 um in diameter x Abundant , pinkish blue in color cytoplasm x Very fine and diffusely granular cytoplasm with irregular peripheral border x Multiple nuclei may be visibleor the nucleus may show multilobulation x N:C ratio is 2:1 to 1:
- Mature megakaryocyte x 40 to 120 um in diameter x Cytoplasm contains coarse clump of granules aggregating into little bundles, which bud off from the periphery to become platelets x Nucleus is multilobulated x N:C ratio is 1:
- Platelet (thrombocyte) x 1 to 4 um x Cytoplasm: light blue to purple , very granular x No nucleus ( Steinenger et al.,) Maturation stage Cytoplasmic granules Cytoplasmic tags Nuclear fragments Thrombocytes visible Megakaryoblast Absent Present Single nucleus, fine chromatin nucleus No Promegakaryocyte Few Present Double nucleus No Megakaryocyte Numerous Usually absent Two or more nucleus No metamegakaryocte Aggregated absent Four or more nucleus Yes Note : In differentiating the maturation stages of the megakaryocytic cells, emphasis should be placed on the cytoplasmic appearance rather than the number of nuclei or the chromatic structure (Steinenger). RODAKS ORDER OF MATURATION (from least to most mature) Progenitors – resemble lymphocytes and cannot be distinguished by wright stained light microscopy a. Burst forming unit-Meg (BFU-Meg)= diploid and able to perform mitosis b. Colony forming unit – Meg (CFU-Meg)= diploid and able to perform mitosis c. Light density CFU-Meg (LD-CFU-Meg) = cannot divide but it retains DNA replication and cytoplasmic maturation for endomitosis Precursors a. MK-1 / megakaryoblast b. MK-II / promegakaryocyte c. MK-III/ Megakaryocyte As endomitosis proceeds, megakaryocyte progenitors leave the proliferation phase and enter terminal differentiation (maturation) .. T tr^ T bone (^) marrow : (^) Osteoclast - resembles (^) Mega Karyocyte
FEATURES OF THE THREE TERMINAL MEGAKARYOCYTE DIFFERENTIATION STAGES
MK-I MK-II MK-III
% of precursors 20 25 55 Diameter 14 to 18 um 15 to 40um 30 to 50um Nucleus Round Indented Multilobed Nucleoli 2 to 6 variable Not visible Chromatin Homogenous Moderately condensed Deeply and variably condensed N:C ratio 3:1 1:2 1: Mitosis Absent Absent Absent Endomitosis Present Ends Absent Cytoplasm Basophilic Basophilic and granular Azurophilic and granular Alpha – granules Present Present Present Dense granules Present Present Present Demarcation system Present Present Present Platelet UltraStructure PLATELETS / THROMBOCYTES Also known as thrombocytes with a diameter of 2 to4 um or 1 to 4 um with a volume approximately 6 to 7.5 fL and have a discoid shape. With Wright’s stain, platelets have a light violet-purple granular appearance and look like “specks of dust” Platelets are produced directly from the megakaryocyte cytoplasm Each megakaryocyte produces between 2000 to 4000 platelet(Rodaks), (1000-4000 Steinenger) Average platelet counts are slightly higher in woman than in men On a Wright-stained wedge-preparation blood film, platelets are distributed throughout the red blood cell monolayer at 8 to 20 cells per 100x field. ( 7 to 21 per OIF =RODAKS) Maturation time: 5 days Life span of 8 to 11 days or 9 to 12 days. At the end of their life span, platelets are phagocytized by the liver and spleen and other tissues of the mononuclear phagocytic system. 2/3 of the platelets are on the circulation 1/3 of the platelets are found on the spleen The platelet is composed about 60% protein, 30 % lipid, 8 % carbohydrate, various mineral, water and nucletotides. Platelet is anatomically divided into four areas: 1. Peripheral zone, 2. Sol-gel zone, 3. Organelle zone, 4. Membranous system
1. Peripheral zone – composed of the membranes and is responsible for platelet adhesion and aggregation a. Glycolayx – outer surface, fuzzy coating , primarily composed of glycoproteins including coagulation factors V,VIII and fibrinogen. b. Plasma membrane- bilayer of asymmetrically distributed phospholipids c. Submembranous area- where messages from external membrane are translated into chemical signals causing activation and physical change in platelet Glycocalyx A Fluffy coat This glycocalyx is unique among the cellular components of the blood. It is composed of plasma proteins and carbohydrate molecules that are related to the coagulation, complement, and fibrinolytic systems. The glycoprotein receptors of the **glycocalyx mediate the membrane contact reactions of platelet adherence, change of cellular shape, internal contraction, and aggregation. (Turgeon)
- Sol gel zone** a. Microfilaments – actin and myosin, which upon stimulation of platelet will interact to form actomyosin (thrombosthenin), a contractile protein for platelet contraction b. Microtubules – tubulin, maintains platelet disc shape Directly beneath the cell membrane is a series of submembrane filaments and microtubules that form the cellular cytoskeleton. In addition to providing the structure for maintaining the circulating discoid shape of the cell , the cytoskeleton also maintains the position of the organelles. A secondary system of microfilaments is functional in internal organization and secretion of blood coagulation products, such as fibrinogen. Micromegakaryocytes -Dwarf megakaryocytes -Lost their ability to undergo endomitosis -Myeloproliferative disorders -Resemble lymphocytes -Distingushing feature: Cytoplasmic blebs Phsphatidylserine- Platelet membrane phospholipid flips from the inner surface to the plasma surface on activation and serves as the assembly point for coagulation factors White clots: Largely composed of Platelets and Von williebrand factor Microparticles: Bud off of platelets after their exposure to strong agonists undergo^ last^ stageEhdomitosis^ to Personal ) reps PSOM
- 8, shape contraction scam aggregation membrane contact (^) rxn shape (^) actomyosin - - contraction organelles (^) tubulin -_ (^) Shape
C. Intravascular Component
- The key component in intravascular hemostatis are platelets and biochemical (procoagulants) in the plasma. Hemostasis, the arresting of bleeding, depends on several components. The four major components are the vascular system, platelets (thrombocytes), blood coagulation factors, and fibrinolysis and ultimate tissue repair. Three other, less important, components are the complement and kinin systems as well as serine protease inhibitors. (Turgeon) PRIMARY HEMOSTATIS - primary hemostatis is initiated by the exposure of platelets to the subendothelial connective tissue components blood vessels (Collagen, microfilaments, basement membranes). VASCULAR RESPONSE x Vascular injury to a large or medium-size artery or vein requires rapid surgical intervention to prevent exsanguination. When a smaller vessel, such as an arteriole, venule, or capillary, is injured, contraction occurs to control bleeding. This contraction of the blood vessel wall is called vasoconstriction. x Minimal interactions leading to platelet activation or clot formation occur between the circulating blood and intact endothelial surfaces. However, disrupted endothelial cells release thromboplastic substances that can initiate coagulation. Collagen, in particular, initiates contact activation of factor XII, thereby initiating blood coagulation. x Blood vessel endothelial functions are a. Angiogenesis = synthesis of stromal components b. Coagulation = vascular tone regulation c. Inflammation = special metabolic functions (blood vessel will undergoe vasodilation) d. Immune responses Vascular integrity or the resistance to vessel disruption requires three essential factors. These factors are circulating functional platelets , adrenocorticosteroids , and ascorbic acid. A Lack these factors produces fragility of the vessels, which makes them prone to disruption Endothelial Prothrombotic-Antithrombotic Balance Prothrombic Antithrombic Platelet activating factor Tissue factor Vwf Plasminogen activator Inhibitor- 1 Other coagulation factors (e.g factor V) Prostacylin Thrombomodulin Tissue plasminogen activator Urokinase Heparin like molecules Endothelial Vasoconstrictor-Vasodilator Balance Constrictor Dilator Endothelin- 1 Angiotensin- 2 Vasoconstrictor Prostaglandins Prostacyclin Nitric oxide PLATELET RESPONSE x Platelets have an average diameter of 2 to 4 mm, with younger platelets being larger than older ones. In contrast to megakaryocytes platelets have no nucleus. The cytoplasm is light blue, with evenly dispersed, fine red-purple granules. x An inactive or unstimulated platelet circulates as a thin, smooth-surfaced disc. This discoid shape is maintained by the microtubular cytoskeleton beneath the cytoplasmic membrane. x Platelets are extremely sensitive cells and may respond to minimal stimulation by forming pseudopods that spontaneously retract. Stronger stimulation causes platelets to become sticky without losing their discoid shape; however, changes in shape to an irregular sphere with spiny pseudopods will occur with additional stimulation. This alteration in cellular shape is triggered by an increase in the level of cytoplasmic calcium. x Agonists that lead to platelet activation are varied and include a nucleotide (ADP), lipids (thromboxane A2, platelet-activating factor), a structural protein (collagen), and a proteolytic enzyme (thrombin). Platelet Adhesion 9 When vascular injury occurs, platelet come in contact with subendothelium (collagen, fibronectin). vWf binds to glycoprotein Ib/IIb or the GP Ib/IX/V complex on platelet surface 9 If vascular injury exposes the endothelial surface and underlying collagen, platelets adhere to the subendothelial collagen fibers, spread pseudopods along the surface, and clump together (aggregate). Platelet adhesion to subendothelial connective tissues, especially collagen, occurs within 1 to 2 minutes after a break in the endothelium. Platelet Activation 9 Morphological and functional change in plates 9 Cyclooxygenase(from the platelets) metabolizes arachidonic acid to form prostaglandin enderoperoxides, which are converted to thromboxane A2 (a vasoconstrictor and a platelet stimulator, causing platelet secretion and aggregation) 9 Aspirin inhibits cyclooxygenase pathway(remission after 7 to 10days) Platelet secretion 9 Following activation,platelets will undergoes a shape change most probably caused by contraction of microtubules 9 From a disk shape to spherical shape with the extrusion of numerous pseudopods 9 Platelet granules move to the center of the platelets and fuse with the open canalicular system connected to the outside of the platelet; in this way the content of the granules are extruded to the outside I Intrinsic^ ) (^) collagen t X functional platelets^ ✓^ ✓ (^) ✓ Adreno corticosteroids Apc CAP ascorbic acid DTS ATTC ttnbrombin TXAZ collagen Plt -2 (^) Endothelial ↳ ↳^ SPHreleases^ TXAZRADP b^ surfaceProthrombin surface (^) ↳ Matt (^) .ofdzml6Pla/Ha ) V (^) Wf for^ collagen GPIBHXIV CAPT cyclooxygenase L^ pit^ ) Arachidonic^ tr acid t prostaglandin ↳ TXA
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Platelet aggregation 9 Simultaneously with platelet release, platelet stimulating agents (collagen, ADP, epinephrine, thrombin) bind to the platelets, causing then to adherence to one another 9 platelet stimulating agents such as collagen, ADP, epinephrine , thrombin bind to platelets, causing them to adhere to one another. Fibrinogen binds to GP IIb/ IIIa receptors on adjacent platelets and joins them together in the presence of ionized calcium 9 fibrinogen is necessary as cofactor for platelet aggregation 9 Bridges formed by fibrinogen in the presence of calcium produce a sticky surface on platelets. This results in aggregation. If these aggregates are reinforced by fibrin, they are referred to as a thrombus Platelet Plug Consolidation and Stabilization Fibrinogen, under the influence of small amounts of thrombin, provides the basis for this consolidation and stabilization. This process involves the precipitation of polymerized fibrin around each platelet. The result is a fibrin clot that produces an irreversible platelet plug. Vascular injury →exposes subendothelium and vasoconstriction→platelet adhesion→ platelet aggregation→ platelet plug formation → consolidation of platelets →fibrin stabilization SUMMARY OF MOST IMPORTANT SUBSTANCES SECRETED BY PLATELETS AND THEIR ROLE IN HEMOSTASIS ROLE IN HEMOSTASIS SUBSTANCE SOURCE FUNCTION Promote coagulation HMWK Alpha granules Contact activation of intrinsic coagulation pathway Fibrinogen Alpha granules Converted to fibrin clot formation Factor V Alpha granules Cofactor in fibrin clot formation FactorVIII:Vwf Alpha granules Assist platelet adhesion to subendothelial to provide coagulation surface Promote aggregation ADP Dense granules Promote platelet aggregation Calcium Dense granules Promote platelet aggregation Platelet factor 4 Alpha granules Promote platelet aggregation Promote vasoconstriction Serotonin Dense granules Promotes vasoconstriction at injury site Thromboxane A2 Membrane phospholipids Same Promote vascular repair Platelet derived growth factor Alpha granules Promotes smooth muscle growth for vessel repair Beta thromboglobulin Alpha granules Chemotactic for fibroblasts to help in vessel repair Other systems affected Plasminogen Alpha granules Precursor to plasmin, which induces clot lysis Alpha 2 antiplasmin Alpha granules Plasmin inhibitor; inhibits clot lysis C1 esterase inhibitor Alpha granules Complement system inhibitor PLATELET ACTIVATION PATHWAYS G proteins ¾ Controls the cellular activation for all cells at the inner membrane surface Eicosanoid ¾ Called as prostaglandin, cyclooxygenase, or thromboxane pathway ¾ It is triggered by G proteins Inositol triphosphate- Diacylglycerol ¾ Second G protein dependent activation SECONDARY HEMOSTATIS COAGULATION FACTORS / CLOTTING FACTORS NUMERAL PREFERRED NAME SYNONYMS I Fibrinogen II Prothrombin Prethrombin III Tissue factor Tissue thromboplastin IV Calcium Ionized calcium V (^) Proaccelerin Labile factor Accelerator globulin VII (^) Proconvertin, Stable factor Serum prothrombin conversion accelerator (SPCA), Autoprothrombin I VIII Antihemophilic factor (AHF) Antihemophilic factor A Antihemophilic globulin (AHG) Platelet cofactor I IX Plasma thrombopastin component (PTC) Christmas factor Antihemophilic factor B Platelet cofactor II X Stuart-Prower factor Stuart factor Prower factor Autoprothrombin III XI Plasma thromboplastin antecedent Antihemophilic factor C XII (^) Hagemen factor Glass factor Contact Factor XIII Fibrin stabilizing factor Laki- Lorand factor Fibrinase Plasma transglutaminase Fibrinoligase HMWK Fitzgerald factor Contact activation cofactor Williams factor Flaujeac Factor Pre Kallikrein Fletcher factor (PTA) FIBRINOGEN Most concentrated <100mg/dL = Prolong PT and APTT Platelet aggregation Absorbed, transported and released by Alpha granules Increase 10 mg/dL/decade in the elderly Ouren's disease/ Parahemophilia Hemophilia A/ Classic hemophilia Hemophilia B/ Christmas disease Hemophilia C/ Rosenthal syndrome Ashkenazi Jews FXII: No bleeding tendency 5M Urea Solubility Test/ Duckert's test BV (^) injury →^ TF →^ Thrombin^ PARI^ &^ PA^ Ra^ C^ Plt^ )^ =^ COAT^ p^ It Lil b (^) Bz (6101lb 11119 ) fibrinogen & VWF^ =p^ It^ agg^. CATE Plt →^ Plt GP (^) 1lb 11119 Fibrinogen O --
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NOMENCLATURE FOR FACTOR VIII of the International Committee on Thrombosis and Hemostasis VIII/vWF ¾ The entire molecule as it circulates in the plasma. Composed of VIII:C and VIII: vWF VIII: vWF ¾ Portion of molecule responsible for binding to endothelium and supporting normal platelet adhesion and function. Tested by bleeding time VIII: C ¾ Portion of molecule acting in intrinsic system as cofactor to factor IXa (with Ca++) in the conversion of factor X to Xa. Tested by APTT. VIIIC:Ag ¾ Antigenic property of procoagulant portion as measured by immunologic monoclonal antibody techniques VIIIR:Ag ¾ Factor VIII-related antigen, which is a property of thqe large vWF portion of the molecule and measured by immunologic techniques of Laurell rocket or immunoradiometeric assay. VIII: RCo ¾ Ristocetin (an antibiotic no longer used therapeutically) cofactor activity, which is factor VIII-related activity required for aggregation of human platelets with ristocetin in in vitro aggregation studies. SUBSTITUTION/ MIXING STUDIES Defiiency PT APTT TT SUBSTITUTION STUDIES NORMAL PLASMA
ABSORBED
PLASMA
AGED SERUM
I Abn Abn Abn C C NC II Abn Abn N C NC NC V Abn Abn N C C NC VII Abn N N C NC C VIII N Abn N C C NC IX N Abn N C NC C X Abn Abn N C NC C XI OR XII N Abn N C C C Fresh Plasma All coagulation factors are present Aged Plasma Factors V and VIII are absent Fresh Serum Factors I,V,VIII and XIII are absent, only ≤20% Factor II is detected Aged serum Factors I,II,V,VIII, and XIII are absent Adsorbed plasma Factors IX, X, VII, II are absent . . signingagain I f " Ebay mexoea.rs W x x x
MUST KNOW IN ORDER TO ASSESS COAGULATION SUSBSTITUTION STUDIES
1. Factors according to pathway A. Common pathway = B. Intrinsic pathway = C. Extrinsic pathway= 2. Factors/Pathway according to different coagulation test A. PT = B. APTT= C. Reptilase and Thrombin time (TCT)= D. Stypven time= **EXAMPLE CASES: a. Fibrinogen deficiency b. Factor IX deficiency c. Factor VII deficiency d. Factor XIII deficiency
- PT abnormal, PTT normal , TCT normal =
- PT abnormal , PTT abnormal , TCT abnormal =
- PT normal, PTT abnormal, TCT normal =
- PT normal, PTT normal, TCT normal = MIXING STUDIES a. Lupus anticoagulant / circulating anticoagulant b. Fibrinogen deficiency c. Factor II deficiency d. Factor VII deficiency
- Adsorbed plasma not corrected, Aged serum corrected , Fresh plasma corrected=
- Adsorbed plasma not corrected, Aged serum not corrected, fresh plasma corrected =
- Adsorbed plasma corrected, Fresh serum not corrected, fresh plasma corrected =
- Fresh plasma not corrected, Fresh serum not corrected, Adsorbed plasma not corrected = COAGULATION CASCADE Fibrinogen group/thrombin sensitive group Vitamin K dependent group**^ Contact group 9 Factor I, V, VIII, XIII 9 Consumed during coagulation 9 Absent in serum 9 Not absorbed by barium sulfate or aluminum hydroxide 9 Increases in inflammation, pregnancy, stress and fear, oral contraceptives 9 Factor VII, IX ,X, II 9 Requires vitamin K 9 Not consumed during coagulation 9 Absorbed by barium sulfate or aluminum hydroxide 9 Increases in pregnancy, and oral contraceptives 9 Factor XII, XI, HMKW, PK 9 Not consumed during coagulation 9 Not absorbed by barium sulfate or aluminum hydroxide NOTE - Vitamin K catalyzes an essential posttranslational modification of the prothrombin group proteins: gamma-carboxylation of amino-terminal glutamic acids INHIBITORS OF COAGULATION
- Protein C – major inhibitor of blood coagulation. Activated protein C is a strong anticoagulant and degrades factor Va and VIIIa and stimulates fibrinolysis by inactivating plasminogen activator inhibitors
- Protein S – servers as cofactor with protein C. 40 % - free form and active , 60% - bound to compelement c4 Binding protein and not active
- Antithrombin C – major inhibitor of thrombin. Enhanced by heparin
- Heparin cofactor II- inhibits also thrombin
- Alpha 2 macroglobulin- forms complex with thrombin, kallikrein, and plasmin thus inhibiting their activities
- Extrinsic pathway inhibitor (EPI) – Also called as lipoprotein associated coagulation inhibitor (LACI) , inhibits the VIIa-tissue factor complex
- C1-inhibitor- principle inactivator of factor XIIa and plasma kallekrein
- Alpha 1 antitrypsin- weak inhibitor of thrombin and factor Xa and Xia
- Activated protein C inhibitor- inhibit the activity of protein C
- Thrombin + thrombomodulin(endothelial surface) - modifies the action of thrombin to act more as inhibitor by inactivating protein C 10, 5, 2, 1 12, 11, 9, 8 3 and 7 Extrinsic and Common Pathway Intrinsic and Common Pathway Common Pathway Fibrinogen (^) Reptilase time -unaffected by Heparin . Inhibits main serine protease i
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SPECIMEN COLLECTION AND PROCESSING FOR HEMOSTATIS TESTING
I. Method of collection a. Two syringe method b. Evacuated tube technique
- These two methods are preferred because it requires that a few volume of blood should be discarded first then the next volume of blood is collected, this is done to prevent possible contamination of tissue fluid that contains thromboplastin which somehow alters results obtain from clotting test II. Special considerations and Information a. Anticoagulant- 0.105 to 0.109M (3.2% )buffered Sodium citrate b. Ratio of Blood to anticoagulant – ( 9:1) c. Citrate is the anticoagulant of choice for platelet function testing except for the glass bead retention test, which require heparin. d. Specimen processing for hemostasis testing should be done at 37 ‘C e. Labile factors (V and VIII) deteriorate at RT and increased pH f. Factors VII and XI prematurely activates when place at a cold temperature (4’C) g. Aspirin and other NSAID’s inhibit cyclooxygenase pathway
- Platelet function test : 1 week abstinence from the following drugs
- Bleeding time test : 24 hours aspirin intake is prohibited h. Platelet poor plasma (PPP) – centrifuged at 2000 x g for 10 minutes (steinenger), Centrifuged at 1500 x g for 15 minutes ( Rodaks ) , centrifuged at 2500 x g for 20 minutes (Turgeon) i. Platelet rich Plasma (PRP) – centrifuged at 60 - 100 x g for 10 minutes (steinenger), Centrifuged by 50 x g for 30minutes(Rodaks ) j. Coagulation specimen should be centrifuged within 1 hour of obtaining the sample k. Specimen processing for PT is valid within 24 hours l. Specimen processing for APTT is valid for 4 hours m. The PT and APTT , TCT are prolonged when Fibrinogen level is ≤100mg/dl(Rodak) , or less than 80mg/dl(Brown) n. The size of glass test tube used for PT and APTT is 12 x75mm(Steienenger) , or 13x 100mm(Brown) PRP (Platelet rich Plasma) can be obtained by centrifuging WB collected from 3.2% Sodium Citrate at 50g x for 30 minutes it contains 200,000 to 300,000 /ul platelets (200 to 300 x10^9 /L platelets) it is used for platelet aggregometry or platelet function test PPP(Platelet Poor Plasma) can be obtained by centrifuging WB collected from 3.2% Sodium Citrate at 1500g x for 15 minutes it contains less than 10,000/ul platelets it is used for clot-based coagulation test (E.g PT and APTT) FACTORS THAT INTERFERE WITH THE VALIDITY OF CLOT BASED TEST RESULTS Problem Solution Blood collection volume less than specified minimum PT falsely prolonged; recollect specimen Hematocrit >55% (polycythemia) Adjust anticoagulant volume using formula and recollect specimen using new anticoagulant volume Clot in specimen All results are affected unpredictably; recollect specimen Visible hemolysis PT falsely shortened; recollect specimen Icterus or lipemia Measure PT using a mechanical coagulometer. Heparin therapy Use reagent known to be insensitive to heparin or one that includes a heparin neutralizer such as polybrene Lupus anticoagulant PT result is invalid; use chromogenic factor X assay instead of PT. Incorrect calibration, incorrect dilution of reagents Correct analytical error and repeat test. 500 is mins 50 30 MIN AF agg re (^) geometry function tests = Clot based coagulation short draw^ : riffraff :^ FT Hemolysis :^ pre activation of cells^ :^ Ft G -- O
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LABORATORY TESTS FOR PRIMARY HEMOSTASIS
1.PLATELET COUNT
NV= 150- 400 x10 9 / L , 150 to 450 x10 9 (Rodak’s ) Thrombocytosis : PV, idiopathic thrombcythemia ,CML, splenectomy Thrombocytopenia: thrombocytopenia purpura, aplastic anemia, acute leukemia, pernicious anemia, Gaucher’s disease Dilution : 1: Squares: count 25/25 squares or 10 tertiary squares of the center square Microscope: Phase contrast microscope Formula : Platelet count x Dilution factor Area counted x 0.1 cm (depth) Manual platelet count Procedure (RODAK’S 5 th^ Ed.)
- Make a 1:100 dilution by placing 20 mL of well-mixed blood into 1980 ul of 1% ammonium oxalate in a small test tube.
- Mix the dilution thoroughly and charge the chamber. (Note: A special thin, flat-bottomed counting chamber is used for phase-microscopy platelet counts.)
- Place the charged hemacytometer in a moist chamber for 15 minutes to allow the platelets to settle.
- Platelets are counted using the 40x objective lens (400x total magnification). The platelets have a diameter of 2 to 4 um and appear round or oval, displaying a light purple sheen when phase-contrast microscopy is used. The shape and color help distinguish the platelets from highly refractile Dirt and debris. “Ghost” RBCs often are seen in the background.
- Count the number of platelets in the 25 small squares in the center square of the grid. The area of this center square is 1 mm^2. Platelets should be counted on each side of the hemacytometer, and the difference between the totals should be less than 10%. 6.calculate the platelet count
- The accuracy of the manual platelet count should be verified by performing a platelet estimate on a wright stained peripheral blood. NOTE! A. If fewer than 50 platelets counted on each side = repeat procedure and dilute blood to 1: B. If more than 500platelets counted on each side = repeat procedure and dilute blood to 1: DETERMINATION A. Direct o Reese ecker/ Toncantin’s method – sodium citrate, bicarbonate, brilliant cresly blue, formalin o Guy and leake – sodium oxalate, bicarbonate, crystal violet o Brecker – Cronkite – 1% sodium oxalate, uses phase contrast microscope o Unopette – 1 % ammonium oxalate as diluting fluid to lyse rbc and allows platelets to form pseudopods. - dilution factor is 1: - platelet count = initial platelet count x 1000 B. Indirect (Smear) o Dameshek o Fonio’s o Olek’s ¾ 1 platelet per 10 - 40 RBCs ¾ 3 - 10 platelets per 100 RBCs (or in 1 OIF) ¾ 5 - 20 platelets per 200 RBCs ¾ A normal blood smear should demonstrate approximately 8 to 20 platelets per field SMEAR: platelet estimate (factor x20,000) , counted on 10 OIF Platelet estimate Report platelet as 0 - 49 000 / Ul Marked decreased 50,000-99,000 / ul Moderate decreased 100,000-149,000 / ul Slight decreased 150,000-199,000 / ul Low normal 200,000-400,000 /ul Normal 401,000-599,000 /ul Slight increased 600,000-800,00 /ul Moderate increased Above 800,000 /ul Marked increased Manual Platelet count Normal Platelet count + prolonged BT 9 Qualitative platelet abnormality 9 Primary vascular abnormality 9 Von Willebrand’s syndrome Lowplatelet count + Normal BT 9 Autoimmune thrombocytopenia Low Platelet count + very prolonged BT 9 Simultaneous quantitative and qualitatitve platelet deficiency Significant Platelet Values
- less than 100,000/ul = abnormally low
- 30,000-50,000/ul = bleeding possible with trauma
- less than 30,000/ul = spontaneous bleeding possible
- less than 5,000/ul = severe spontaneous bleeding PICS
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5. CAPILLARY FRAGILITY TEST/RUMPEL- LEEDE TEST/HISS METHOD
Inflate BP cuff to a point hallway between the systolic and diastolic pressure (never exceed 100mmHg), maintain pressure for 5 minutes Remove BP cuff and wait for 5 to 10 minutes before proceding, count petechiae Positive test is found on thrombocytopenia, decreased fibrinogen and in vascular purpura Grade Description Numbers of petechiae 1+ Few petechiae on the anterior part of the forearm 0 - 10 2+ Many petechiae on the anterior part of the forearm 10 - 20 3+ Multiple petechiae over the wholearm and back of the hand 20 - 50 4+ Confluent petechiae on the arm and back of the hand 50 and above
6. GLASS BEAD RETENTION TEST An in vitro test for platelet adhesion Principle: as the anticoagulated blood passes through a glass bead column, normal platelets and vWF adhere to the glass bead surface that will result to a decrease in platelet count from the original platelet count. Uses 3 anticoagulated blood DECREASED PLATELET RETENTION INCREASED PLATELET RETENTION
- Glanzmann’s thrombasthenia
- Von willebrand disease
- BSS
- Chediak Higashi
- Aspirin administration
- Thrombotic disorders- such as venous thrombosis and pulmonary embolism
- Pregnancy
- Hyperlipidemia 7. BLEEDING TIME ( Test for Platelet Adhesion) In vivo measure of primary hemostasis The bleeding time test was first described by Duke This test provides an estimate of the integrity of the platelet plug and thereby measures the interaction between the capillaries and platelets. The bleeding time test was the original test of platelet function,although it is now largely replaced by near-patient analysis of platelet function using the PFA- 100 Test is affected if the platelet count is <75,000 x 10^9 /L As the platelet count drops below 100 × 10^9 /L, the bleeding time increases progressively from a normal of 3 to 8 minutes to more than 30 minutes. (Turgeon) A prolonged bleeding time in a patient with a platelet count greater than 100 × 10^9 /L indicates either impaired platelet function or a defect of subendothelial factor. The blood is blotted on the filter paper every 30 seconds Method Reference value Duke method-skinpuncture 2 to 4 minutes Ivy method – uses BP cuff inflated at 40mmHg 3 to 6 minutes Modified template method 6 to 9 minutes Anticoagulant: Heparin Earlobe .
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LABORATORY TEST FOR SECONDARY HEMOSTASIS
1. CLOTTING TIME
Measures period required for free formation of blood to clot after it has been removed from the body Capillary blood method N.V = 2-4 minutes
- Slide and drop
- Capillary tube/ Dale and Laidlaw’s Whole blood/ Lee and White method( Tilt tube method) N.V = 5-15 minutes
- uses glass tube 13x100mm in size 2. PROTHROMBIN TIME NV 10- 1 2 Seconds / 12.6 to 14.6 seconds (Rodaks 5th^ Ed.) Determine extrinsic and common pathway factor deficient Citrated blood →Centrifuged →PPP PPP + PT reagent PT reagent: tissue thromboplastin + CaCl 2 Begin the time for clot formation after the addition of calcium chloride The test is done at 37 ‘C If the procedure is performed in duplicate, the duplicate values must be within 10% of their mean or the test is repeated for a third time It is used to monitor Warfarin/Coumadin therapy
- PT reagents, often called thromboplastin or tissue thromboplastin, are prepared from recombinant or affinity-purified tissue factor suspended in phospholipids mixed with a buffered 0.025 M solution of calcium chloride. A few less responsive thromboplastins are organic extracts of emulsified rabbit brain or lung suspended in calcium chloride.
- Recently, however, newer generation thromboplastin reagents have been developed that are based on purified, recombinant human tissue factor that has been reconstituted into phospholipid vesicles Results are reported as International Normalized Ratio (INR) INR = Patient PT x 100 Control PT The closer the ISI is to 1, the more sensitive the reagent is the higher the ISI, the less sensitive the reagent INR CONDITIONS 2.0-2.5 DVT, high risk surgery 2.0-3.0 Hip surgery,femur fracture 2.0-3.0 DVT, Pulmonary embolism , transient ischemic attack 2.5-3.5 Mechanical/ prosthetic heart valves 2.0-4.5 Recurrent DVT and pulmonary embolism, myocardial infarction , arterial disease The ISI is a calibration parameter that defines the responsiveness of the reagent relative to a World Health Organization(WHO) International Reference Preparation, which by definition has an ISI of 1.0. 3. ACTIVATED PARTIAL THROMBOPLASTIN TIME NV 20-45 seconds Determines Intrinsic and common pathway factor deficient Monitors unfractionated heparin therapy Citrated blood →centrifuged →PPP PPP + APTT reagent PTT reagent contains = activator, Calcium chloride , and Phospholipids Activator such as kaolin, celite, silica and ellagic acid Phospholipids = which was historically extracted from rabbit brain, is now produced synthetically Begin the time for clot formation after the addition of calcium chloride The test is done at 37 ‘ C If the PTT is performed manually, the test should be done in duplicate, and the two results must match within 10%. It is standard method for monitoring unfractionated heparin therapy The test is affected and will give a prolong results in the presence of specific inhibitors/Lupus anticoagulant , Fibrin degradation products or paraproteinemia such as Multiple myeloma The PTT reagent contains phospholipid (previously called partial thromboplastin) and a negatively charged particulate activator such as silica, kaolin, ellagic acid, or celite in suspension. The phospholipid mixture, which was historically extracted from rabbit brain, is now produced synthetically 4. STYPVEN TIME/ RUSSEL VIPER VENOM TIME NV 6-10 Seconds From the viper ( Vipera ruselli ) venom / from the Daboia russelii viper(Rodak’s 5th^ edition) It bypasses coagulation by directly activating factor X Determines coagulation factor deficiency in the common pathway PPP + Stypven reagent(platelin-calcium chloride) It is now an obsolete test The diluted russel viper venom test (DRVVT) is used to detect and confirm lupus anticoagulant PT^ EXWdr DIP 10- 20-
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PLATELET DISORDERS
I. QUANTITATIVE PLATELET DISORDERS
A. THROMBOCYTOPENIA
- Decreased platelet production- Decreased megakaryocyte in the bone marrow. Congenital hypoplasia Aplastic anemia Fanconi syndrome Ineffective thrombopioesis Infiltration of the bone marrow by malignant cells TAR syndrome ( Thrombocytopenia with absent radii ) Acquired hypoplasia of megakaryocytes due to drugs, radiation, chemotherapy, or infectious agents Hereditary macrothrombocytopenia (e.g Alport’s syndrome)
- Decreased platelet survival time due to increased or decreased consumption Increased platelet destruction : Immunologic : Idiopathic thrombocytopenic purpura (ITP) – majority cases is due to viral infection Drug induced immunologic thrombocytopenia- antibiotics, hypnotics, analgesics, heavy metals, diuretics, etc. - Generally both the drugs and the antibody must be present in the system at the same time for destruction of platelets Post transfusion purpura – occurs 7 to 10 days after transfusion, due to anti – PIA or anti-HPA-1a(platelet antibodies). The recipient’s plasma is found to contain alloantibodies to antigens on the platelets or platelet membranes of the transfused blood product, directed against an antigen the recipient does not have. Isoimmune neonatal thrombocytopenia/Neonatal autoimmune thrombocytopenia – rare hemolytic disease of newborn. Result of transplacental transfer of maternal antiplatelet antibodies produced in response to fetal antigen inherited from the father. It is associated to a mother with ITP or SLE. Increased platelet consumption : Non immunologic: Thrombotic thrombocytopenic purpura (TTP) – rare disorder ,the exact cause is unkown. The development of TTP seems to be directly related to the accumulation of ultra-large von Willebrand factor (ULVWF) multimers in the plasma due to deficiency of the von Willebrand factorcleaving protease known as a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13). DIC HUS
- Increased platelet sequestration on the spleen
- Dilution of platelet count by multiple blood transfusions o Extensive blood transfusion often is accompanied by thrombocytopenia, the degree of which is directly proportional to the number of units transfused Immunologic Idiopathic thrombocytopenic purpura (ITP) Drug induced immunologic thrombocytopenia Post transfusion purpura (PTP) Isoimmune neonatal thrombocytopenia Non immunologic Thrombotic thrombocytopenic purpura (TTP) DIC HUS Destroy . consume
B. THROMBOCYTOSIS
- Reactive (secondary) thrombocytosis o Moderate increase, usually asymptomatic o Generally responds when the underlying disorder is treated like recovery from major surgery, splenectomy, after childbirth and acute blood loss o The term reactive thrombocytosis is used to describe an elevation in the platelet count secondary to inflammation, trauma, or other underlying and seemingly unrelated conditions. o Reactive thrombocytosis is not associated with thrombosis, hemorrhage, or abnormal thrombopoietin levels.
- Autonomous thrombocytosis o Marked increase, associated with thrombotic/ hemorrhagic complication o Primary or autonomous thrombocytosis is a typical finding in four chronic myeloproliferative disorders (CML, Polycythemia vera, Essential thrombocythemia, and Primary Myelofibrosis) II. QUALITITAVE PLATELET DISORDERS Qualitative platelet disorders can be attributed to adhesion, aggregation, or secretion defects. Release defects are the largest group of platelet function disorders. This condition is caused by abnormalities of signal transductase from membranes, abnormal internal metabolic pathways, or abnormal release mechanisms. **A. HEREDITARY / INHERITED PLATELET PROBLEMS
- Platelet adhesion problems** Von Willebrand’s disease (Vwd) 9 With five major subtypes 9 Inherited as both an autosomal dominant types and autosomal recessive trait 9 Normal with ______ 9 Abnormal aggregation with
Bernard – Soulier syndrome 9 Autosomal recessive trait 9 Platelets lack gp1b or the gp I/IX/V complex 9 Presence of giant platelets 9 Normal with ________________ 9 Abnormal aggregation with
CLASSIFICATION OF VON WILLEBRAND’S DISEASE Type Description 1 Partial quantitative deficiency of vWF 2 Qualitative deficiency of Vwf 2A Decreased platelet-dependent vwF function and selective deficiency of high molecular weight multimers 2B Increased affinity to platelet glycoprotein1b 2M Decreased platelet dependent vWF function with high molecular weight multimers present 2N Markedly decreased binding of factor VII to vWF 3 Complete deficiency of wWF ADP, Collagen, Epinephrine Ristocetin ADP, Collagen, Epinephrine Ristocetin
T (^) F -
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VASCULAR DISORDERS (PRIMARY HEMOSTASIS DISRODERS)
¾ Generally, the platelet count is normal, as are most platelet function and coagulation studies. The bleeding time and tourniquet test are usually abnormal Hereditary hemorrhagic telangiectasia / Osler- Weber-Rendu Syndrome x Most common inherited disorder x Inherited as autosomal dominant trait x Localized dilation of the walls of the small blood vessels of skin and mucous membranes; walls of the affected blood vessels are thin and lack of smooth muscle. x Telangiectasias are thin, dilated vessels. With a lesion ranges from pinpoint to 3mm in size and are red violet in color. Hereditary Hemangioma / Kasabach – Meritt syndrome x Disorder associated with tumors composed of blood vessels that commonly swell and bleed at the surface Ehlers Danlos Syndrome x Increased vascular fragility x Hyperextended joints and hyperplastic skins x Autosomal dominant x The defect may lie in a peptidase enzyme that converts procollagen to collagen Marfan syndrome x Increased vascular fragility x Autosomal dominant x Long extremities (long arm and legs) Pseudoxanthoma elasticum x Autosomal recessive x Connective tissue elastic fibers in small arteries are calcified and structurally abnormal x Subarachnoid and gastrointestinal bleeding are the most common causes of death Senile Purpura x Loss of elasticity of the skin x Usually due to aging, (elderly persons) x The aging process brings about a degeneration of collagen, elastin, and subcutaneous fat Scurvy x Deficiency of ascorbic acid x Acquired defect in the synthesis of collagen and hyaluronic acid Henoch-Schonlein purpura x Immunologic damage to endothelial cells (specific allergic purpura) x Gastrointestinal hemorrhage and joint swelling occur with purpuric rash x Most common in children and often follows upper respiratory infections PRINCIPAL HEREDITARY AND ACQUIRED BLEEDING DISORDERS ASSOCIATED WITH VASCULAR ABNORMALITIES ABNORMALITY HEREDITARY ACQUIRED Connective Tissue defects Ehlers Pseudoxanthoma elasticum-Danlos syndrome Vitamin C deficiency (Scurvy) Senile Purpura Corticosteroid purpura Aging Cushing’s disease Altered vessel wall structure Hemorrhagic telangiectasia Congenital Hemangiomata Diabetes Mellitus Amyloidosis Endothelial Damage Infectious purpura
- Bacterial: TB, Scarlet fever, typhoid fever, diphtheria, endocarditis, others
- Viral: small pox, influenza, measles, others
- Ricketssial: Rocky mountain spotted fever
- Protozoal: malaria Autoimmune vascular purpura
- Allergic purpuras: Henoch-Schonlein
- Drug induced purpuras: Quinine, Procaine, Penicillin, Aspirin, sulfonamides, sedatives , coumarins Miscellanoues abnormalities causing purpura secondary to vessel damage Waldenstrom’s macroglobulinemia Kaposi’s sarcoma Certain skin diseases Hemochromatosis Snake venom .
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COAGULATION DISORDERS
A. CLOTTING FACTOR DEFICIENCIES
FACTOR INHERITED COAGULOPATHIES ACQUIRED COAGULOPATHY
INHERITANCE
PATTERN
COAGULOPATHY
I
Autosomal recessive Autosomal dominant Afibrinogenemia Dysfibrinogenemia Severe liver disease DIC Fibrinolysis II Autosomal recessive Prothrombin deficiency Liver disease Vitamin K deficiency Anticoagulant therapy V Autosomal recessive Factor V deficiency (Owren’s disease) Severe liver disease DIC Fibrinolysis VII Autosomal recessive Factor VII deficiency Liver disease Vitamin K deficiency Anticoagulant therapy VIII x-linked recessive autosomal dominant Hemophilia A vWD
DIC
Fibrinolysis IX x-linked recessive Hemophilia B Liver disease Vitamin K deficiency X Autosomal recessive Factor X deficiency Liver disease Vitamin K deficiency Anticoagulant therapy XI Autosomal recessive Hemophilia C (common in eastern European, jewish descent / Ashkenazi jews) XII Autosomal recessive Factor XII deficiency XIII Autosomal recessive Factor XIII deficiency Liver disease DIC Fibrinolysis PreKallikrein Autosomal recessive Fletcher trait HMWK Autosomal recessive Fitzgerald trait CLINICAL MANIFESTATIONS OF COAGULATION FACTOR DEFICIENCIES Type of Bleeding Coagulation factor deficiency Easy bruising Factors II, VII, IX Hematoma Factors II, VII, IX Mucosal bleeding Factors II, VII, IX, XI Hemarthrosis Factors X, VII, IX Post- surgical bleeding Factors I,II,V,VII,VIII,IX,X,XI,XIII Intracranial bleeding Factors VII, VIII, IX, XIII Delayed wound healing Fibrinogen, Factor XIII Umbilical cord bleeding Factors X, XIII Miscarriage Fibrinogen, Factor XIII Thrombosis ABNORMAL FIBRINOGENS Asymptomatic/ No bleeding tendencies Factor XII, Prekallikrein, HMWK B. CIRCULATING ANTICOAGULANTS AND INHIBITORS ¾ Prolonged APTT and PT and NOT CORRECTED by the addition of normal fresh serum or plasma ¾ Inactivate an activated coagulation factor or block interaction between coagulation factors and platelet ¾ They can be IgM, IgG, or IgA ¾ Two group : a. the specific inhibitors against specific coagulation factors and b. non-specific inhibitors such as the LUPUS INHIBITOR – anti phospholipid antibody, Paraproteins and FDP’s. Test: a. Platelet neutralization test b. Dilute russel viper venom test (DRVVT) c. silica-based partial thromboplastin time (PTT), d. kaolin clotting time (KCT) e. Dilute thromboplastin time (DTT, also named tissue thromboplastin inhibitor test, TTI). C. LIVER DISEASE ¾ Associated with multiple factor deficiency without the presence of D-dimer ¾ Liver is one of the major site for coagulation factor synthesis ¾ Factor VII is the first coagulation factor to exhibit decreased activity in liver disease ¾ A more specific marker of liver disease because it is not affected by Vitamin K deficiency ¾ Prothrombin time (PT) Serves as a sensitive early marker for mild liver disease that can detect factor VII deficiency and Vitamin K deficiency ¾ In end-stage liver disease, the fibrinogen level may fall to less than 100mg/dl which is a mark of liver failure Delayed bleeding at the incision site Increased risk of thrombosis Most common hereditary thrombotic disorder Prothrombin 20210 mutation: 2nd most common hereditary thrombotic disorder
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