Documente Academic
Documente Profesional
Documente Cultură
2009-2010
DEFINITIE
Proces de formare a coagulului sanguin la locul de injurie a unui vas de sange pt a stopa pierderile de sange
Proces rapid, localizat si foarte bine reglat Sangerari anormale sau tromboze pot sa apara cand elemente specifice ale acestui proces lipsesc sau sunt disfunctionale
Elemente cheie
Plachete
Factori ai coagularii Sistemul fibrinolitic
DIDACTIC:
HEMOSTAZA PRIMARA dop plachetar instabil HEMOSTAZA SECUNDARA coagul fibrino-plachetar stabil
FIZIOLOGIC 4 faze:
Reducere sangerare
Agregare plachete pt formare dop plachetar Secretie eliberare continut plachetar = hemostaza primara
Plachetele sunt activate la locul injuriei pt a forma dopul plachetar initial ce asigura efectul hemostatic si stopeaza sangerarile mici si moderate Raspuns functional plachete:
ADEZIUNE depunere plachete pe matrixul subendotelial AGREGARE coeziune plachete-plachete SECRETIE eliberare continut granule ACTIVITATE PROCOAGULANTA amplificarea formarii de trombina
Interactioneaza cu factorii eliberati de plachete Activeaza cascada coagularii pornind de la nivelul plachetelor activate
Previne hemoragia
Interventie mecanisme de control (t-PA) limiteaza formarea dopului fibrinoplachetar doar la nivelul injuriei
Cascada coagularii
Conversia in cascada a unor serii de proenzime inactive in enzime active formare trombina Trombina converteste fibrinogenul plasmatic solubil in fibrina insolubila
FACTORII COAGULARII
1.
FACTORII COAGULARII
2. Cofactori solubili:
procoagulanti
F V proaccelerina F VIII antihemofilic A F von Willebrand molecula carrier pt FVIII Proteina S anticoagulant, cofactor clivare si inactivare Va, VIIIa
3. Factori de contact:
F XI activat in vitro de F XII, in vivo nu necesita obligatoriu factorii de contact, poate fi activat de trombina de pe supraf plachetara F XII - Hageman Responsabili de activarea de HMWK(kininogen) - Fitzgerald contact a coagularii Prekalikreina- Fletcher
Factorul tisular TF (III, tromboplastina tisulara) factor major de initiere coagulare in asociere cu VIIa
Inhibitorul caii factorului tisular- rol inhibitor pt fact Xa si VIIa/TF Antitrombina III ATIII- blocheaza situsurile active ale trombinei,
fact Xa si IXa
Proteina C sinteza hepatica, activata de complexul trombinatrombomodulina, actioneaza ca si cofactor pt proteina S in inactivarea factorilor Va, VIIIa
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trombina
CELULA TF+
TF, receptor pt F VII
VII
VIIa/TF cx
X IXa VIIIa Xa Va II IIa
Activare IX
XIa
Tr activat
ADP adrenalina Tromboplastina intrinseca
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Caile coagularii
Doua cai de formare a fibrinei Intrinseca Initiata de suprafete incarcate negativ Extrinseca Initiata de injuria tisulara Ambele converg catre cale finala comuna Protrombina Trombina Fibrinogen Fibrina coagul
Controlul coagularii
Antitrombine (e.g., antitrombina III) Proteina C and S Sistemul fibrinolitic Plasminogen plasmina produsi de dgradare ai fibrinei (PDF) D-dimer reprezentant major al PDF
Sistemul fibrinolitic
Fibrinoliza
http://www.si.mahidol.ac.th/department/biochemistry/home/md/courseware_202/lec/hemostasis_lec.pdf
Reglarea fibrinolizei
http://www.si.mahidol.ac.th/department/biochemistry/home/md/courseware_202/lec/hemostasis_lec.pdf
Dezechilibru hemostaza
coagulare antifibrinoliza
defect
Hemoragii
fibrinoliza
anticoagulare
defect
Tromboze
Tulburari hemoragice
Anomalii vasculare
Anomalii plachetare
CID
Plasma Componenta noncelulara a sangelui Ser Plasma fara fibrinogen si Alti factori ai coagularii
Evaluare de laborator
Timp vasculo-plachetar
- numar trombocite - teste functionale agregare plachetara activitatea ristocetinei - timp de sangerare - testul garoului (Rumpel-Leede)
NUMAR TROMBOCITE Numarare manuala imprecisa, consumatoare de timp Metode automate - rapid, precis Metode optice Flow cytometrie N = 150,000 - 450,000 /mcL - trombocitopenia risc crescut de sangerare - trombocitoza tromboze
TESTE FUNCTIONALE PLACHETARE Raportul numarului de plachete pt evaluare functionala raportul intre numarul de plachete dintr-o proba de sange, inainte si dupa stimularea cu un agonist (ADP, epinefrina) evalueaza capacitatea de agregare corelat bine cu agregometria
Acid arahidonic. Trombina ADP Epinephrine Collagen Ristocetin- necesita FWv si receptori membranari integrii
Agregometria plachetara Clasica plasma imbogatita in plachete (obtinuta prin centrifugare) stimulata cu ADP, epinefrina, ristocetina evaluare agregate prin turbidimetrie Depinde de functia normala a plachetelor, de concentratia de agonisti nu poate detecta agregate formate din mai putin de 100 Tr Agregometrie sange integral Prin impedanta electrica Light scattering methods Analiza flow-citometrica a marimii particulelor
Expunere plachete sange integral la un flux rapid (5000 to 6000/sec) prin tuburi capilare si monitorizarea scaderii fluxului pe masura formarii dopului hemostatic in centrul unei membrane acoperite cu ADP si colagen
Rezultatul testului exprimat ca timp de inchidere Poate inlocui timpul de sangerare in vivo Test screening pt evaluare functionala Nu este speific unei anomalii
Masoara afinitatea de legare a factorului vW la receptorul plachetar GP Ib Plasare plachete pacient intr-o serie de eprubete in care se adauga concentratii joase de ristocetina (0.4 - 1.2 mg/mL) Se urmareste formarea sau nu a agregatelor in fiecare tub
VWF antigen - ELISA VWF activitate Activitatea cofactorului ristocetinic (VWF:RCo), "gold standard" pt activitatea VWF
TIMPUL DE SANGERARE Metoda Duke (lobul urechii) Tehnica: dezinfectare lob ureche sau pulpa deget inelar, intepatura de 3-4 mm adancime, tamponare cu hartie de filtru din 30 in 30 de secunde pana la oprirea sangerarii. Normal = 2-4 minute
Metoda Ivy (antebrat) Normal = 4 10 minute Test de sange ce urmareste cat de repede se opreste sangerarea de vase mici Slab reproductibil, invaziv, consumator de timp Cu toate acestea - screening test de prima intentia in clinica
Valori crescute trombocitopenie, CID, boala Bernard-Soulier , trombastenia Glanzmann, aspirina, vasculopatie, rar coagulopatie
1.
PROBA VENTUZEI Tehnica: decompresiune a unei suprafete cutanate cu ajutorul unei palnii de sticla sau ventuze conectate la o pompa aspiratoare si manometru cu mercur - se determina momentul de aparitie si numarul de petesii aparute ca rezultat al decompresiunii. Rezistenta capilara normala nu apar petesii pana la o decompresiune de 20-25 mm Hg la adult, 30 mm Hg copil. PROBA RUMPELL-LEEDE Tehnica: tensiometru aplicat deasupra cotului si se ridica presiunea la o valoare intre presiunea sistolica si cea diastolica timp de 5 minute. Rezistenta capilara normala nu apar petesii. Fragilitate capilara cu atat mai mare cu cat numarul si intinderea petesiilor sunt mai mari.
Intrucat rezistenta si permeabilitatea capilara depind de integritatea morfologica si functionala a vasului dar si de numarul si calitatea trombocitelor, proba ventuzei si Rumpell-Leede sunt pozitive atat in vasculopatii cat si in afectiuni 35 plachetare.
Cascada coagularii
PT, INR
TT
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Timpul de tromboplastina partiala activata (aPTT) cefalina (tromboplastina partiala) + plasma cercetata expusa la suprafete incarcate negativ (activata) , aceasta fiind diferenta fata de PTTn=40-60 s APTT deficit factori coagulare IX, XI, VIII, XII, PK, HMWK, sau prezenta inhibitori -aPTT descopera inhibitorii obisnuiti: anticoagulanti lupici, Ac anti-fosfolipid care interfera cu reactivul fosfolipidic folosit in reactie diluarea plasmei scade concentratia inhibitorilor si aPTT tinde sa scada -aPTT folosit pt monitorizarea terapiei cu heparina: aPTT=1,5-2,5x timp referinta
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este singurul test care se lucreaza pe ser si singurul la care valorile scazute sunt patologice evidentiaza indirect deficite factori coagulare XI, IX, VIII
Mecanism: protrombina reziduala din ser se transf in trombina in prezenta tromboplastinei calcice. Deficit al formarii protrombinazei (IX, XI, VIII) face sa ramana cant mare de protrombina care in prezenta tromboplastinei calcice formeaza cheagul mai rapid.
CALE INTRINSECA
Suprafata incarcata negativ (in vitrosticla, caolin, dextransulfat) PK, HMWK
T Howell=1,12,1 min
CALE EXTRINSECA
Factorul tisular
XII
XIIa
VII
VIIa-
Ca2+
XI
XIa
Ca2+
IX IXa
TCP (30)
TENAZA Ca2+ PL membranar X Xa PROTROMBINAZA Ca2+ PL membranar
TQ/TP (11-15 )
VIII
VIIIa
Va
II
IIa
Ia
Teste de corectie
constau in refacerea timpului Howell si aPTT cu reactivi ce contin factorul considerat deficitar Reactivii folositi sunt: plasma normala proaspata contine toti factorii (PN) plasma normala proaspata adsorbita pe sulfat de bariu contine F VIII si XI (PAD) ser vechi contine F XI si IX (SV) cefalina in suspensie (C) daca se lucreaza T Howell, aici participa si Tr
eprubeta Reactiv Corectia testului
E1
PN
E2
PAD
E3
SV
E4
C Deficit de factor
+ + + +/-
+ + -
+ + -
VIII
Hemofilia A
IX
Hemofilia B
XI
Hemofilia C
Fp3
Inhibitori protrombinaza
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II. TROMBINOFORMAREA
timpul Quick / timp protrombina = plasma de cercetat (citratata, fara Tr post centrifugare la 2500 g, 15 min) + tromboplastina calcica n= 11-15 sec. evalueaza factorii II, V, VII, X, trombino, fibrinoformarea cale extrinseca - deficit II, V, VII, X, deficit vit K, hipo/afibrinogenemii INR= International Normalized Ratio protrombine time ratio (PTR)=timp protrombina pacient/timp martor (PTR =1 la n, =1,5-2,5 la cei cu anticoagulante orale)
VII
T QUICK
X
II V
FIBRINOGEN
Testul Koller = vitamina K parenteral la subiecti TQ + determinare TQ TQ normal deficit aport sau absorbtie vitamina K TQ alungit defect de utilizare
+ Ca tromboplastina
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III. FIBRINOFORMAREA timpul de trombina = timp coagulare plasma oxalatata + exces trombina= timp de conversie fibrinogen fibrina timpul de reptilaza
reptilaza coaguleaza fibrinogenul si este insensibila la anticoagulanti in prezenta inhibitori polimerizare fibrina, normal in caz anticoagulanti antitrombinici - deficit sau exces (inhiba activitatea trombinica) de fibrinogen; fibrinogen n cu anomalii fct.; inhibitori fmare fibrina: substante heparin-like (neoplazii), exces PDF plasma normala corecteaza TT in hipo si afibrinogenemii, partial in fibrinoliza acuta
TIMP TROMBINA
TIMP REPTILAZA
DIAGNOSTIC
HIPOFIBRINOGENEMIE
N
N
DISFIBRINOGENEMIE
ANTICOAGULANTI CIRCULANTI
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Retractia cheagului
metoda de explorare a timpului trombodinamic investigheaza indirect si hemostaza primara, depinde de activarea trombocitara, se desfasoara normal in functie de numarul si functia trombocitelor recoltare 5 ml sange, eprubeta in baie de apa la 37 grade, se apreciaza retractia cheagului la 2 si la 24 ore prin cantitatea de ser expulzat n= 30% ser expulzat la 2 ore si 45% dupa 24 ore varianta: apreciere retractie pe lama timpul necesar pt aparitia unei picaturi de ser pe lama cu o pic sange n=30 min
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EVALUAREA FIBRINOLIZEI
Extrinsec: t-PA, urokinaza Plasminogen Intrinsec: factor XIIa, HMWK, kallikrein Exogeni: streptokinaza Fibrina, fibrinogen Plasmina Fibrina, fibrinogen, produsi degradare
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viteza inalta, deoarece contin inhibitor-1 al plasminogenului) diluata cu apa distilata pt a scadea puterea inhibitorilor fibrinolizei + acid acetic precipitare fractiune euglobuline (factori ai coagularii, t-PA, complex activator tisular plasminogen-inhibitor1 al activatorului tisular al plasminogenului) dizolvare precipitat in mediu tamponat coagulare prin adaos de trombina cheag euglobulinic liza cheagului euglobulinic in 60 minute interpretare: TLCE - sindrom fibrinolitic supraacut < 15 min, acut 15-30 min, subacut 30-60 min nu poate face diferenta intre un sindrom fibrinolitic primar (crestere t-PA, scadere inhibitor t_PA) si unul secundar (CID)
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PDF D-dimeri
PDF D-dimeri --
PDF D-dimeri
CID
FIBRIN OLIZA PRIMAR A
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Proteinele reglatorii
Antitrombina-III: inhibitor natural al coagularii Sintetizata de ficat, inhiba activitatea trombinei, factorilor IXa, Xa, XIa, XIIa, plasminei Inhibitia trombinei de catre AT-III este accelerata de heparina Proteina C: proteaza vit K dependenta Inactiveaza Va, VIIIa si accentueaza fibrinoliza Deficit - risc tromboembolic Determinari imunoelectroforetice sau ELISA Proteina S: cofactor proteina C, Determinare imunologica sau functionala
Thrombocytopenia
Von Willebrands disease Drugs (Aspirin, NSAIDs, high dose penicillins, etc.) Cirrhosis, Uremia, PLTs dysfunction
Heparin
Excessive Warfarin
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TESTE SCREENING:
Numar trombocite Timp partial tromboplastina activata / PTT, aPTT INR, timp protrombina/TQ Timp trombina Timp sangerare Fibrinogen, dimeri Retractia cheagului
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Laboratory Tests of Hemostasis by Phase Test Purpose Formation of initial platelet plugs Platelet count Bleeding time Quantifies platelet number Screens for overall adequacy of platelet adhesion and aggregation on injured vascular surfaces Evaluates adequacy of platelet responsiveness to physiologic stimuli that activate platelets (eg, collagen, adenosine diphosphate, arachidonic acid) Patterns are abnormal in hereditary or acquired platelet functional disorders Measures total concentration of plasma VWF protein Evaluates distribution of VWF multimers in plasma (eg, large multimers are missing in type II variants of VWD)
Platelet aggregation
Test
Purpose
Formation of initial platelet plugs Ristocetin agglutination Screens for large multimers of VWF in plasma (often done as part of routine laboratory evaluation for VWD)
Laboratory Tests of Hemostasis by Phase Test Purpose Formation of fibrin PT PTT Screens for the factors in extrinsic and common pathways (factors VII, X, and V; prothrombin; and fibrinogen) Screens for the factors in intrinsic and common pathways (prekallikrein; high mol wt kininogen; factors XII, XI, IX, VIII, X, and V; prothrombin; and fibrinogen) Determines activity as a percentage of normal Evaluates the last step of coagulation (thrombin cleavage of fibrinogen to fibrin) Is prolonged by heparin activation of antithrombin and in conditions resulting in qualitative fibrinogen abnormalities or hypofibrinogenemia If it is normal and thrombin time is prolonged, provides presumptive evidence that a plasma sample contains heparin because reptilase time is not affected by heparin activation of antithrombin Quantifies plasma fibrinogen, which is increased in acute phase reactions and decreased in severe liver disease and severe DIC
Reptilase time
Fibrinogen level
Fibrinolysis Clot stability during 24-h incubation in saline and in 5M urea Plasminogen activity 2-Antiplasmin
Plasma D-dimer
Causes lysis of clots in saline if fibrinolytic activity is excessive or in 5M urea if factor XIII is deficient Should be done in patients with defective wound healing or frequent miscarriages Quantifies plasma plasminogen, which is decreased in patients with congenital early-onset venous thromboembolism (rare) Quantifies plasma level of this fibrinolysis inhibitor, which is reduced in patients with excessive bleeding due to increased fibrinolysis (rare) Screens for DIC Decreased levels when plasmin has acted on fibrinogen or fibrin in vivo (eg, in DIC) Superseded by plasma D-dimer assay Is measured with a monoclonal antibody latex agglutination test or with an ELISA If high, indicates that thrombin has been generated in vivo with resultant deposition of fibrin, activation of the cross-linking enzyme, factor XIII, and secondary fibrinolysis Is useful in the diagnosis of in vivo thrombosis (eg, deep venous thrombosis, pulmonary embolism), especially the sensitive ELISA version