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Blood Coagulation - Screening assays and single factor assessment -
Jørn Dalsgaard Nielsen Thrombosis Centre Gentofte Hospital Copenhagen, Denmark JDN
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SUBENDOTHELIAL TISSUE
ENDOTHELIAL CELLS ADP Ca++ Serotonin Prostacyclin Nitric oxide Inhibition PF4 Activated platelet Tromboxane A2 Activation Activation of monocytic cells, induced by endotoxin, cytokines, etc. Aktivation of monocytic cells, induced by endotoxin, cytokines, etc. PF3 GP Ib-IX Platelet adhesion og activation GP IIb-IIIa IL-1 TNF GP Ib-IX ¤ von Willebrand factor ¤ Collagen Tissue factor Released from TNF or IL-1-activated endothelial cells F VII F VIIa TFPI Inactivates F VIIa ¤ F X ¤ tissue factor -kompleks F IX F X Endothelial damage F XIa PF3, Ca++, F VIIIa F Xa F XIa F XI ¤ HMWK---- F XII---- PK ¤ HMWK---- F XIIa Contact activation Thrombomodulin F VIIIi PS F VIII PC PCa F V Kallikrein F Vi Prothrombin PF3, Ca++, F Va t-PA Thrombin PAI-1 Inhibits serine proteases Plasmin Plasminogen F XIII Fibrinogen α2-antiplasmin Antithrombin Fibrinogen/-Fibrin degradation products Proteoglycans Heparin Cofactor II Polymerizing fibrin Inhibits thrombin F XIIIa Crosslinked fibrin
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Is testing of single factors necessary in patients with suspected haemostatic dysfunction ?
It depends on who you are addressing A surgeon: Will the patient bleed ? Can I stop bleeding with fresh-frossen plasma ? A haematologist: Single factor assessment is often necessary to establish a correct diagnosis
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Indications for evaluation of haemostatic function
Clinical problem Biochemical defect? Biochemical defect Clinical problem? Bleeding Thromboembolism Coagulation Fibrinolysis Platelets Endothelium Screening Further screening Single factor assay Prophylaxis/ treatment indicated? or not? Abnormal test result Further screening Single factor assay
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The challenge of evaluation of clotting abnormalities
In vitro assessment of haemostasis is difficult because the important interaction between the endothelium and blood components cannot be evaluated in a single assay. So-called ”global tests” can be used to test the haemostatic capacity of blood components (plasma and blood cells) but not the influence of antithrombotic properties of the endothelium. Thrombelastography may give a clue of a clotting defect, platelet dysfunction, or hyperfibrinolysis but will not give the final diagnosis.
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Increased in patients with clotting defects
Thrombelastography Increased in patients with clotting defects Decreased in patients with platelet dysfunction or defect fibrin formation Amplitude Reaction time TEG Start Minutes
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Thrombelastography LA and HIT-2 are associated with a high risk of thrombosis A shortened reaction time might, therefore, be expected but is not seen because the thrombotic predisposition is provoked by endothelial dysfunction TEG
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The challenge of evaluation of clotting abnormalities
As global tests of haemostasis neither give a consice diagnosis nor results that reliably reflect the clinical problem, more specific assays are often needed for the evaluation of thrombotic and haemorrhagic disturbances of the haemostatic system. However, separation of the complex network of reactions may result in a number of other pitfalls and impede a comprehensive view.
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The challenge of evaluation of clotting abnormalities
Among laboratory testing, coagulation assays are the most influenced by the inaccurate standardization of the pre-analytical phase. Clotting times are influenced by: time of tourniquet placement (<60 sec recommended) needle size (19-22 gauge recommended) citrate concentration ( mM recommended) incomplete filling of tubes (PT<80%, APTT<90%) platelet count (<10*109/l recommended) haemolysis and lipaemia temperature and G-force during centrifugation temperature and duration of storage until testing
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The challenge of evaluation of clotting abnormalities
Screening methods of coagulation should optimally be sensitive to all coagulation defects. This is not the case but by combination of simple procedures we can get close to the final diagnosis.
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Exploring coagulation
The present theory of the function of the coagulation system is based on numerous studies performed in the 20th century. The history of the discovery of clotting factors and development of assays may help understanding the use of screening assays of coagulation.
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The theory of blood coagulation Year 1900: the ’four factor’ theory
Known factors: Fibrinogen Prothrombin Thromboplastin Calcium Cellular damage Prothrombin + Ca Fibrinogen Thromboplastin Thrombin Fibrin Hammerstein. Hoppe-Seylers Zeitschrift für physiologische Chemie 1899; 28: 98. Morawitz, P. Ergebnisse der Physiologie biologischen Chemie und Experimental Pharmakologie 1905; 4: 307.
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The theory of blood coagulation Year 1935: the ’Quick’ test
Determination of the clotting time of citrated plasma after addition of thromboplastin and calcium chloride Prothrombin + Ca Fibrinogen Thromboplastin Thrombin Fibrin Quick AJ. J Biol Chem 1935; 109: LXXIII
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The theory of blood coagulation Year 1947: Factor V
Cellular damage Prothrombin + Ca Fibrinogen Factor V Thromboplastin Thrombin Fibrin Owren PA. Acta Med Scand 1947; Suppl: 194
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The theory of blood coagulation Year 1947: Factor V
Cellular damage Prothrombin + Ca Fibrinogen Factor V Thromboplastin Thrombin Factor V deficiency showed to be a rare disease, and the discovery of FV did not explain the puzzle that the standard coagulation test: the Quick test, was normal in most patients with congenital bleeding tendency. Fibrin Owren PA. Acta Med Scand 1947; Suppl: 194
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Mixing assays Whole blood clotting time and plasma clotting time are prolonged in haemophiliac patients and can be normalized by mixing patient blood/plasma with equal parts of normal blood/plasma. Both tests, however, have high CV%.
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First description of APTT
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The theory of blood coagulation Year 1953: APTT
Prothrombin + Ca Thrombin Fibrinogen Fibrin Factor V Thromboplastin Ca++ PT ”Partial thromboplastin” Ca++ Unknown factors Incubation APTT Kaolin Langdell et al. J Lab Clin Med 1953;41:
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The theory of blood coagulation Year 1959: the Roman numerical nomenclature
Factor Synonyms I Fibrinogen II Prothrombin III Thromboplastin IV Calcium V Accelerator globulin; proaccelerin; labile factor VI Factor V derivative (not used now) VII Proconvertin; stable factor; autoprothrombin I VIII Antihaemophilic factor A; platelet cofactor 1 IX Plasma thromboplastin component (PTC); Christmas factor; antihaemophilic factor B; autoprothrombin II; platelet cofactor 2 X Stuart-Prower factor XI Plasmathromboplastin antecedent (PTA) XII Hageman factor XIII Fibrin stabilizing factor suggested by an international committee under the chairmanship of Dr. IS Wright
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The theory of blood coagulation Year 1964: the cascade scheme
Surface contact Problems XII XIIa XI XIa IX IXa ? VII ? VIII VIIIa ? X Xa V Va II IIa I Ia (fibrin) Macfarlane, RG. Nature 1964; 202: 498
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The theory of blood coagulation Year 1975: the classic coagulation system
Internal pathway Surface contact External pathway Tissue factor XII XIIa XI XIa IX IXa VIIa VII Ca++ Phospholipid, Ca++, VIII X Xa X Phospholipid, Ca++, V II IIa I Ia (fibrin) Austen DEG & Rhymes. A laboratory manual of blood coagulation
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The theory of blood coagulation discoveries of the last decades
The major in vivo importance of the external pathway Acceleration of coagulation by positive feed-back mechanisms Inibitory mechanisms of blood coagulation
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The theory of blood coagulation today
Tissue factor VII VIIa
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EXPRESSION OF TISSUE FACTOR
CONSTITUTIVE e.g.: epithelial cells glial cells INDUCED e.g.: monocytic cells endothelial cells PROHIBITED e.g.: lymphocytes erythrocytes IL-1 TNF- C5a
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The theory of blood coagulation today
Activation by a serine protease, e.g. hepsin Tissue factor VII VIIa XI XIa IX IXa VIII VIIIa X Xa V Va XIII IIa II XIIIa XL-Fibrin Fibrin Fibrinogen
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The theory of blood coagulation today
Activation by a serine protease, e.g. hepsin Tissue factor XII ? VII VIIa XI XIa IX IXa VIII VIIIa X Xa V Va XIII IIa II XIIIa XL-Fibrin Fibrin Fibrinogen
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Endothelial damage Zn2+ F XIa F XI ¤ HMWK---- F XIIa F XII----
Activated platelet Endothelial damage Zn2+ F XIa F XI ¤ HMWK---- F XII---- PK ¤ HMWK---- F XIIa activation Kallikrein urokinase prourokinase t-PA PAI-1 Plasmin Plasminogen
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Natural inhibitors of blood coagulation
Tissue factor TFPI VII VIIa XI XIa AT IX IXa HC-II VIII VIIIa Endothelial cell PCa X Xa PS XIII V Va PC IIa II XIIIa TM XL-Fibrin Fibrin Fibrinogen
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The classic coagulation system
APTT Surface contact Prothrombin time Tissue factor XII XIIa XI XIa IX IXa VIIa VII Ca++ Phospholipid, Ca++, VIII X Xa X Phospholipid, Ca++, V II IIa Thrombin time I Ia (fibrin) Austen DEG & Rhymes. A laboratory manual of blood coagulation
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Clotting defects and bleeding
Coagulation factor deficiencies seldom cause bleeding if the level of the deficient factor is >40%. APTT is normal when the level of coagulation factors is >40%. Therefore, APTT determined in a mixture of equal parts of normal plasma and plasma from a haemophiliac patient will be normal. Unless an inhibitor is present.
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Antibodies against coagulation factors
Two types: Alloantibodies: Patients with hereditary coagulopathy may develope antibodies against the deficient factor when treated with plasma-derived or recombinant factor concentrates Autoantibodies: Aquired antibodies, most often against factor VIII and typically in patients with autoimmune diseases, malignancy and in women during pregnancy and post partum. In half of the cases no underlying disease can be found. Incidence: 1-5 per
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APTT-based inhibitor test
Add APTT reagents Determine APTT mix Patient plasma Normal plasma
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APTT-based inhibitor test
Patient plasma Normal plasma mix Haemophilia Factor: <1% % >50% APTT: normal Patient plasma Normal plasma mix Patient with inhibitor Factor: <1% % <50% due to excess of antibody APTT: prolonged
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Treatment of bleeding in patients with antibodies against coagulation factors
In some patients (low responders) the neutralizing effect of the antibody can be overcome by increasing the dose of factor concentrate In patients with high titers of antibody recombinant factor VIIa can be used to obtain haemostasis
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The ”by-passing effect” of factor VIIa
FVII FVIIa FIX TF FXIa FXI INITIATION Platelet activation FIXa AMPLIFICATION FVIIIa FVIII FX FXa FVa FV Prothrombin Thrombin Fibrin Fibrinogen FXIIIa FXIII Cross-linked fibrin FIBRIN FORMATION
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30-year old female with refractory bleeding
Aquired factor VIII deficiency with a progressive inhibitor to factor VIII. Bethesda titer: 5.5 BU.
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Algoritm for evaluation of prolonged APTT
Exclude preanalytical factors causing spuriously prolonged APTT Underfilled tubes, delayed testing, venipuncture above heparin lock etc. Is the patient receiving antithrombotic treatment? E.g. heparin, thrombin inhibitors, vitamin K antagonists, fibrinolytics Defect fibrin formation? Determine fibrinogen concentation thrombin time If not – then test for inhibitors Lupusinhibitor (Thrombophilia) Antibodies against a coagulation factor (Haemophilia, aquired/cong.)) If neg. inhibitor test: Coagulation factor deficiency Contact factor deficiency (No bleeding) Deficiency of other clotting factors (Haemophilia)
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Algorithm for evaluation of prolonged APTT
Explore possible AC treatment: Heparin: Thrombin time Vitamin K antagonist: INR Fibrinogen < 3 M Yes Explore hypofibrinogenaemia No Corrects APTT Pt:NP 1:1 mix immediate APTT Pt:NP 1:1 mix Incub 2h APTT Fails to correct APTT Fails to correct APTT Corrects APTT Phospholipid dependent Inhibitor present Factor deficiency Procoagulant factor deficiency Lupus inhibitor Fails to correct APTT Pt:APTT-reagent Incub 10 minutes Phospholipid independent Corrects APTT Contact factor deficiency Specific inhibitor
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Prolonged preincubation with APTT reagent
PK-deficient plasma Asmis et al. Thromb Res 2002;105:463-70
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Algorithm for evaluation of prolonged APTT
Explore Thrombin time: Heparin? INR: Vitamin K antagonist? Fibrinogen > 3 M No Explore hypofibrinogenaemia Yes Corrects APTT Pt:NP 1:1 mix immediate APTT Symptoms Bleeding Pt:NP 1:1 mix Incub 2h APTT None Thombosis Fails to correct APTT Fails to correct APTT Corrects APTT Phospholipid dependent Inhibitor present Factor deficiency Procoagulant factor deficiency Lupus inhibitor Fails to correct APTT Pt:APTT-reagent Incub 10 minutes Phospholipid independent Corrects APTT Contact factor deficiency Specific inhibitor
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Evaluation of 177 consecutive cases of prolonged APTT
Results Chng et al. 2005
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Evaluation of 177 consecutive cases of prolonged APTT
No obvious cause Factor deficiencies: 15 % LA Chng et al. 2005
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Factor XIII deficiency
In FXIII deficiency the APTT, PT and thrombin time are normal. Moderate to severe FXIII deficiency can be diagnosed by the clot solution test. A fibrin clot prepared from patient plasma is placed in 8 M urea. Dissolution of the clot within 24 hours is suggestive of FXIII deficiency.
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Jørn Dalsgaard Nielsen E-mail: jdn@dadlnet.dk
Blood coagulation Screening assays and single factor assessment Jørn Dalsgaard Nielsen Thrombosis Centre Gentofte Hospital Copenhagen, Denmark
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