Understanding haemostasis

Symposium: Haematology

Understanding haemostasis

To interpret coagulation tests, it is necessary to have an understanding of the normal process of coagulation and which aspects of this are reflected in the common tests.

Mary Mathias

Normal coagulation

R Liesner

The first response to interruption of the integrity of the vascular endothelium is that platelets adhere to the exposed collagen, aggregate and are activated.1 Platelets adhere to damaged endothelium via the surface glycoprotein Ib/IX, which is constantly expressed on platelets, and von Willebrand factor (vWF), which acts as a co-linking protein to the endothelium (Figure 1). Collagen is a potent platelet activator and causes the release of thromboxane A2 and the expression of other important surface glycoproteins such as IIb/IIIa. Abnormal or absent platelet glycoproteins results in severe hereditary bleeding disorders, and a reduction or abnormality of vWF also leads to an abnormality of primary haemostasis in that binding of the platelets to each other and to the vessel wall is impaired. The other function of vWF is as a carrier protein for factor VIII (FVIII), and a significantly reduced vWF level correlates with a low level of FVIII (Figure 1.) Following adhesion to collagen as the primary event, platelets become activated and undergo a significant change in shape, forming pseudopodia and increasing their surface area. Activation leads to the release of granule contents including thromboxane A2, ADP, factor V (FV) and vWF, all of which further promote the coagulation process. The phospholipid bilayer of the platelet membrane undergoes a ‘flip-flop’ process whereby the negatively charged poles of the lipids are externalized, creating an ideal environment for the activation of the coagulation proteins in secondary haemostasis. This is now believed to be

Abstract The normal haemostatic process relies on the presence of normal vascular endothelium, a normal number of functioning platelets and appropriate levels of the coagulation proteins involved in both the propagation and the limitation of clot formation. Our ability to detect abnormalities in the haemostatic process relies initially on taking a thorough bleeding history and on coagulation screening tests. It is vital to understand the limitations of these screening tests and when it is appropriate to perform further investigations and when to seek advice from a haematologist. This review outlines the haemostatic process, and discusses the relevance of abnormal coagulation screen results, the indications for diagnostic coagulation tests,and the pitfalls of bleeding disorders that are not reflected by coagulation screens.

Keywords child; haemostasis; platelet; thrombophilia

Introduction One of the most important aspects of understanding haemostasis is understanding the limitations of the blood tests performed to investigate it. Blood coagulation is a dynamic process that involves the endothelium of the blood vessel, the platelets (and other blood cells) and the coagulation proteins. The classical screening tests such as prothrombin time (PT) and activated partial thromboplastin time (APTT) involve taking citrated blood, centrifuging it to remove the platelets, and then activating coagulation and measuring the time that the plasma takes to start to form a clot. It is therefore a totally unphysiological process and while the results of the screen can be helpful, it is crucial to appreciate that a normal screen does not always equate with normal in vivo coagulation. Equally, a normal platelet count tells us that there is a normal number of platelets; it does not tell us anything about how well they are working. It is vital when interpreting coagulation screen results to do it in conjunction with a thorough bleeding history for the child and his or her family and an understanding of the effects of intercurrent illness on the coagulation screen.

GP IIbIIa

Fibrinogen Platelet Platelet granules

GPIbIX receptor vWF Collagen fibres

Subendothelium Mary Mathias BA MBBS MRCP MRCPath is a Consultant at the Haemophilia Centre, Great Ormond Street Hospital For Children NHS Trust, London WC1N 3JH, UK.

Figure 1 Primary haemostasis. von Willebrand factor binds to exposed collagen in the vascular subendothelium and the platelets adhere to von Willebrand factor via glycoprotein Ib/IX. Activated platelets express glycoprotein IIb/IIIa, which binds to fibrinogen. Activated platelets release their granule contents, which further promotes platelet aggregation. GP, glycoprotein; vWF, von Willebrand factor.

R Liesner MA MBBChir MD MRCP FRCPath MRCPCH is a Consultant at the Haemophilia Centre, Great Ormond Street Hospital For Children NHS Trust, London WC1N 3JH, UK.

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Symposium: Haematology

i­nitiated by circulating factor VII (FVII) binding in its activated form (FVIIa) to tissue factor (tF) exposed in the subendothelium. The tF:VIIa complex activates factor X (FX), forming a ‘prothrombinase complex’ that converts small amounts of prothrombin to thrombin. The initial generation of this small amount of thrombin allows the activation of factor XI (FXI), which in turn activates factor IX (FIX) and FVIII. Activated FVIII, FIX and FV form a complex that activates much larger volumes of FX and then thrombin. As there is an amplification effect at each step of this secondary thrombin production pathway, the result is a huge thrombin ‘burst’. Thrombin then allows the polymerization of fibrinogen to form insoluble fibrin, which is subsequently cross-linked by factor XIII to form a stable, lysis-resistant clot. A defect in the secondary pathway (such as occurs in haemophilia A or B) does not prevent the initial small amount of thrombin production but it prevents the thrombin burst, so that primary haemostasis is achieved but secondary consolidation of the clot cannot occur. This can be reflected in the natural history of a bleeding episode in a boy with haemophilia; following a relatively trivial injury, there may be no immediate problem with haemostasis, but after an interval of a few hours the initial inadequate clot breaks down (Figure 2).

binds to the surface of the clot and when activated by tissue plasminogen activator (tPA) causes disassembly of the fibrin to small, soluble fragments called fibrin degradation products. D-dimers, which are commonly measured to assess the degree of a fibrinolytic process, are one type of fibrin degradation product. In the same way that coagulation is limited to the site of tissue injury, there are safeguards that prevent fibrinolysis becoming a systemic process. Circulating tPA has a very short half-life and most is in an inactive form bound to its inhibitor plasminogen activator inhibitor-1. Circulating plasmin is similarly neutralized by α2-antiplasmin. Thrombin:thrombomodulin also causes the activation of a carboxypeptidase called thrombin activatable fibrinolysis inhibitor. This binds to fibrin, preventing the binding of plasminogen and thereby slowing the fibrinolytic process (Figure 3).

Coagulation screening tests A coagulation screen should include PT, APTT, thrombin time (TT) and a fibrinogen level. A platelet count as part of the full blood count should also be available. These samples should be taken freely from a peripheral vein via a butterfly or cannula (if central access is being used it is vital that an adequate volume of blood is discarded – 2–3 ml). Difficult venepuncture is likely to result in inaccurate or abnormal results (most commonly the sample is activated; i.e. the coagulation process has been initiated) and will need to be repeated. Erroneous results due to

Inhibition of coagulation To prevent uncontrolled propagation of the clot, there are a number of pathways in place to limit fibrin formation and ultimately to allow the fibrin to be gradually reabsorbed as part of the normal wound healing process. Thrombin, when bound to the cell membrane protein thrombomodulin, causes the activation of protein C, which in conjunction with its co-factor protein S cleaves FV and FVIII, rendering them inactive. Thrombin also binds to antithrombin, leading to conformational changes such that thrombin is no longer a procoagulant. In addition to these ‘breaking’ processes, the fibrinolytic pathway is activated as soon as fibrin is generated. Plasmin FVIIIa:FV:FIXa

FVIIIa:FV:FIXa

FIX

PC:PS

FX TM

FXa:Ca2+:PL

Thrombin

AT

FIX

PAI-1 Plasminogen tPA

TM FXI

FX

FXIa

Plasmin

α2AP

Fibrin TAFla

TF:VIIa

FDP

2+

FXa:Ca :PL Prothrombin

Thrombin

Figure 3 Inhibition of coagulation. When bound to the membrane protein thrombomodulin, thrombin activates proteins C and S, which cleave and inactivate factors V and VIII, inhibiting the coagulation process. Thrombin:thrombomodulin also activates thrombin activatable fibrinolysis inhibitor, which inhibits the breakdown of fibrin to its degradation products. Antithrombin binds to thrombin and prevents its procoagulant function. The fibrinolytic process is inhibited by plasminogen activator inhibitor-1 and α2-antiplasmin. PC, protein C; PS, protein S; TM, thrombomodulin; AT, antithrombin; PAI-1, plasminogen activator inhibitor-1; α2AP, α2-antiplasmin; TAFIa, activated thrombin activatable fibrinolysis inhibitor; TF, tissues factor; tPA, tissue plasminogen activator; PL, phospholipid; thick lines, inhibitory effect; broken lines, indirect action.

Plasminogen tPA Plasmin

Fibrinogen

Fibrin

D dimers

Figure 2 Secondary haemostasis. Exposed tissue factor binds to factor VII, producing an initial small amount of thrombin that triggers an amplifying cascade, resulting in a thrombin ‘burst’ that converts enough fibrinogen to fibrin to form a stable clot. TF, tissues factor; tPA, tissue plasminogen activator; PL, phospholipid.

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Symposium: Haematology

poor sampling technique are common in paediatric practice. Heel-prick or finger-prick sampling is not suitable for coagulation testing. PT reflects abnormalities of factors II, V, VII and X in that a deficiency of any of these factors results in a prolongation of the PT. APTT similarly detects a deficiency of factor VIII, IX, XI or XII. If there is an isolated prolongation of the PT or APTT, the coagulation laboratory should automatically undertake mixing studies, in which the patient’s plasma is mixed with an equal volume of pooled normal plasma to see whether this corrects the abnormality. If it does, the patient sample has a deficiency of one or more of the PT- or APTT-based factors. If there is no correction, an inhibitory process is occurring in vitro that must be further defined. TT reflects the time taken to convert fibrinogen to fibrin, and an isolated prolongation of TT is likely to be due to either an abnormality or a deficiency of fibrinogen. Fibrinogen results can be calculated in several ways, but in most routine laboratories they are derived from the PT (an unreliable method that is subject to PT abnormalities) or the Clauss fibrinogen test, which is the gold-standard method and measures fibrinogen activity. All of these screening tests are now performed on automated machines that allow maximal throughput in busy laboratories and reduce human error; however, they are only as uniform as the reagents used to run the tests and different reagents may detect abnormalities to differing degrees. For example, a laboratory that receives only paediatric blood samples is likely to choose a machine/reagent combination with the maximal sensitivity to mild coagulation factor deficiencies, as it will deal with a significant number of pre-operative screening samples from children with no previously known bleeding disorder or haemostatic challenge, and it is vital that no mild factor deficiencies are missed. An isolated abnormality of any of these screening tests is much less common than a combination of abnormalities that tends to reflect an acquired coagulopathy or the presence of an anticoagulant. Table 1 lists the causes of isolated and combined abnormalities of the screening tests. These tests can alert the clinician to a potential factor deficiency, but on their own they cannot provide a precise diagnosis. The first step after the finding of an abnormal coagulation screening result is to repeat the test. A significant proportion of abnormal results are normal when repeated, and even when a child is difficult to bleed it is always worth doing this to confirm that there was no sampling error. To minimize venepunctures, it is sensible to confer with a haematologist and to take enough blood on the second occasion to cover the supplementary tests that may be required. It is also important to understand what a coagulation screen cannot detect. It is a relatively common situation that children with rare but severe bleeding disorders present to their local hospital after a bleeding episode, but following a normal coagulation screen and platelet count the parents are reassured that there is no problem and they are discharged. Screening tests detect most problems, but the exceptions (Table 1) occasionally cause life-threatening bleeding. If a child has a significant history of provoked or unprovoked bleeding and the coagulation screen is normal, it is always worth seeking advice from a haemophilia comprehensive care centre about referral and further testing.

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Interpretation of coagulation screen results PT

APTT

TT

Possible abnormality

↑

N

N

• Factor VII deficiency (extrinsic pathway)

N

↑

N

N

N

↑

↑

↑

N

N ↑

↑ ↑

↑ ↑

N

N

N

• Liver disease • Vitamin K deficiency • Deficiency of factor VIII (due to haemophilia or vWD), factor IX, XI or XII or contact factors (intrinsic pathway) Lupus anticoagulant or other • coagulation factor inhibitor (prolongation does not correct with normal plasma) • Hypofibrinogenaemia • Dysfibrinogenaemia • Deficiency of factor II, V or X (common pathway)  Vitamin K deficiency • Liver disease • • Massive transfusion • Oral anticoagulants • Heparin • Disseminated intravascular coagulation • Large amounts of heparin • Severe hypofibrinogenaemia All tests normal but history of bleeding – consider: • Mild vWD or haemophilia • Factor XIII deficiency • Platelet disorder

PT, prothrombin time; APTT, activated partial thromboplastin time; TT, thrombin time; N, within normal range; ↑, prolonged; vWD, von Willebrand disease.

Table 1

Normal ranges At birth, even a healthy, term infant does not have a mature pattern of coagulation.2 There are many differences from the adult situation, and indeed from a healthy 6-month-old. All of the ­vitamin K-dependent coagulation factors (II, VII, IX, X, protein C and protein S) are physiologically low at birth and increase gradually to the normal levels at around 6 months (except protein C, which may not increase to the adult normal range until the early teens). Plasminogen levels are also below the adult level until around 6 months, as is FXI. Neonatal thrombin has more sialic acid ­residues than ‘normal’ thrombin, which leads to a falsely raised TT in neonates (this can persist up to around 8 weeks and is an in vitro phenomenon). It is therefore vital that a coagulation screen or factor assay in a premature, newborn or young baby is interpreted in the context of the normal range for that age group. Although normal ranges are quoted in the literature, the gold standard for a laboratory 319

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Symposium: Haematology

handling a significant number of paediatric samples is to establish a local normal range.

Abnormal platelet function Abnormalities of platelet number are easily diagnosed from the full blood count. A low platelet count in a child is usually the result of intercurrent illness or is iatrogenic (e.g. post-­chemotherapy). Thrombocytopenia in association with deranged coagulation is likely to reflect disseminated intravascular coagulation, for which it is usually easy to identify a cause. The causes of isolated thrombocytopenia are beyond the scope of this review. Provided the platelets that are present are functionally normal, spontaneous bleeding problems are relatively rare in an otherwise well child unless the platelet count falls below 30×109/litre. Abnormal platelet function can occur with normal or low platelet counts. The commonest cause of mild platelet function abnormalities is acquired, in the form of exposure to non-­steroidal anti-inflammatory drugs (NSAIDs) or aspirin. Aspirin irreversibly inhibits the enzyme cyclo-oxygenase, thereby impairing the ­synthesis of thromboxane A2, which has potent platelet aggregating properties. After the child has stopped taking aspirin, its effect it continues for around 7 days because of the lifespan of the average platelet. NSAIDs have a similar effect to aspirin and require the same washout period.

Acquired inhibitors of coagulation If the APTT is not corrected by a 50:50 mix with normal plasma, there is some form of inhibitor present. In most cases in children, this is lupus anticoagulant. The other situation in which a prolonged APTT is not corrected by normal plasma is when a boy with haemophilia develops an alloantibody to infused FVIII or FIX.

Diagnostic tests When a repeatable abnormality of the coagulation screen has been identified, it must be followed-up to clarify the problem, but the extent of further investigation varies according to the clinical scenario. For example, a premature neonate with sepsis on the paediatric ICU is extremely unlikely to have a normal coagulation screen. However, if is established that the screening tests are corrected by normal plasma, it may not be necessary to perform detailed factor assays and on the occasions when these are done, they usually reflect globally low levels of coagulation factors secondary to sepsis and liver immaturity. In contrast, a 6-year-old boy with prolonged APTT on pre-operative screening for tonsillectomy, and with no previous haemostatic challenges, requires factor VIII, IX, XI and XII testing and a von Willebrand disease (vWD) screen before surgery can proceed. There is a significant risk that this boy may have haemophilia or vWD (the commonest hereditary bleeding disorder), and the absence of a significant bleeding history or a family history cannot be used to exclude a problem. Factor assays may not to be readily available in district general haematology laboratories and if a persistent coagulation screen abnormality has been detected in this setting it is worth considering referring the child to a specialist centre. A cogent reason for doing this is that, in a paediatric haemophilia centre, the volume of blood required to conduct diagnostic bleeding tests is likely to be significantly smaller than in a centre predominantly running adult samples. It is good practice to undertake comprehensive bleeding investigations in a child referred for a haemostasis opinion, as more than one abnormality may be present. A typical group of tests would be factors II, V, VII, VIII, IX, X, XI and XII, with von Willebrand antigen, ristocetin co-factor activity and collagen-binding assay.3

Hereditary platelet defects A pattern of significant bleeding in a child with normal coagulation, particularly if the bleeding is mucosal, should alert the physician to the possibility of a hereditary platelet defect.5 The two severest forms, which are also the least common, are Glanszmann’s thrombasthenia (GT) and Bernard Soulier syndrome (BSS). The platelet count is normal in GT but tends to be low in BSS and declines further in the face of intercurrent bleeding. In both these conditions, bleeding is usually severe after a haemostatic challenge and is often spontaneous; it tends to be mucosal, but joint bleeds also occur in GT. The diagnosis of GT and BSS can be made with a small volume of blood by platelet flow cytometry for platelet glycoproteins Ib/IX (BSS) and IIb/IIIa (GT). This is a useful technique in babies, as formal platelet aggregometry requires 20 ml of blood. Platelet aggregometry and platelet nucleotide content and release are highly specialized tests that are performed on children in only a few centres in the UK. They are time-­consuming for laboratory staff and rely on venepuncture having been straightforward and the blood sample reaching the laboratory preferably within 1 hour. The results are profoundly abnormal in GT and BSS, but the other hereditary platelet abnormalities tend to give more subtle patterns of abnormalities and should be interpreted only by a haematologist with experience in this area. These abnormalities are usually described as storage pool disorders or nucleotide release defects and most are associated with bleeding only in the context of trauma or surgery. ◆

Thrombophilia testing There has been increasing awareness in recent years of hereditary conditions associated with an increased risk of thromboembolism, and many haematology laboratories can now screen for these. Thromboembolic disease in children remains very rare and is almost always associated with intercurrent illness and/or the presence of a central venous catheter. Thrombophilia screens should not be performed at the time of the acute event, as the levels of the natural anticoagulants tend to decline; it is almost always advisable to postpone the tests until the appropriate period of anticoagulation is finished.4 Interpretation of the results of these screening tests can be difficult and they may need to be discussed with a haematologist. It is important to remember that factor V Leiden is present in around 1/20 of the healthy Northern European population and prothrombin gene mutation in around 1/50; their presence should therefore not be over-interpreted.

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References 1 Goodnight SH, Hathaway WE. Disorders of hemostasis and thrombosis: a clinical guide, 2nd edn. New York: McGraw-Hill, 2001 1: 3–19. 2 Williams MD, Chalmers EA, Gibson BES. Guideline: the investigation and management of neonatal haemostasis and thrombosis. Br J Haematol 2002; 119: 295–309.

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3 Bolton-Maggs PHB, Perry DJ, Chalmers EA, et al. Rare coagulation disorders-review with guidelines for management from the United Kingdom Haemophilia Centre Directors Organisation. Haemophilia 2004; 10: 593–628. 4 Walker ID, Greaves M, Preston FE. Guideline: investigation and management of heritable thrombophilia. Br J Haematol 2001; 114: 512–28. 5 Bolton-Maggs PHB, Chalmers EA, Collins PW. A review of inherited platelet disorders with guidelines on their management on behalf of the UKHCDO. Br J Haematol 2006; 135: 603–33.

• Haemostasis is limited to the immediate site of vascular injury by the natural inhibitors of coagulation and by the fibrinolytic process, which is initiated by thrombin and fibrin. • Heel-prick or finger-prick sampling is not suitable for coagulation testing. • Abnormal tests must always be repeated. • Results must be interpreted with the appropriate normal range for age. • Abnormal coagulation screens must be followed-up by the appropriate factor assays. • Thrombophilia testing should not be performed at the time of an acute thrombotic event. • Abnormal coagulation tests in children are most often related to intercurrent illness or poor sampling. • A thorough bleeding history is as important as a coagulation screen. • Life-threatening hereditary bleeding disorders can exist with a normal coagulation screen.

Practice points • Primary haemostasis involves the binding of platelets to exposed collagen in the subendothelium of damaged vessels. • Secondary haemostasis is the process of activation of coagulation factors leading to the production of thrombin.

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