The application of meta-analysis of randomized studies (‘evidence-based medicine’) to diagnostic laboratory testing has been limited,² and hemostatic testing is no exception. It is therefore not possible yet to claim evidence-based validation, in its strict sense, for many of the principles discussed below. However, the writings of many expert clinician–scientists over the years are the best guide we have to these principles, and should certainly form a starting-point for further analyses.

Physiology of hemostasis applied to diagnosis

The clinical approach to the patient who may have a hemostatic disorder is informed by knowledge of the physiology of hemostasis. Hemostatic reactions operate in a clock-like sequence, the first two phases being termed ‘primary’ and ‘secondary’ hemostasis.

A careful clinical history and examination (see below) can tentatively locate the potential defect in one of these phases, guiding the selection of initial investigations. The pretest probability of a defect involving primary hemostasis rises if abnormal bleeding follows a ‘mucosal’ pattern (see below), while a history of muscle or joint bleeding increases the likelihood of a coagulation deficiency. Disorders of the regulatory protein C pathway tend to manifest as venous thromboembolism. Abnormalities of the final phase of hemostasis, fibrinolysis, tend to contribute to bleeding in specific clinical settings, for example disseminated intravascular coagulation (DIC) and hepatic failure.

To assist this diagnostic thinking, it helps to keep in mind a simplified map of the hemostatic system, whatever knowledge of its complexity one possesses (or not, as the case may be). These simple maps are caricatures: readers are referred to fuller versions ^3,⁴ and to other chapters in this volume.

Primary hemostasis: formation of the platelet plug ⁵ (Fig. 31.1)

Platelets, highly structured and excitable anucleate cellular bodies, circulate in the blood at a concentration of 150–400 × 10⁹/l.

Fig. 31.1 Platelet adhesion and aggregation at sites of vascular damage (schematic).

Platelet adhesion

Vessel wall damage provokes a variety of signals from damaged or activated vascular endothelial cells and/or exposure of the underlying subendothelial matrix. Platelets first adhere to the signaling site via adhesive ligands. The dominant interaction is between platelet membrane receptor glycoprotein Ib-IX and the giant polymer von Willebrand factor (vWF), particularly its most adhesive high molecular weight forms.

Platelet aggregation

Adherent platelets flatten and activate membrane fibrinogen receptors (glycoprotein IIb-IIIa) that bind plasma fibrinogen (Fb). The resulting syncytial platelet aggregate provides a reactive surface composed of platelet membranes. This activated membrane flips inside out, exposing the negatively charged phospholipid, phosphatidylserine (PS). By binding the tenase and prothrombinase complexes, PS allows coagulation reactions to proceed. This altered (‘activated’) platelet membrane is sometimes termed platelet factor 3.

Secondary hemostasis: generation of fibrin clot by the coagulation pathway (Fig. 31.2)

Unless underpinned by a fibrin net, primary platelet plugs disintegrate under the shear stress of flowing blood. The complex coagulation pathway that generates fibrin can be divided into three substages:

Fig. 31.2 Thrombin generation in vivo: coagulation factor interactions mediated by cell surfaces (schematic).

Clot regulation and removal: the protein C and fibrinolytic pathways

Two further systems regulate and eventually remove the clot (in the context of tissue repair and neoangiogenesis) (also see Chapter 28):

Clot regulation: the protein C system ⁶ and antithrombin

Thrombin formed around healthy vascular endothelial cells puts a brake on coagulation by binding to a receptor, thrombomodulin, which retargets it to protein C. Thrombin/thrombomodulin activates PC, which inactivates FVa and FVIIIa, slowing thrombin formation. To target FVa and FVIIIa in their membrane complexes, aPC needs a co-factor, protein S.

Thrombin activity is restricted to the platelet surface and the fibrin matrix of the clot by a conformation-dependent inhibitor, antithrombin (AT)⁷ that inactivates fluid-phase thrombin. To work efficiently, AT must bind to heparin-like proteoglycans on healthy endothelial cells.

Fibrinolysis: the plasmin system ⁸

Clots contain the seeds of their own destruction in the form of a clot-bound protein, plasminogen. This is cleaved by tissue plasminogen activator (tPA) secreted by healthy vascular endothelial cells, or urokinase on the surface of macrophages, to the fibrinolytic enzyme plasmin. Plasmin cleaves fibrin into fibrin degradation products, notably the D-dimer fragment specific to cleavage of cross-linked fibrin.⁹ This removes the clot, and activates matrix metalloproteinases that initiate remodeling of vessels (angiogenesis).

One problem in the investigation of coagulation is the artificial nature of available laboratory tests, which commence by separating plasma from the very cell surfaces crucial to in vivo hemostasis, particularly platelets. These absent membranes are then simulated by adding back tissue extracts or recombinant proteins with properties similar (but rarely identical) to the physiologic substrates. In addition, as is seen below, the coagulation ‘screen’ – the crucial bridge between the clinical perception that something is wrong and its laboratory definition – invokes an obsolete model of hemostasis. A clear mental distinction must be made between the current model of in vivo hemostasis and the older model applied in vitro.

The clinical approach to the patient with a possible hemostatic disorder

If you prick us, do we not bleed?

William Shakespeare, ‘The Merchant of Venice’, III.i.

The question of a possible hemostatic disorder occurs in two main settings. An individual is referred because they have presented with, or self-reported, clinical phenomena suggesting excess bleeding. Investigation can proceed in a structured elective style. In the second case, excess bleeding occurs acutely in a patient undergoing treatment in the hospital, emergency department or surgical theater. The tempo, urgency and completeness of the diagnostic work-up (before recourse to therapeutic action) are then different, but the principles are shared.

Experts writing about the investigation of possible bleeding disorders unanimously stress the importance of a carefully taken history.¹⁰^–¹² They also recommend specific questions, answers to which alter the pretest probability of a bleeding disorder. The discussion below draws on this consensus. Similarly, key findings on clinical examination may aid the diagnostic process, although they are less frequent than narrative clues.

It must be conceded that these narrative and clinical signs have not been formally tested, either singly or in clusters, for their relative value in predicting the presence of hemostatic disorders. Such testing has refined and simplified the use of clinical clues in other contexts,¹³ and may be of future benefit in hemostasis. Until such clarification becomes available, the shared insight of experienced clinicians is our best guide.

History

The role of the history-taker is to determine if the patient’s account is consistent with excessive bleeding. After initial open questioning related to the presenting complaint, a systematic inquiry is made with the help of key questions intended to elicit quantitative information about the bleeding in question. People (including doctors) tend to overestimate, by eye, volumes of blood lost from the body, so it is more informative to focus questioning on the duration of a bleeding episode and what had to be done about it.

Key questions

Surgical challenges

Dental surgery

Surgical trauma to the incompressible tooth socket sitting in the fibrinolytic milieu of the oral cavity is a stiff challenge to the hemostatic system. Useful questions about the effect of extractions focus on the duration of bleeding and the actions compelled by it. Compare the two accounts in Box 31.1. The second account gives a much clearer indication of excessive blood loss. Most people (in the UK, at least) are disinclined to make an early return to the dentist without a pressing reason.

Other types of surgery

Questions about blood loss after circumcision and tonsillectomy are traditional, but the timing and selectivity of the former, and decreasing popularity of the latter, mean that only a small minority of individuals (or their parents) will give a useful response. As in the case of dental extraction, questions should focus on duration of bleeding and subsequent medical actions.

Many individuals are referred for investigation of a possible bleeding disorder as a result of excess blood loss after major surgery, although the commonest cause of this is purely ‘surgical’ – a transected blood vessel evading the hemostat. Large and/or late wound hematomata or generalized ‘oozing’ from a tissue surface or organ bed are more likely to indicate a hemostatic disorder. The patient’s own recall of these events is likely to be hazy, and documentation of the amount, duration and clinical reaction to peri- and postoperative bleeding should be sought in the patient’s medical records.

Epistaxis

Nosebleeds are a universal experience in childhood, so the usefulness of enquiring about them (nearly everyone will recall some) depends on the questions asked. In bleeding disorders, epistaxes tend to run ‘like a tap’ rather than to drip; require a bowl to catch the blood rather than a tissue; and resist arrest (or reroute via the mouth) on pinching the nares. Frequently recurring epistaxes that provoke multiple nasal cauterizations also increase the possibility of a bleeding disorder.

Gastrointestinal or urogenital bleeding

Rectal bleeding compels a search for colorectal disease even if a systemic bleeding disorder is present. Coumarin-induced rectal bleeding has led to the early detection and cure of cancers. Similar action must follow hematemesis, hematuria or vaginal bleeding. Occasional prolonged episodes of spontaneous hematuria occur in hemophilia, sometimes in mildly affected individuals.

Menstruation

As with other perceptions of bleeding symptoms by both sexes, women accustomed only to their own menstrual loss may not regard it as abnormally heavy. Bleeding for >7 days per month, bleeding that regularly ‘breaks through’ sanitary protection, the need to wear both tampons and pads (or double pads), and the need to protect the bed with a towel, or to cancel social engagements due to bleeding, are reliable indications of menorrhagia. Questioning should be sensitive and preceded by an explanation of its relevance.

Bruising

A sizeable minority of the population will answer ‘yes’ to ‘do you bruise easily?’ and many older people bruise the sun-thinned skin of their hands and forearms, so this question is not helpful. A semi-quantitative approach is useful: the bruises can be compared to some common object (in the UK the 50 pence coin, about 2 cm in diameter). Having frequent bruises larger than this is significant. Most normal (’simple’) bruises occur on the outer surfaces of the upper arms and thighs, ‘bumpers’ in contact with the environment: bruises on the trunk, neck or face, or on the inner aspects of limbs are more significant, as are palpable bruises (hematomata). Solar or simple bruises are rarely pathologic.

A patient complaining of easy bruising who cannot show a single bruise at the time of the consultation, or one who agrees that there are fewer days with bruises than days without,¹¹ is less likely to have a bleeding disorder. Thrombocytopenic purpura crop around the ankles, where venous pressure is highest, and are more likely to be perceived as ‘a rash’ than as bruises.

’Third space’ bleeds

In the hemophilias (inherited and acquired), over-anticoagulation with heparins or coumarins, and other systemic bleeding disorders, the presenting complaint may be hemorrhage into joints, muscles, or other deep tissue compartments. These events may not be perceived as bleeds by the patient or even by the attending clinical team, since they present with pain, swelling, nerve entrapment or other space-occupying features rather than with evident blood loss. By mimicking tumors, or presenting as acute monoarthritis, they may provoke biopsy or drainage attempts with potentially catastrophic results. In the context of anticoagulant therapy, failure to recognize such bleeds, and consequent ‘pushing on’ with heparin or warfarin, is equally dangerous.

The first-line clinicians called upon to deal with these events are rarely experienced in their recognition, so the best protection lies in local in-service education and guidelines, together with constant availability of hematologic advice and the freedom to access it.

Summation and duration of bleeding episodes

All types of blood loss should be summated. A patient with a credible history of significant bruising and epistaxis is more likely to have a bleeding disorder than one with bruising alone. Bleeding symptoms that go back to childhood or adolescence are likely to be inherited, and prompt a family history, while if recently developed they point to an acquired cause and a general enquiry for systemic disease.

Pattern of bleeding

A ‘mucosal’ pattern of bleeding episodes (epistaxis, menorrhagia, bleeding after dental surgery) may guide the initial investigation towards platelet and vWF analysis since it suggests a problem with primary hemostasis. Presentation with hemarthrosis or other third-space bleeds is classical in hemophilia. However, this is hardly a clear distinction, since hemophilia also causes mucosal hemorrhage and dental disasters: stating that menorrhagia is more likely in primary bleeding disorders than in hemophilia is tautologic. In general, it is necessary to perform at least screening tests (see below) of both primary hemostasis and coagulation in people who bleed too much.

Drug history

A full list of all prescribed or over-the-counter medication (including herbal and other complementary medicines) taken by the individual should be compiled. Aspirin remains the most prevalent agent causing bleeding symptoms and abnormal platelet function test results: in addition to being prescribed widely for its antithrombotic effect, it is a component of many preparations on sale to the public. Some of these preparations have names that advertize the presence of aspirin (e.g. Aspro®) while others (e.g. Nurse Sykes’ Powders®) do not: a full list of such products is given in the British National Formulary.14 Other non-steroidal anti-inflammatory agents share the aspirin effect. Antibiotics, major tranquillizers and antidepressive agents may all be associated with bleeding via antiplatelet function effects. Platelet function testing should be performed first with the patient taking the drug, then 2 weeks after stopping, in order to demonstrate its effect.

Family history

A reliable family history entails documenting a pedigree chart including all known family members with their names and dates. The key questions illustrated above are asked about each member in turn, seeking confirmation of any said to have a bleeding tendency. Any described as having hemophilia should, if possible, be traced to the Hemophilia Center carrying out their care: a relative famous for ‘hemophilia’ often turns out to have no evidence of the disorder at all.

Taking a family history of this quality is time-consuming, may take more than one session, and suits the elective better than the emergency setting. It often extends the individual’s historical knowledge of their family beyond its limit. Furthermore, a negative family history excludes nothing, since many bleeding disorders, including severe hemophilia, occur sporadically.

Clinical examination

Skin

The whole skin surface should be inspected for purpura and bruising, documenting the distribution, size and age of lesions and correlating them with the clinical history. Palpation of bruises will detect hematomata, while palpable purpura suggests vasculitis. Close attention should be paid to the ankles, where venous and capillary pressure is highest: petechiae first appear here in thrombocytopenia, and signs of venous or arterial insufficiency may be evident. Large bruises (ecchymoses) typical of hemophilia or anticoagulant overdose may be found tracking into dependent parts of the body such as the scrotum.

The surface of lesions should be inspected. Edema may indicate the urticarial component of anaphylactoid purpura. Lesions of hereditary hemorrhagic telangiectasia may be seen in finger pulps and ear lobes, spreading over the face in later life. Bruises with abrasions or thermal trauma, that follow the outline of a blunt object, or are associated with other signs of abuse or self-harm may indicate non-accidental injury or factitious bruising.

Scars should be examined. Keloid formation might rule out a skin bleeding time. In Ehlers–Danlos syndrome they pucker like tissue paper on sideways compression, and may show central breakdown with fresh exudation. Poor scar quality may also be seen in hypo- or afibrinogenemia.

Non-hemorrhagic lesions mistaken for signs of bleeding include cherry-red Campbell de Morgan spots, stretch marks, livedo reticularis and Majocchi’s purpura or other ‘dermatological’ purpuras.

Mucosae

The oral cavity should be inspected for the petechiae or ‘blood blisters’ of ‘wet’ thrombocytopenia. Gingival bleeding is usually associated with gingivitis. Oral hemorrhage in the hemophilias occurs at sites of minor trauma or dental surgery, and may consist of a small but persistent bleeding point, a friable oozing clot, or a tumor-like sublingual swelling.

Musculoskeletal system

Joints should be examined for warmth, effusion, synovitis, reduced range of movement and misalignment. Muscle groups should be examined for wasting and contractures. These signs of cumulative damage due to hemarthrosis and intramuscular hematomas are characteristic of the hemophilias, but are also seen in rare disorders such as type 3 von Willebrand disease (vWD), deficiency or severe recessive disorders such as homozygous factor XIII, factor VII or factor X deficiency. Intramedullary hemorrhage of the long bones is a feature of afibrinogenemia and α2-antiplasmin deficiency, both very rare: it mimics lytic bone disease.¹⁵

Nervous system

Evidence of nerve compression injuries may be evident combined with damage to the musculoskeletal system identified above. Retinoscopy should be performed in all patients with purpura, particularly involving the oral mucosa: retinal hemorrhages indicate active CNS bleeding and the need for urgent therapy.

Active bleeding

The postoperative or traumatized patient with excessive bleeding should be examined for the signs itemized above, but sites of blood loss should be directly observed if possible. External losses, including those via surgical drains, should be assessed: dilution with tissue exudate can exaggerate blood losses. Similar overestimation can occur in hematuria. If in doubt in either of these situations, a hemoglobin estimate on the drain fluid or urine can be helpful. Tracking hematomata should be sought. All intravascular access points, together with other skin incisions pre- or postdating the main episode of blood loss, should be inspected for evidence of bleeding or rebleeding after earlier closure. Fresh bleeding from such sites is a sign of DIC in its consumptive phase.

On defining the pretest probability of a bleeding disorder

Using information from the history and examination the clinician can work out a broad pretest probability (e.g. low, moderate or high) that the patient has a clinical bleeding disorder. The accuracy and precision of the history and examination described above in defining this pretest probability have not been tested by methods that have provided such information in other contexts.¹³ Such studies are feasible and desirable in bleeding disorders, but even in their absence, a rational estimate of pretest probability is a crucial step towards interpreting the results of laboratory testing. Without it, tests of hemostasis can be frankly misleading.

Screening tests of hemostasis: two warnings

Armed with an estimate of pretest probability, the next step is to perform screening tests of hemostasis to generate further data capable of increasing or decreasing it.

On venipuncture

This requires a blood sample, which should be taken by an expert venipuncturist – especially in the case of a child – from a peripheral vein with minimal venous stasis. On no account should the jugular, subclavian or femoral veins be approached if there is any possibility of a bleeding disorder. Many inexperienced clinicians seem drawn to perform a ‘femoral stab’ on patients covered in bruises: this can result in a massive compartment bleed in the femoral triangle. Multiple attempts to obtain samples from the antecubital fossa can likewise result in severe bleeds. An expert hand is vital.

On screening tests

These tests ‘screen’ hemostasis, not people – a source of considerable misunderstanding and futile testing. They do not meet the epidemiological standard of true screening tests because they are not sensitive or specific enough to screen a population for bleeding disorder. They only work in concert with the history and examination as described above.

The 250 ‘clotting screen’ requests typically made every day in a large teaching hospital represent educational failure. This futile attempt to screen the population entering hospital for surgery (or other intervention) for bleeding risk depends partly on misinterpretation of the ambiguous term ‘screen’. Even more misleading – and potentially wasteful – is the lazy application of the term ‘thrombophilia screen’ to detailed testing for inherited and acquired thrombophilia. When the term ‘screen’ is unavoidable, it is used below strictly to refer to tests performed as the result of a clinical history of bleeding or thrombosis.

Initial screening tests, usually applied whatever the pattern of abnormal bleeding, consist of a multiparameter blood count including the platelet count, and coagulation tests: a prothrombin time (PT), activated partial thromboplastin time (APTT), and sometimes a thrombin clotting time (TT).

If the pretest probability of a bleeding disorder is possible or probable, normal results in these initial tests should be followed by a skin bleeding time estimation or whole blood platelet function analysis. The need for further platelet function tests, specific assays of hemostatic proteins or genes, or further clinical tests for systemic disorders depends in part on the results of ‘global’ tests of hemostasis, but should also proceed if the full history is convincing, even if initial tests are normal. Below, tests of primary hemostasis and coagulation are grouped together for coherency, but they are also ranked into ‘screening’ and ‘diagnostic’ categories.

Laboratory investigation of hemostasis

Tests of primary hemostasis

Screening tests

The platelet count

Methods. In the current laboratory, platelet counting is performed on an anticoagulated venous blood sample as part of the multiparameter ‘full blood count’ generated by automated cytometers. Current systems count particles of platelet-like size (2–37 µm³) by electrical aperture impedence or laser light scattering. To censor ‘noise’ at the low end and red cells at the high end of this range, devices fit a lognormal distribution curve to this raw count or otherwise manipulate it to calculate the reported platelet count.

The validity of the platelet count accordingly depends on instrument standardization, calibration and quality control: details of these procedures can be found elsewhere.¹⁵ Because instruments count particles by size, blast cell fragments (in acute leukemia) or schistocytic red cells (in thrombotic thrombocytopenic purpura) may lead to overestimation, and large platelets (in immune thrombocytopenia or myelofibrosis) to underestimation, of the true platelet count.

A commoner source of error in platelet counting is ethylenediaminetetraacetic acid (EDTA)-induced platelet clumping, an in vitro artifact confirmed by microscopy of a blood film of EDTA-anticoagulated blood and a recount in citrate-anticoagulated blood. A low platelet count should also be checked by examining the specimen tube for clot formation.

Normal and abnormal platelet counts. The normal (‘Gaussian’) reference range for the concentration of platelets in venous blood (’the platelet count’) is 150–400 × 10⁹/l. By definition, 5% of normal individuals have platelet counts outside this range. To regard and investigate asymptomatic individuals with isolated, stable, mild thrombocytopenia (100–150 × 10⁹/l) as if they had a disease may be to confound ‘Gaussian’ and ‘diagnostic’ concepts of normality.¹ However, evidence to justify abandoning this seemingly unproductive practice is lacking.

By contrast, in a sick patient, falling platelet counts in the range 150–400 × 10⁹/l, or even from >400 × 10⁹/l into the normal range, may indicate the early, reversible stages of dangerous hemostatic disorders (e.g. DIC or heparin-induced thrombocytopenia). A falling platelet count in the normal range may also be a clue to the presence of sepsis, falciparum malaria or other systemic diseases. Any fall of >50 × 10⁹/l in a 24-h period should alert the hematologist and be communicated to the clinical team.

Correlating the platelet count with the clinical situation. The action taken in response to the finding of a low platelet count depends on the presence or risk of bleeding, since the two are not always correlated. In many patients with immune thrombocytopenia (ITP), clinical bleeding may be minor or absent even at very low counts (<10 × 10⁹/l), and precipitant therapy may not be necessary. However, the presence of mucosal bleeding in ITP indicates early therapy.

Lesser degrees of thrombocytopenia (20–50 × 10⁹/l) are dangerous when combined with reduced platelet function (e.g. antiplatelet agents, myelodysplasia, myelofibrosis); abnormal coagulation (e.g. DIC); leukemia (e.g. acute promyelocytic leukemia); cerebral vasculopathy in sickle cell anemia, or with severe anemia of any cause. In these situations, aggressive therapy including intensive platelet transfusion support is often needed.

When confronting a reduced platelet count, an apparently simple variable, potential laboratory error or artifact must be sought, and the platelet count must be placed firmly in the clinical context. These are core principles in all hemostatic testing.

Platelet function testing

If a history of excess bleeding suggests a defect in primary hemostasis but the platelet count is normal, or insufficiently reduced to account for it (>100 × 10⁹/l), tests of platelet function are indicated. Recent technological developments have changed the range and sequence of tests applied for this purpose. It is logical first to perform ‘global’ tests of platelet function: skin bleeding time and whole blood platelet function analysis. If either or both give results consistent with abnormal platelet function, further definition of the defect by platelet aggregometry and other tests should be attempted. The limited sensitivity of these methods, and the myriad defects that can occur in the platelet’s parallel activation, transduction and secretion systems,¹⁶ often mean that no definitive diagnosis can be made outside a research laboratory. A degree of diagnostic uncertainty is tolerable, however, because therapeutic modalities for disorders of primary hemostasis tend to be broadly applicable across them all.

Whole blood platelet function analysis. Many workers have attempted to develop devices that mimic (and therefore test) the linked phases of platelet adhesion and aggregation in uncentrifuged whole blood.¹⁷ Recent automated devices appear to accomplish this in a valid and reproducible way. By eliminating the need to prepare platelet-rich plasma, they reduce both sample volume and the time needed to do the test. These methods appear to be more sensitive to subtle platelet function defects, possibly because they eliminate ex vivo platelet activation during centrifugation.

The most widely used device is the PFA-100® (Dade-Behring)¹⁸ which draws a citrated blood sample through paired filters impregnated with platelet agonists and measures the time taken by resulting platelet aggregates to occlude them, providing two numerical end-points. One filter contains collagen and adenosine diphosphate (ADP): occlusion of this (at the high shear rate achieved by the device) is dependent on vWF–platelet gpIb/IX interaction and therefore the vWF content of the blood sample. The second filter combines collagen with epinephrine, and the rate of occlusion tests platelet granule function and signal transduction. Occlusion of the collagen/epinephrine filter is also very sensitive to the effect of aspirin and other antiplatelet agents. Use of this device is now widespread in laboratories testing for vWD and platelet function disorders. PFA-100 analysis, where available, is also tending to replace the skin bleeding time for evaluating the response of primary bleeding disorders to therapy with desmopressin, sources of vWF, or platelet concentrates.¹⁸

The skin bleeding time (SBT)

Previously a major criterion for the diagnosis of defects in primary hemostasis, prolongation of the skin bleeding time (even in its most reliable form, the Ivy template method) lost some of this status after being shown to lack sensitivity, reproducibility and operator-independence ¹⁹ in general use. In expert hands it can still produce useful evidence in equivocal cases,¹¹ and it remains part of the constellation of tests helpful in the diagnosis of vWD, although PFA-100 analysis is probably more sensitive.¹⁸

The Ivy method is recommended. A sphygmomanometer cuff is applied above the elbow and kept inflated to 40 mmHg to increase distal capillary pressure uniformly. Using a disposable device (e.g. Simplate®), a 5 mm long × 1 mm deep incision is made on the volar surface of the forearm, and a stopwatch started. Emerging blood is traditionally lifted off with the edge of a Whatman® filter paper, without applying pressure to the incision. The watch is stopped when the cut stops bleeding, and the time recorded as the SBT. Using this technique, an SBT >10 min is abnormal, indicating a primary bleeding disorder.

Measuring the size of the blots or further observation of the cut for rebleeding are no longer thought to add useful data. The procedure is uncomfortable for many patients, especially children, and leaves a small scar even if properly dressed with skin closures. Patients should be warned about this before giving valid consent to the procedure. After one instance of reflex withdrawal of the forearm that extended the cut from 5 mm to 3 cm, the author always keeps one hand gently but firmly on the patient’s wrist when doing this test.

Diagnostic tests

Classical platelet aggregometry

This elegant but demanding technique was introduced by Born.²⁰ Platelet-rich plasma (PRP) prepared by centrifugation of citrated blood is subsampled into warmed plastic cuvettes, stirred, and exposed to platelet agonists in doses that provoke aggregation of normal platelets. Consequent aggregation (or the lack of it) is detected by increasing light transmission through the cuvette, the time-course and extent of which are recorded on paper in the form of a curve. Interpretation combines inspection of the shape of this curve with a value for % aggregation, 100% being taken as the difference in light transmission between stirred PRP and buffer solution (‘blank’).

Platelet agonists (collagen, thrombin, epinephrine, arachidonate) cause aggregation by binding to receptors on the platelet surface and provoking the platelet release reaction, or by serendipitous interaction with the gpIb/IX receptor and vWF in the patient plasma (ristocetin). The combined response of an individual’s platelets to a panel of these reagents may form a pattern characteristic of a specific disorder (e.g. Glanzmann’s thrombasthenia), a narrow differential diagnosis (e.g. Bernard–Soulier disease versus vWD), or a broad class of disorders (e.g. δ- and α-storage-pool disorders). Aggregometry is therefore indispensable in the diagnosis of severe platelet function disorders, but requires expert performance and interpretation.

In practice, the investigation of individuals with convincing histories of excess bleeding is often frustrated by normal findings using classical aggregometry. This suggests that the technique is relatively insensitive to mild platelet function disorders. In about half such cases, PFA-100® analysis (see above) detects a prolonged closure time, usually of the collagen–epinephrine filter.

Investigations of platelet granule structure and function

Measurement of the release of effector molecules from δ-granules (e.g. ADP by the luciferase method, or ¹⁴C-serotonin release) or α-granules (e.g. β-thromboglobulin by ELISA) after platelet stimulation by agonists may further define disorders with findings on aggregometry or whole blood analysis consistent with storage pool disorders.

Transmission electron microscopy of platelets may demonstrate abnormal granule number or ultrastructure.

Fluorescence-activated cell sorting (FACS) analysis of platelets labeled with monoclonal antibodies specific for cell membrane epitopes (e.g. glycoproteins Ib/IX and IIb/IIIa) are useful in the diagnosis of Bernard–Soulier disease and Glanzmann’s thrombasthenia respectively.

These techniques straddle the boundary between diagnostic and research laboratory methods. They are appropriate only for reference laboratories that have sufficient expertise, technical resources and experience to correctly interpret their results.

Coagulation tests

Coagulation screening tests

This venerable set of simplified bioassays is performed on platelet-poor plasma centrifuged from a citrated sample of blood. In the modern laboratory, coagulometers detect the assay end-point of fibrin clot formation by mechanical or optical means.

Prothrombin time (PT) and the international normalized ratio

As invented by Quick,²¹ a source of tissue factor (’thromboplastin’: an aqueous extract of mammalian brain or, increasingly, a recombinant version)²² is added to citrated test plasma at 37°C and the mixture recalcified. Maximal stimulation of the clot initiation (‘extrinsic’) pathway results in clot formation in 12–15 s.

The PT depends on: 1) concentrations and activity of coagulation factors VII, X, V, II and fibrinogen in the test plasma; and 2) the sensitivity of the chosen thromboplastin to these activities and their inhibition. The PT is more sensitive to early-acting factors, particularly FVII, than to FII and fibrinogen (Fig. 31.3).

Fig. 31.3 Sequence of reactions in the prothrombin time (PT) test.

The end-point (clot formation) is timed and compared to the mean result obtained testing normal plasmas. If the PT (performed for diagnosis) is prolonged, a 50 : 50 mixing test (see below for the APTT) can be performed to indicate whether factor deficiency or inhibition is more likely to be responsible, although inhibitors affecting the PT alone are rare.

The PT is reported as a time (the control time coreported) or, increasingly, as a ratio (PT test plasma: PT normal plasma). Ratios obtained with different thromboplastins are transformed (‘normalized’) by the international sensitivity index (ISI) assigned to the test thromboplastin to correct for its sensitivity to factors VII, X and II by comparing its performance to that of an international reference thromboplastin.²³ The transformed ratio is reported as the international normalized ratio (INR):

where MNPT = the geometric mean PT of the population (in practice, 20 normal plasma samples).

The INR was introduced to harmonize coumarin anticoagulation, but it also functions as the prothrombin time for diagnosis ratio (PTDr) if a suitably sensitive thromboplastin is used. The closer the ISI of a thromboplastin to 1.0, the more likely it is to be reliable in both settings.

Interactions between thromboplastins and automated coagulometers performing the PT introduce more complexity. A large laboratory should not only determine its own reference range for the INR/PTDr, but also the ‘system ISI’ of its coagulometer/thromboplastin combination(s).²⁴

INR >1.2 indicates a defect in the TF/VII clot initiation pathway to thrombin. This could be due to deficiency or inhibition (anticoagulant or antibody) of any or all of factors VII, X, V, II, or fibrinogen (if <1 g/l) (Box 31.2). Coumarin therapy rapidly reduces FVII levels and the INR is sensitive to FVII. The INR is therefore used to monitor coumarin therapy (therapeutic range 2–4).

Box 31.2 Causes of ↑ INR/PTDr (>1.2)

Hepatocellular disease

Vitamin K malabsorption (cholestasis, steatorrhea)

Vitamin K deficiency (neonates, anorexia, hospitalization, intensive care)

Vitamin K antagonist (coumarins, e.g. warfarin)

Inherited deficiency of FII, FVII or FX

Disseminated intravascular coagulation

Inhibitors to FII, FVII or FX (autoantibodies or anticoagulants (e.g. hirudin, heparin))

Activated partial thromboplastin time (APTT)

This mini-assay of the clot amplification (‘intrinsic’) pathway was introduced by Langdell et al.²⁵ A phospholipid reagent that mimics the activated platelet surface (i.e. rich in PS,²⁶ although to supraphysiologic levels)²⁷ is incubated with test plasma at 37°C. Erratic contact activation is over-ridden by adding a strong activator such as kaolin, and the mixture recalcified. Sequential reactions provoked by contact activation in the presence of PS result in clot formation in 30–40 s.

The clotting end-point is measured by a coagulometer and expressed as the APTT in seconds, compared to the locally derived reference range. The APTT is also expressed as a ratio (APTTr) when used as a monitoring test for therapy with unfractionated heparin (UFH), and the use of this ratio in diagnostic work is acceptable.

This end-point depends on: 1) the concentrations and activities of contact factors prekallikrein, high-molecular-weight kininogen (HMWK) and factor XII; 2) the concentrations and activities of coagulation factors XI, IX, VIII, X, V, II and fibrinogen; and 3) the sensitivity of the whole test system to these activities and their inhibition. The APTT is more sensitive to contact and early-acting coagulation factors than to prothrombin and fibrinogen (Fig. 31.4). Elevated acute-phase proteins (factor VIII and fibrinogen) shorten the APTT and may obscure the effect of mild deficiencies and inhibitors in pregnancy and sepsis.

Fig. 31.4 Sequence of reactions in the activated partial thromboplastin time (APTT) test.

The APTT is an important test in three clinico-pathologic situations. First, it detects inherited and acquired hemophilia A and B because of its sensitivity to factors VIII and IX, and their inhibition, in plasma. Second, it detects the presence of ‘lupus-like’ (phospholipid-dependent) inhibitors because of its sensitivity to PS in the test reagent. Third, it detects the presence and concentration of heparin in plasma, and is therefore used to monitor unfractionated (but not low-molecular-weight) heparin therapy.

A reagent/coagulometer system sensitive to all three of these variables should be employed if possible, but the key function of the APTT – its role in the coagulation screen – is to detect hemophilia. Sensitivity to factor VIII and IX (i.e. the ability to detect levels of either factor below 45 IU/dl) is paramount, the practical convenience of a multifunctional APTT notwithstanding. A reference laboratory might opt for different APTT systems for different roles, rather than compromise any of them.

APTT correction tests

The sensitivity of the APTT to inhibitors of coagulation dictates that further information is gained by repeating the APTT on a 50 : 50 mixture of test plasma and normal pooled plasma. This correction test should be performed in most instances of prolonged APTT or APTTr not due to heparin therapy or contamination (see below).

A prolonged APTT due to contact or coagulation factor deficiency corrects on addition of an equal volume of pooled normal plasma to the test sample. For example, the factor IX content of a normal pool suffices to bring the mixture up to a level giving a near-normal APTT, even if the test plasma has <1% normal factor IX. In contrast, an inhibitor in the test plasma will inactivate coagulation factors in the added normal plasma, preventing correction.

‘Correction’ has been defined as the APTT of the mixture ‘being near to that of normal’²⁸ but using current reagents, most authors imply that correction of the APTT in a 50 : 50 mix means ‘to normal’^12,^29,³⁰ (i.e. APTTr <1.2). As with all coagulation tests, local criteria based on the performance of reagents and coagulometers and the experience of expert laboratory staff should be determined and applied. Since full, partial or absent correction (i.e. all possible results of the test) will all lead to more sensitive assays of coagulation factors and inhibitors, correction tests only indicate where to start.

If the clinical situation and/or screening tests suggest a possible factor VIII inhibitor, a modified method reflects inhibitor kinetics and identifies mild but clinically significant inhibitors. Unlike phospholipid-dependent antibodies, allo- and autoimmune anti-FVIII antibodies may need incubation with the target proteins for up to 2 h at 37°C to inhibit them. Extended incubation of the mixture therefore avoids illusory correction of a slow-acting inhibitor. An extra control (test and normal plasmas incubated separately and mixed just before testing) corrects for loss of factor VIII activity due to incubation alone.³¹

Classic mixing experiments

Correction studies employing absorbed plasma reagents, aged serum, or plasmas from patients with severe deficiencies, are popular thought experiments in practical examinations in hematology. Laboratories that have the time, staff and expertise to prepare, store, and maintain stringent quality control of a library of such reagents still find their differential correction of patient APTT a rapid route to specific diagnosis.¹¹ However, these requirements, and incompatibility with the automated coagulometers used to confirm the specific diagnosis of coagulation factor deficiencies, mean that they are no longer part of current routine practice. In any event, findings in classic mixing experiments must always be confirmed by specific factor assays.

Diagnostically misleading APTT prolongation

APTT sensitivity extends to components of the test plasma that are clinically irrelevant (prekallikrein, HMWK and factor XII) and the frequent contamination of coagulation samples with heparin from intravenous lines. The APTT is therefore prone to diagnostic false positives when used as an indiscriminate screening test in hospital practice, potentially causing delay and cost while the cause of a long APTT is tracked down. For these reasons, and its sensitivity both to states causing severe bleeding (hemophilia) and dangerous thrombosis (lupus anticoagulant), the APTT can only be interpreted in the light of the clinical history (Box 31.3).

APTT detects UFH but not low-molecular-weight heparins (LMWH). APTTr monitors UFH therapy (therapeutic range APTTr 1.5–2.5). Samples taken via heparin-flushed lines are useless: heparin cannot be flushed out.

Thrombin time (TT)

This simple test (Fig. 31.5) adds thrombin to test plasma and times the resulting clot end-point. TT is expressed in seconds and compared to a normal control range (e.g. TT 15 s, control = 11 s). An abnormal TT (>15 s) is due to: 1) deficiency of fibrinogen; 2) an inhibitor capable of inhibiting exogenous thrombin (e.g. heparin, hirudin); or 3) inhibition of fibrinogen polymerization due to an abnormal fibrinogen molecule (dysfibrinogenemia) or interfering substances (fibrin/fibrinogen degradation products, paraproteins) (Box 31.4). Some laboratories add calcium to the thrombin solution used in the TT to narrow the normal range and improve reproducibility, but this entails a loss of sensitivity to dysfibrinogens and is not recommended.

Fig. 31.5 The thrombin time (TT) test reaction.

Box 31.4 Causes of ↑ TT (>3s >control)

Afibrinogenemia

Hypofibrinogenemia

Dysfibrinogenemia

Inhibitors of fibrin polymerization: paraproteins, fibrin–fibrinogen degradation products (FDPs)

Heparin (unfractionated)

Heparin interference with coagulation screening tests: the reptilase time

Heparin contamination is common in samples from wards, theaters and intensive care units – anywhere that intravenous access devices are used for blood sampling. It is therefore reasonable to exclude it before pursuing a diagnosis of DIC (see below), which it mimics. Reptilase (the venom of Bothrops atrox) clots fibrinogen in a heparin-insensitive way while retaining sensitivity to other abnormalities that prolong the TT. A reptilase time (normal 10–12 s) is therefore a helpful and necessary adjunct to the coagulation screen.

Fibrinogen assay

Measuring the concentration of fibrinogen in plasma can be regarded as an extension of the initial coagulation screen: the PT and APTT are relatively insensitive to moderate hypofibrinogenemia and a prolonged TT requires explanation if heparin contamination has been excluded by a reptilase time. The most reliable method for the automated routine laboratory is the Clauss method,³² a parallel-line bioassay based on the TT performed on serial dilutions of patient plasma and control. Fibrinogen estimates ‘derived’ from automated PT or APTT analysis can be misleading in the very states (e.g. DIC) in which fibrinogen assay is most useful, and are not recommended.

Fibrin–fibrinogen degradation products (FDPs) and D-dimer assay

In several clinical situations it is helpful to detect the presence of plasmin-digested cleavage products of cross-linked fibrin and fibrinogen termed fibrin–fibrinogen degradation products (FDP). An elevated FDP concentration (>100 mg/ml) suggests DIC (see below) or rarer primary fibrinolytic states.

A variety of commercial immunoassays use polyclonal antibodies to detect and quantify molecules expressing fibrinogen epitopes, for example by coated latex bead agglutination. These include native fibrinogen and its direct plasmin cleavage products, as well as fragments that signify plasmin digestion of intravascular fibrin. This lack of specificity necessitates testing serum produced by ex vivo clotting in the presence of an inhibitor of fibrinolysis (e.g. aprotinin) to prevent ex vivo generation of FDP, requiring a separate and specific FDP sample tube.

Recently developed assays employ monoclonal antibodies that recognize the D-dimer fragment produced by plasmin digestion of cross-linked fibrin (i.e. thrombus).¹¹ The increased sensitivity and specificity of this assay allows detection of the relatively low levels of D-dimer circulating in the presence of deep vein thrombosis and pulmonary embolism (venous thromboembolic disease, VTED). D-dimer assay combined with the pretest probability estimate derived from a clinical scoring system is useful in the diagnosis of VTED.³³ Since the D-dimer assay retains sensitivity to DIC and uses the citrated coagulation screen sample it has largely replaced older polyspecific assays for serum FDP.

Logical use of the coagulation screen

Combining the results of the PT, APTT and TT tests, and using them as a logical ‘circuit tester’ (see Fig. 31.6) maximizes the information provided by the coagulation screen, particularly when considered with the platelet count. The logic of the coagulation screen combined with platelet testing has been expressed in algorithmic form³⁴ and as a web-based interactive computer program,³⁵ but is probably straightforward enough to keep in one’s head.

Fig. 31.6 The coagulation screen as a ‘circuit tester’.

For example, if the APTT ratio is increased and corrects to normal with 50 : 50 normal plasma, but the INR, TT and platelet count are normal, the probable deficiency is restricted to one of the factors tested only by the APTT: FXII, FXI, FIX or FVIII. The exact deficiency is determined by specific assays of single factors, starting with a factor VIII assay because this is the commonest cause of severe hemophilia (see Box 31.5).

Box 31.5 Logical use of the clotting screen and pretest probability

A 66-year-old male bled for 48 h after surgery. Excess bleeding was noted from surgical drains and as a wound hematoma. Pretest probability of a bleeding disorder (from history and examination) ∴ = high

Platelet count = 245 × 10⁹/l

APTTr ↑ (1.9) corrects with 50 : 50 normal plasma.

INR and TT normal.

∴ There is a deficiency (it corrects) of one or more factors tested only by the APTT: a contact factor, or clotting factors VIII, IX or XI.

Pretest probability suggests FVIII, FIX or FXI deficiency: all can cause excess bleeding after surgery. Contact factor deficiencies do not cause excess bleeding.

Specific activity assays of these factors are performed, starting with FVIII-C (the commoner of the two potentially severe deficiencies).

Result: FVIII:C = 190 IU/dl (normal 50–150 IU/dl)

FIX:C = 13 IU/dl (normal 50–150 IU/dl)

∴ The patient has mild hemophilia B (Christmas disease). This may not become manifest as bleeding until a major surgical challenge occurs, perhaps for the first time in later life. In the context of surgery no hemophilia is ‘mild’.

∴ therapy is needed to raise the FIX level.

A single gene lesion typically reduces the function of a single coagulation factor, and therefore usually prolongs a single coagulation screen test. Exceptions to this rule of thumb are, in the first case, genetic disorders causing combined factor deficiencies (e.g. FV + FVIII deficiency),³⁶ and in the second case, severe FX, FV, prothrombin or fibrinogen deficiencies; these are all rarities.

In contrast, systemic diseases or drugs alter the synthesis, postsynthetic processing or function of several clotting factors and may reduce the platelet count. They are therefore reflected by abnormalities of several screening tests. For example, hepatocellular failure: 1) reduces the plasma concentration of all coagulation factors; 2) induces a hyperfibrinolytic state further consuming them; and 3) is often associated with portal hypertension causing splenic pooling of platelets.

The dangerous clinical disorder of hemostasis designated DIC also causes abnormalities in several screening tests because coagulation factors and platelets are consumed by chaotic activation of the whole hemostatic system. The APTTr may be misleadingly low in DIC, either because DIC is occurring on the background of an acute phase response (in sepsis or pregnancy) or due to circulating activated coagulation factors. Whenever the clinical situation and/or the pattern of abnormalities seen in the coagulation screen are consistent with DIC, an FDP or D-dimer assay (see above) should be added to the screen to detect the high levels of fibrin/fibrinogen degradation products characteristic of DIC. A fibrinogen assay should also be done to guide supportive transfusion therapy.

Heparin, by blocking the function of several clotting factors, mimics the effect of these serious global disorders of hemostasis by prolonging all three coagulation screening tests. It mainly confounds when samples for coagulation screening are drawn from vascular access devices flushed with heparin to keep them patent. Although such contamination can be partly excluded by performing a reptilase time test (see above), this causes delay in critical situations. It is better to establish a general rule that all coagulation samples must be taken by direct venipuncture.

Disorders of hemostasis that may not affect screening tests

von Willebrand disease (vWD). This common disorder may prolong the APTT, but this depends on a secondary effect on FVIII/vWF binding. Clinically significant type I vWD is often associated with a normal APTT. If the pretest probability of a bleeding disorder is moderate or high, whole blood platelet function (see above) should be measured. This procedure will also ensure that platelet function disorders, also ‘silent’ in the coagulation screen, are not missed. vWF assays should be done if whole blood platelet function analysis is not available.

Mild but clinically significant deficiencies of coagulation factors. Depending on reagent/coagulometer system sensitivities, mild deficiencies (i.e. factor levels 10–40 IU/dl) may not be detected by the APTT. When normal coagulation screening tests and platelet function are found in the context of a high pretest likelihood of a clinical bleeding disorder, exclusion of factor VIII, factor IX and factor XI deficiencies finally depends on specific assays (see Box 31.6).

Box 31.6 Final words on screening tests of hemostasis

If the clotting screen or full blood count is abnormal, refocus the history and examination (e.g. to look for malignancy or hepatic disease).

Clinical findings always take precedence over negative laboratory screening.

If there is moderate or high pretest probability of a bleeding disorder, ignore negative screening tests and proceed to assays of specific coagulation factors and platelet function.

If these in turn find nothing, refer the problem to an expert in the field of hemostasis.

Factor XIII deficiency. This transglutaminase stabilizes fibrin polymer by catalyzing fibrin cross-linking. Factor XIII deficiency, a rare autosomal recessive disorder, presents classically as bleeding from the umbilical cord stump or delayed bleeding after surgical challenge.

Clots formed from the plasma of individuals with severe factor XIII deficiency (<0.03 units/ml) differ from normal by dissolving in 5M urea, monochloroacetic acid or 2% acetic acid. This simple screening test should be applied to individuals with a high pretest probability of a bleeding disorder who give negative results with all other tests, supplemented by more sensitive immunoassays for factor XIII.

Diagnostic coagulation tests

Specific assays of individual clotting factors

The final diagnostic step in the investigation of an individual with a potential bleeding disorder is assay of the biological activity and/or molecular concentration of individual coagulation factors in their plasma. Knowledgeable interpretation of the results of screening tests will narrow the range of likely defects and the first assays performed should follow this logic. This is preferable to ‘shotgun’ analyses because coagulation assays are expensive tests that consume the scarce resource of expert technical attention. Clearly, the labeling of an individual as having an inherited severe coagulation disorder has such profound implications for them, their family and the healthcare system that the investigation must attain the highest possible degree of certainty at this stage.

Functional bioassays

The most clinically relevant assay of a coagulation factor tests its ability to promote clot formation in human plasma – its ‘activity’. Testing function in a biological system (e.g. human plasma) is termed bioassay. In exchange for clinical relevance, the complexity of bioreagents imposes limits on accuracy and reproducibility. To keep these limits within tolerable bounds the use of a hierarchy of plasma standards to calibrate and control assays is essential, as is constant participation in quality control exercises. These are discussed in the next section.

The activity of a coagulation factor is determined by bioassay, in which the potency of the patient’s plasma – its ability to correct the prolonged clotting time of plasma missing the factor in question – is compared to that of a plasma standard. Since the plasma standard has a known content of the factor, the unknown content of the patient’s plasma can be calculated by comparison. The resulting activity is indicated by the suffix [:C], for example factor VIII:C.

If the calibration trail of the assay standard leads eventually to the current International Standard, the result can be expressed in international units (IU). Strictly, the activity of a coagulation factor in plasma should be expressed as IU/ml (normal 0.5–1.5 IU/ml), but is often expressed as IU/dl (normal 50–150 IU/dl) in order to match the intuitive convention of ‘per cent normal’.

One-stage assays. The simplest assay design is the one-stage method which depends on correction of the clotting time (e.g. using the APTT as the ‘marker system’ in a FVIII:C assay) of a plasma from which the factor in question is absent. This reagent is termed the substrate plasma. Individuals with severe inherited deficiencies remain a major source of substrate plasma (which accordingly is a potential infection risk), although the availability of commercial reagents reliably depleted of single coagulation factors by immunoadsorption has broadened the applicability of one-stage assays. As a result, because of their conceptual simplicity, adaptability to automation, and use of everyday (i.e. APTT, PT) reagents, one-stage assays are commoner in practice than more complex designs, except in special situations.

All one-stage assays share the same design apart from their marker system, which may be the PT (for prothrombin, FV:C, FVII:C and FX:C assays) the APTT (for FVIII:C, FIX:C, FXI:C or contact factor assays) or snake venom clotting times (Taipan for prothrombin (not in the context of coumarin therapy), Russell’s viper for FX:C).

Serial dilutions (at least three) of patient plasma are added to substrate plasma. The same is done with the assay standard. Clot end-points are measured for each dilution.

The manual method plots clotting times on the vertical axis against dilution on the horizontal axis, using logarithmic graph paper. This results in two ‘curves’ (which should form parallel straight lines) – a standard curve and an unknown (patient) curve. From the standard curve, a horizontal line (‘of equal potency’) is drawn to where it intercepts the unknown curve: a vertical line dropped to the horizontal axis from this point marks the potency of the patient plasma relative to the standard. Full descriptions of this method, including the requirements for parallelism on which its validity rests, can be found elsewhere.³⁷^–³⁹

Even when controlled and standardized, it remains a bioassay with irreducible limits on precision and reproducibility. A single assay can only be relied upon to give an answer within 20% of the ‘true’ value (the idealized mean value of an infinite number of tests). This is one basis (the other being the variation of plasma FVIII levels in individuals) of the common rule that at least three assays (reducing the error to ± 10%) are required to define the severity of hemophilia. To reduce the error to ± 2.5% would theoretically require 64 assays.³⁷ These sources of within-laboratory error are compounded by inter-laboratory variance, which in external quality control exercises can give a CV (coefficient of variation) of 30–50%.⁴⁰

In modern routine laboratories the potency is calculated mathematically by computer modules linked to automated coagulometers.

Two-stage assays. This form of assay differs by eliminating the substrate plasma and phospholipid reagent of the APTT-based assay: instead, a reaction mixture is prepared (containing excess FIX and FX) in which the formation of factor Xa is proportionate to the concentration of FVIII:C or FIX:C in dilutions of unknown and assay standard plasmas as above. FXa thus generated is measured by its action in a second reaction mixture (using a clot end-point) or directly by its action on a chromogenic substrate (see below). Potency calculations are then carried out as for one-stage assays.

Chromogenic assays. Rather than using a clotting end-point in a two-stage assay as above, the availability of synthetic amidolytic substrates sensitive to factor Xa enable the use of a color reaction detectable and quantifiable by spectrophotometry. This chromogenic method has been adapted to measure several hemostatic enzymes and their inhibitors, and is highly compatible with automation.⁴¹ One theoretical advantage of the chromogenic method is that it directly measures the first product (FXa) of the FVIII/FIX interaction, rather than requiring the participation of several other factors in the production of a clotting end-point. This increases the precision and reproducibility of the assay. In addition, lack of dependence on phospholipid reagents makes the chromogenic method more reliable in the assay of therapeutic concentrates, both in the vial and in the patient.²⁷

Immunoassays

Immunoassays use poly- or monoclonal antibodies to detect and quantify coagulation proteins. Except in the special case of ‘functional’ epitopes (i.e. immunologic determinants linked so closely to the functional site of a molecule that they are assumed to act as a reliable marker of its activity), immunoassays detect the presence of a protein rather than its function. Immunoassays may therefore give normal results in disorders due to loss-of-function mutations that result in normal or slightly reduced amounts of protein (e.g. type 2A vWD). However, immunoassay remains a cornerstone of diagnosis in vWD because of the continued lack of a reliable non-surrogate functional assay of vWF.

The ability to stick purified monoclonal antibodies to plastic or latex surfaces, together with ubiquitous chromogenic reactions derived from the alkaline phosphatase/anti-alkaline phosphatase (APAAP) method, have transformed immunoassay technology. Enzyme-linked immunosorption assay (ELISA) and recent latex agglutination assays are rapidly supplanting the older radiometric and immunoelectrophoretic methods (e.g. Laurell electroimmunoassay).⁴² Such immunoassays are most often used in the diagnosis of vWD and thrombophilia.

Molecular diagnosis in disorders of hemostasis

The diagnostic chain may now be continued to DNA sequencing and the direct detection of gene mutations or deletions affecting genes encoding coagulation proteins, particularly carrier detection and antenatal diagnosis in the hemophilias. However, gene analysis has not yet become part of the standard clinical diagnostic criteria for coagulation and platelet disorders as it has for some types of thrombophilia.

The problem of testing the fibrinolytic system

The fibrinolytic system is of vital importance to the organism, and scientific investigation and exploitation of its components has led to important therapeutic agents with major impact in common life-threatening thrombotic disorders. None the less, it remains the ‘Cinderella’ of hemostatic testing.⁴³ Partly, this is due to the evanescent nature of most clinical disturbances of fibrinolysis. Compared to chronic disorders of coagulation, the fibrinolytic system rarely ‘sits still’ for long enough to study it. In addition, a fibrinolytic equivalent to the coagulation screen does not really exist: the euglobulin clot lysis time (ECLT) is insensitive and poorly reproducible. Immunoassays of key fibrinolytic enzymes and inhibitors are available, but interpreting their results in thrombotic syndromes is difficult and currently unproductive.⁴³ In acute clinical situations fibrinogen and D-dimer assays will give clues to the presence of hyperfibrinolysis. α2-Antiplasmin deficiency, the only well-documented inherited bleeding disorder attributed to chronic excess plasmin activity, does not declare itself in any screening test: clinical suspicion should lead to measurement of α2-antiplasmin by ELISA.⁴⁴

Monitoring hemostasis using global tests

The tests discussed above (except the PFA-100 system) require separation of citrated plasma or platelet-rich plasma from venous blood samples, resulting in an ex vivo derivative that differs in two crucial ways from the circulating in vivo blood.

Firstly, key cellular messengers, calcium ions, are removed from the milieu. Secondly, the complex interactions between coagulation factors and the cell membranes of platelets, leukocytes and red cells are negated and substituted by a phospholipid reagent that is unphysiological in both composition and dose.

Given these test features, it is perhaps fortunate that generally reliable, clinically useful inferences can be based on their use. However, it is widely perceived that validated test systems capable of measuring events in whole blood, complete with its cellular elements, could generate information that correlates better with clinical reality. Such test systems are referred to as global tests of hemostasis.

The ideal coagulation test

The ideal global assay would measure the dynamics of thrombin generation, detect whole blood clot formation and/or fibrin formation, and evaluate the structure and stability of the formed clot.

Such an assay should improve determination of the baseline phenotype, and therapeutic monitoring in an individual with a bleeding disorder. It should also help in diagnosis when conventional tests have given normal or equivocal results, as happens in up to 20% of individuals with a credible history of excess bleeding on challenge.

This ideal test does not yet exist, but automated methods for measuring (a) thrombin generation, and (b) whole blood coagulation with thromboelastography/thromboelastometry are currently the most promising candidates for the role.

Calibrated automated thrombin generation measurement

In the calibrated automated thrombin (CAT) assay, a fluorogenic thrombin substrate is incubated with test plasma and coagulation is activated by tissue factor. After recalcification, a computer converts automated continuous measurements of fluorescence into a quantitative dynamic profile of thrombin generation (Fig. 31.7).

Fig. 31.7 A quantitative dynamic profile of thrombin generation.

Thrombin generation measurements are hypersensitive to pre-analytic variables, so meticulous blood sampling procedures are vital. Many laboratories add corn trypsin inhibitor (CTI – a direct and potent inhibitor of coagulation factor XIIa) to the blood sampling tubes in order to avoid or minimize spontaneous contact activation. This step reduces assay variability.

Interpretation and clinical feasibility

Three informative variables can be measured from the thrombin generation curve: lag time, time to peak thrombin and endogenous thrombin potential (Fig. 31.7). The course of thrombin generation comprises an initiation phase before the first thrombin is detected (the lag time), and an amplification phase during which the maximum rate of thrombin generation occurs.

Thrombin generation then reaches a peak and the time required to reach this point is described as time-to-peak thrombin. The total amount of thrombin generation (= area under the curve) is frequently called the endogenous thrombin potential (ETP).

In principle, abnormal thrombin generation curves are characterized by 1) a prolonged or shortened lag time, 2) reduced or increased peak thrombin, or 3) reduced or elevated ETP.

The lag time is primarily determined by: levels of free tissue factor, tissue factor pathway inhibitor, factor VII, factor IX and fibrinogen. The amplification phase of thrombin is highly dependent on the number and function of platelets. Hence, the higher the platelet count in the sample, the higher the maximum rate and acceleration of thrombin generation.

Males seem to have a lower [ETP]thrombin production than females. In addition, thrombin generation increases with age and decreases as a result of low temperature.

Thrombin generation has been used for laboratory phenotyping a variety of bleeding disorders. The best characterized hemostatic dysfunction described by thrombin generation profiles is hemophilia A.

The current categories of mild, moderate and severe hemophilia A are based simply on the assayed level of factor VIII in plasma. They are not an absolute guide to an individual’s bleeding severity and need for therapy. The rate specific characteristics of thrombin generation have been reported to more accurately reflect the clinical heterogeneity of hemophilia. In particular, thrombin generation has been documented as predictive in distinguishing milder phenotypes of severe hemophilia despite similar low levels of factor VIII (e.g. less than 1% of normal).

Thrombin generation profiles have also been used for monitoring the hemostatic response to treatment. Hemophilia A is treated by substitution with a factor VIII concentrate, but about 20% of patients consequently develop alloantibody inhibitors against factor VIII. These patients can be treated with inhibitor ‘bypassing agents’ such as recombinant factor VIIa or plasma-derived activated prothrombin complex concentrates (FEIBA), but conventional assays cannot measure the hemostatic effect of these bypassing agents. Thrombin generation measurement has been used to monitor substitution with these agents. The overall experience with thrombin measurements as a surrogate of hemostatic efficacy is still rather limited, but preliminary results appear promising.

Outside hemophilia, thrombin generation has proven advantageous for illustrating the hemostatic potential of prothrombin complex concentrates compared with fresh frozen plasma for reversal of vitamin K antagonist therapy.

Finally, thrombin generation is an elegant method for illustrating the impact of various types of anticoagulation. Heparin, direct thrombin inhibitors and indirect or direct factor Xa inhibitors compromise thrombin generation by prolonging the lag phase and reducing peak thrombin and ETP.

Whole blood thromboelastometry

Thrombelastography (TEG®) and thromboelastometry (ROTEM®) provide a visualization of continuous viscoelastic changes occurring during whole blood clot formation. Both devices consist of a cup into which the sample (whole blood, platelet-rich or -poor plasma) and reagents are placed and a pin which sits in the centre of the cup when the device is running. Reduced movement of the pin during clot formation is registered with specialized computer software and visualized on a computer providing a coagulation signal similar to that of traditional thrombelastography (Fig. 31.8A).

Fig. 31.8 (A, B) (A) Standard whole blood thromboelastometry profiles and parameters. The clotting time (CT/r) reflects the initiation of clot formation, whereas the clot formation time (CFT/k) illustrate the dynamic of clot formation. The maximum amplitude is the maximum clot formation (MCF). As illustrated, in a condition of severe Hemophilia A, patients have a delayed and slot clotting process. (B) Illustrate the first derivative of the standard whole blood thromboelastometry profile. The velocity profile enhance the dynamic abnormality of hemophilia as illustrated by a reduced maximum velocity (MaxVel) of clot formation.

Interpretation and clinical feasibility

Traditional thrombelastographic parameters include clotting time (r, CT), clot formation time (k, CFT), and maximal clot formation (MA, MCF) as depicted in Fig. 31.8. Thromboelastometry also assesses clot strength and fibrinolysis levels of MA/MCF (Fig. 31.8).

In principle, abnormal thromboelastographic/thromboelastometry profiles are characterized by 1) a prolonged or shortened r or CT, 2) prolonged k or CFT, or 3) a compromised or elevated MA or MCF.

The digital raw signal from the TEG or ROTEM analyzers can be differentiated to velocity profiles of whole blood clot formation and dynamic coagulation parameters illustrating the propagation phase of clotting can be derived (Fig. 31.8B) such as the maximum velocity (MaxVel) of clot formation and the time until the occurrence of the maximum value (t, MaxVel).

TEG® and ROTEM® have been validated as bedside monitoring tools for diagnosing perioperative coagulopathies and guiding optimal hemostatic intervention. Recent studies have documented that routine use of TEG or ROTEM can reduce unnecessary transfusion of allogeneic blood products and optimize goal-directed hemostatic intervention with coagulation factor concentrates. Several commercial standard assays are available providing activation of the intrinsic pathway (e.g. with kaolin) or extrinsic pathway (with tissue factor) as well as fibrinogen sensitive assays and assays to neutralize heparin.

In addition to the standard assays, sensitive assays have been developed that employ activation with minute (physiological) amounts of tissue factor (as little as 1/50 000th of the concentration used to measure the prothrombin time).

The low tissue factor assay reveals heterogeneity in whole blood coagulation patterns among patients with factor VIII levels <1%, those with less abnormal blood clotting profiles tending to have a less severe bleeding phenotype.

The low tissue factor assay has also been used to illustrated different response patterns to various levels of coagulation factor VIII concentrate. In addition, both in vitro and in vivo studies have demonstrated the ability of thromboelastography to predict the clinical response to bypassing agents in patients with inhibitors.

In the near future, it is possible that thromboelastography may be used to help design individual treatment regimens for patients with hemophilia, with or without inhibitors. This will become increasingly more important as new therapeutic agents become available.

Minimizing error in hemostatic testing, interpretation and process

The hematologist must identify potential sources of imprecision, error or misinterpretation in laboratory testing that could impede correct diagnosis and patient safety. Tests of hemostasis are fertile ground for such errors: no hematologist can afford to be a passive consumer of their results. Areas that require constant vigilance are:

The pre-analytical phase. Traumatic venipuncture, stop-flow blood drawing and suboptimal mixing or filling of sodium citrate-containing coagulation sample tubes are all potential sources of error. Polycythemia (by decreasing the plasma : citrate ratio) and anemia (the reverse effect) can alter results. Coagulation samples should be tested as soon as possible: any sample waiting more than 3 h (>2 h for FVIII:C assays) for analysis will give misleading results. Coagulation bioassays should preferably be done on fresh rather than frozen-thawed plasma, although pressures on the modern laboratory often prevent observance of this rule.

Test methodology. Written standard operating procedures (SOPs) for every test in its repertoire must be held in the laboratory – and used. SOPs must be regularly updated and externally peer-reviewed as part of an inspection by an accrediting agency. Training must ensure observation of SOPs by all workers performing or validating tests during the laboratory 24 h cycle, and by operators of any point-of-care devices under supervision by the laboratory.

Test reagents and calibration: the hierarchy of standards. Bioassays impose a requirement for reference materials, including thromboplastin reagents, assay standards ⁴⁰ and drugs. Plasma or concentrate standards are freeze-dried aliquots of plasma or concentrate with a certified content of the factor in question. These secondary standards are in turn assayed (calibrated) against primary International Standard materials held by national biological standards agencies such as the National Institute for Biological Standards and Controls in the UK, and ultimately the World Health Organization and other international bodies.⁴⁵ Tracing a calibration trail through this hierarchy of standards ensures that results obtained in different laboratories and in different countries are comparable.

Internal quality control and external quality assurance. Laboratories must perform regular internal quality control procedures, particularly after introducing new tests, methods or machines or when established versions give cause for concern. They must also participate in external quality assurance schemes commensurate with their function: a Hemophilia Center laboratory would be expected to participate in an extended scheme that focused on assays of single coagulation factors, the detection and quantification of coagulation inhibitors, etc. Regular participation in the exercises provided by such a scheme registers a ‘running score’ of the performance of a laboratory that indicates the reliability of its results. This cumulative performance indicator must be available for inspection by the users of these results. The collated results of such exercises, published among users as surveys, provide useful information on the performance of current tests.

Potential confounding effects. The influence of age on hemostatic variables must always be considered and age-specific reference ranges consulted, particularly interpreting results obtained in infants and children, whose levels of vitamin K-dependent factors and natural anticoagulants differ from those in adults. Pregnancy, particularly during the third trimester and in the peripartum, is associated with marked changes in the levels of both procoagulant and anticoagulant proteins, with potential under- or over-diagnosis of hemostatic abnormalities. The ABO blood group status of an individual must be considered when interpreting vWF levels, which can also be affected by age, exercise, the acute-phase response, or needle-phobia. vWF levels may also change throughout the menstrual cycle, although this is not a universal finding.