General Structure of the Blood Vessel

The blood vessel wall has three layers: intima, media and adventitia. The intima consists of endothelium and subendothelial connective tissue and is separated from the media by the elastic lamina interna. Endothelial cells form a continuous monolayer lining all blood vessels. The structure and the function of the endothelial cells vary according to their location in the vascular tree, but in their resting state they all share three important characteristics: they are ‘non-thrombogenic’ (i.e. they promote maintenance of blood in its fluid state); they play an active role in supplying nutrients to the subendothelial structures; and they act as a barrier to cells, macromolecules and particulate matter circulating in the bloodstream. The permeability of the endothelium may vary under different conditions to allow various molecules and cells to pass.

Endothelial Cell Function

The luminal surface of the endothelial cell¹ is covered by the glycocalyx, a proteoglycan coat. It contains heparan sulphate and other glycosaminoglycans, which are capable of activating antithrombin, an important inhibitor of coagulation enzymes. Tissue factor pathway inhibitor (TFPI) is present on endothelial cell surfaces bound to these heparans but also tethered to a glycophosphoinositol (GPI) anchor. The relative importance of these two TFPI pools is not known. Endothelial cells express a number of coagulation active proteins that play an important regulatory role such as thrombomodulin and the endothelial protein C (PC) receptor. Thrombin generated at the site of injury is rapidly bound to a specific product of the endothelial cell, thrombomodulin. When bound to this protein, thrombin can activate PC (which degrades factors Va and VIIIa) and a carboxypeptidase which inhibits fibrinolysis (discussed later). Thrombin also stimulates the endothelial cell to produce tissue plasminogen activator (tPA). The endothelium can also synthesize protein S, the cofactor for PC. Finally, endothelium produces von Willebrand factor (VWF), which is essential for platelet adhesion to the subendothelium and stabilizes factor VIII within the circulation. VWF is both stored in specific granules called Weibel Palade bodies and secreted constitutively, partly into the circulation and partly toward the subendothelium where it binds directly to collagen and other matrix proteins. The expression of these and other important molecules such as adhesion molecules and their receptors are modulated by inflammatory cytokines. The lipid bilayer membrane also contains adenosine diphosphatase (ADPase), an enzyme that degrades adenosine diphosphate (ADP), which is a potent platelet agonist (see p. 434). Many of the surface proteins are found localized in the specialized lipid rafts and invaginations called ‘caveolae’, which may provide an important level of regulation.²

The endothelial cell participates in vasoregulation by producing and metabolizing numerous vasoactive substances. On the one hand, it metabolizes and inactivates vasoactive peptides such as bradykinin; on the other hand, it can also generate angiotensin II, a local vasoconstrictor, from circulating angiotensin I. Under appropriate stimulation the endothelial cell can produce vasodilators such as nitric oxide (NO) and prostacyclin or vasoconstrictors such as endothelin and thromboxane. These substances have their principal vasoregulatory effect via the smooth muscle but also have some effect on platelets.

The subendothelium consists of connective tissues composed of collagen (principally types I, III and VI), elastic tissues, proteoglycans and non-collagenous glycoproteins, including fibronectin and VWF. After vessel wall damage has occurred, these components are exposed and are then responsible for platelet adherence. This appears to be mediated by VWF binding to collagen. VWF then undergoes a conformational change and platelets are captured via their surface membrane glycoprotein Ib binding to VWF. Platelet activation follows and a conformational change in glycoprotein IIbIIIa allows further, more secure, binding to VWF via this receptor as well as to fibrinogen. At low shear rates (<1000 s⁻¹) platelet binding directly to collagen appears to dominate.³

Vasoconstriction

Vessels with muscular coats contract following injury, thus helping to arrest blood loss. Although not all coagulation reactions are enhanced by reduced flow, this probably assists in the formation of a stable fibrin plug by allowing activated factors to accumulate to critical concentrations. Vasoconstriction^1,4 also occurs in the microcirculation in vessels without smooth muscle cells. Endothelial cells themselves can produce vasoconstrictors such as angiotensin II. In addition, activated platelets produce thromboxane A₂ (TXA₂), which is a potent vasoconstrictor.

Platelet Function in the Haemostatic Process

The main steps in platelet function⁷ are adhesion, activation with shape change and aggregation. When the vessel wall is damaged, the subendothelial structures, including basement membrane, collagen and microfibrils, are exposed. VWF binds to collagen and microfibrils and then captures platelets via initial binding to platelet GPIb, resulting in an initial monolayer of adhering platelets. Binding via GPIb initiates activation of the platelet via a G-protein mechanism. Once activated, platelets immediately change shape from a disc to a tiny sphere with numerous projecting pseudopods. After adhesion of a single layer of platelets to the exposed subendothelium, platelets stick to one another to form aggregates. Fibrinogen, fibronectin, further VWF released from platelets and the glycoprotein Ib-IX and IIbIIIa complexes are essential at this stage to increase the cell-to-cell contact and facilitate aggregation. Certain substances (agonists) react with specific platelet membrane receptors to promote platelet aggregation and further activation. The agonists include exposed collagen fibres, ADP, thrombin, adrenaline, (epinephrine) serotonin and certain arachidonic acid metabolites including TXA₂. In areas of non-linear blood flow, such as may occur at the site of an injury, locally damaged red cells release ADP, which further activates platelets.

Platelet Aggregation

Platelet aggregation may occur by at least two independent but closely linked pathways. The first pathway involves arachidonic acid metabolism. Activation of phospholipase enzymes (PLA₂) releases free arachidonic acid from membrane phospholipids (phosphatidyl choline). About 50% of free arachidonic acid is converted by a lipo-oxygenase enzyme to a series of products including leucotrienes, which are important chemoattractants of white cells. The remaining 50% of arachidonic acid is converted by the enzyme cyclooxygenase into labile cyclic endoperoxides, most of which are in turn converted by thromboxane synthetase into TXA₂. TXA₂ has profound biological effects, causing secondary platelet granule release and local vasoconstriction, as well as further local platelet aggregation via the second pathway below. It exerts these effects by raising intracellular cytoplasmic free calcium concentration and binding to specific granule receptors. TXA₂ is very labile with a half-life of <1 min before it is degraded into the inactive thromboxane B₂ (TXB₂) and malonyldialdehyde.

The second pathway of activation and aggregation can proceed completely independently from the first one: various platelet agonists, including thrombin, TXA₂ and collagen, bind to receptors and via a G-protein mechanism, activate phospholipase C. This generates diacylglycerol and inositol triphosphate, which in turn activate protein kinase C and elevate intracellular calcium, respectively. Calcium is released from the dense tubular system to form complexes with calmodulin; this complex and the free calcium act as coenzymes for the release reaction, for the activation of different regulatory proteins and of actin and myosin and the contractile system and also for the liberation of arachidonic acid from membrane phospholipids and the generation of TXA₂.

The aggregating platelets join together into loose reversible aggregates, but after the release reaction of the platelet granules, larger, firmer aggregates form. Changes in the platelet membrane configuration now occur; ‘flip-flop’ rearrangement of the surface brings the negatively charged phosphatidyl-serine and -inositol on to the outer leaflet, thus generating platelet factor 3 (procoagulant) activity. At the same time specific receptors for various coagulation factors are exposed on the platelet surface and help coordinate the assembly of the enzymatic complexes of the coagulation system. Local generation of thrombin will then further activate platelets.

Platelets are not activated if in contact with healthy endothelial cells. The ‘non-thrombogenicity’ of the endothelium is the result of a combination of control mechanisms exerted by the endothelial cell: synthesis of prostacyclin, capacity to bind thrombin and activate the PC system, ability to inactivate vasoactive substances and so on. Prostacyclin released locally binds to specific platelet membrane receptors and then activates the membrane-bound adenylate cyclase (producing cyclic adenosine monophosphate or cAMP). cAMP inhibits platelet aggregation by inhibiting arachidonic acid metabolism and the release of free cytoplasmic calcium ions.

Thus platelets have at least three roles in haemostasis:

The Contact Activation System

The contact activation system⁹ comprises factor XII (Hageman factor), high molecular weight kininogen (HMWK) (Fitzgerald factor) and prekallikrein/kallikrein (Fletcher factor). As mentioned earlier, these factors are not essential for haemostasis in vivo. Important activities are to activate the fibrinolytic system, to activate the complement system and to generate vasoactive peptides: in particular, bradykinin is released from HMWK by prekallikrein or FXIIa cleavage. Kallikrein and factor XIIa also function as chemoattractants for neutrophils. The contact activation system also has some inhibitory effect on thrombin activation of platelets and prevents cell binding to endothelium. Recent evidence implicates the contact system in thrombosis via activation by polyphosphate released from platelets.¹⁰

When bound to a negatively charged surface in vitro, factor XII and prekallikrein are able to reciprocally activate one another by limited proteolysis, but the initiating event is not clear. It may be that a conformational change in factor XII on binding results in limited autoactivation that triggers the process. HMWK acts as a (zinc-dependent) cofactor by facilitating the attachment of prekallikrein and factor XI, with which it circulates in a complex, to the negatively charged surface. It has been shown in in vitro studies that platelets or endothelial cells can provide the necessary negatively charged surface for this mechanism and also possess specific receptors for factor XI. The contact system can activate fibrinolysis by a number of mechanisms: plasminogen cleavage, urokinase plasminogen activator (uPA) activation and tissue plasminogen activator (tPA) release. Most importantly from the laboratory point of view, the contact activation system results in the generation of factor XIIa, which is able to activate factor XI, thus initiating the coagulation cascade of the intrinsic pathway.

Tissue Factor

TF is the cofactor for the extrinsic pathway and the physiological initiator of coagulation. It is a transmembrane protein and constitutively present in many tissues outside the vasculature and on the surface of stimulated inflammatory cells such as monocytes and, under some conditions, endothelial cells. Factor VIIa binds to TF in the presence of calcium ions and then becomes enzymatically active. Small amounts of factor VIIa are present in the circulation but have virtually no enzymic activity unless bound to TF. The factor VIIa–TF complex can activate both factor X and factor IX and therefore two routes to thrombin production are stimulated. Factor Xa subsequently binds to TFPI and then to factor VIIa to form an inactive quaternary (Xa–TF–VIIa–TFPI) complex. This mechanism therefore functions to shut off the extrinsic pathway after an initial stimulus to coagulation has been provided.

The Vitamin K-Dependent Factors

The vitamin K-dependent factors group includes coagulation factors II, VII, IX and X. However, it is important to remember that the anticoagulant proteins S, C and Z are also vitamin K-dependent. Each of these proteins contains a number of glutamic acid residues at its amino terminus that are γ-carboxylated by a vitamin K-dependent mechanism. This results in a novel amino acid, γ-carboxyglutamic acid, which by binding calcium is essential in promoting a conformational change in the protein and binding of the factor to negatively charged phospholipid. Because this binding is crucial for coordinating the interaction of the various factors, the proteins produced in the absence of vitamin K (PIVKAs) that are not γ-carboxylated are essentially functionless. The vitamin K-dependent factors are proenzymes or zymogens, which require cleavage, sometimes with release of a small peptide (activation peptide), to become functional. Measurement of these activation peptides has been used as a means of assessing coagulation activation.

Cofactors

Factors VIII and V are the two most labile of the coagulation factors and they are rapidly lost from stored blood or heated plasma. They share considerable structural homology and are cofactors for the serine proteases FIX and FX, respectively; they both require proteolytic activation by factor IIa or Xa to function. Factor VIII circulates in combination with VWF, which is present in the form of large multimers of a basic 200 kDa monomer. One function of VWF is to stabilize factor VIII and protect it from degradation. In the absence of VWF, the survival of factor VIII in the circulation is extremely short (i.e. <2 h instead of the normal 8–12 h). VWF may also serve to deliver factor VIII to platelets adherent to a site of vascular injury. Once factor VIII has been cleaved and activated by thrombin it no longer binds to VWF.

Fibrinogen

Fibrinogen¹¹ is a large dimeric protein, each half consisting of three polypeptides named Aα, Bβ and γ held together by 12 disulphide bonds. The two monomers are joined together by a further three disulphide bonds. A variant γ chain denoted γ′ is produced by a variation in messenger RNA splicing. In the process a platelet binding site is lost and high-affinity binding sites for FXIII and thrombin are gained. The γ′ variant constitutes approximately 10% of plasma fibrinogen. A less common (<2%) γ chain variant ‘γE’ is also produced by splice variation. Fibrinogen is also found in platelets, but the bulk of this is derived from glycoprotein IIbIIIa-mediated endocytosis of plasma fibrinogen, which is then stored in alpha granules, rather than synthesis by megakaryocytes. Fibrin is formed from fibrinogen by thrombin cleavage releasing the A and B peptides from fibrinogen. This results in fibrin monomers that then associate and precipitate forming a polymer that is the visible clot. The central E domain exposed by thrombin cleavage binds with a complementary region on the outer or D domain of another monomer. The monomers thus assemble into a staggered overlapping two-stranded fibril. More complex interactions subsequently lead to branched and thickened fibre formation, making a complex mesh that binds and stabilizes the primary platelet plug.

Factor XIII

The initial fibrin clot is held together by non-covalent interactions and can be deformed and resolubilized. Factor XIII, which is also activated by thrombin, is able to covalently crosslink these fibrin monomers. Factor XIII is a transglutaminase that joins a glutamine residue on one chain to a lysine on an adjacent chain. This loss of resolubility is the basis of the screening test for factor XIII deficiency.

Other Assays

Other assays include measurement of coagulation factors using snake venoms, assay of ristocetin cofactor and the clot solubility test for factor XIII. DNA analysis is becoming more useful and more prevalent in coagulation. However, this requires entirely different equipment and techniques (see Chapter 8).

Waterbaths

A 37°C waterbath is required for manual coagulation tests, incubation steps and the rapid thawing of frozen specimens. Waterbaths set at 37°C should vary by no more than ± 0.5°C because slight variation in temperature will markedly affect the speed of clotting reactions. A waterbath with plastic or glass sides is preferable and some type of cross-illumination helps to determine the exact time of formation and appearance of the fibrin clot. Check that the temperature is 37°C before and during use. Distilled water should be used to fill the waterbath and maintain the water level.

Refrigerators and Freezers

Check that the temperature has not been out of the acceptable range of 4 ± 2°C for refrigerators and −20 ± 2°C or −80 ± 2°C (as applicable) for freezers, rechecking during the day. Records must be kept.

Centrifuges

Check to ensure each machine is clean before and after use. Also do a visual inspection of rotors, buckets and liners for corrosion and cracks. Thorough maintenance records should be kept.

Reagents and Buffers

Attention must be paid to the age and condition of solutions. This is particularly important with the calcium chloride solution. Whenever a solution is prepared it should be correctly labelled and dated. Buffers should be inspected for bacterial growth before use: contamination with microorganisms can cause errors and assay failures as a result of the release of enzymes and other active biological substances into solution. Azide may be added as a preservative to some buffers but should not be used in reagents for platelet studies or ELISA substrates. Chromogenic substrates should be reconstituted with sterile distilled water; contamination with bacterial enzymes may cause para nitro-aniline (pNA) release and yellow discoloration of the reagent. Records of batch numbers and expiry dates should be kept.

Plastic and Glass Tubes

For clotting tests, 75 × 10 mm glass rimless test tubes should be used. Plastic tubes should be used for sample dilutions, storage and reagent preparation.

Pipettes

A range of graduated glass (certified Class A) and automatic pipettes must be obtained. The latter should be accurate and durable. Fluids should not be drawn into the pipette barrels and acids should not be pipetted with instruments containing metal piston assemblies, which may become pitted or corroded. Attention to technique is vital because contamination of reagents with used pipette tips may occur, there may be errors of volume as a result of fluid on the exterior of the pipette tip or the manner of addition of a reagent may alter the results obtained. The amount of fluid drawn into the tip should be inspected visually with each pipetting procedure. Records of pipette accuracy and precision should be kept.

Stopwatches and Stopclocks

Stopclocks are useful for timing incubation periods of several minutes or more, but stopwatches that may be held in the hand, and controlled rapidly, should be used for measuring clotting times and for short incubations. At least four stopwatches are needed unless an automated coagulometer is used.

Automated Coagulation Analysers

A wide variety of automated and semi-automated coagulation analysers are available. The choice of analyser depends on predicted workload, repertoire and cost implications. A thorough evaluation of the current range of analysers is recommended.

Modern analysers distributed in the European Economic Area must be CE marked, certifying that the product has met EU consumer safety, health or environmental requirements.

If coagulation analysers are used, it is important to ensure that their temperature control and the mechanism for detecting the endpoint are functioning properly. Although such instruments reduce observer error when a large number of samples are tested, it is important to apply stringent quality control at all times to ensure accuracy and precision.

Safety

Each laboratory should have its own safety recommendations and procedures to follow in case of accident or contamination (see Chapter 24).

Collection of Venous Blood

Venous blood samples should be obtained whenever possible, even from the neonate. Capillary blood tests require modification of techniques, experienced operators and locally established normal ranges; they are not an easy alternative to tests on venous blood. All blood samples must be collected by personnel who are trained and experienced in the technique. Patients requiring venepuncture should be relaxed and in warm surroundings. Excessive stress and vigorous exercise cause changes in blood clotting and fibrinolysis. Stress and exercise will increase factor VIII, VWF and fibrinolysis.

Whenever possible, venous samples should be collected without a pressure cuff, allowing the blood to enter the syringe by continuous free flow or by the negative pressure from an evacuated tube (see p. 3). Venous occlusion causes haemoconcentration, increase of fibrinolytic activity, platelet release and activation of some clotting factors. In the majority of patients, however, light pressure using a tourniquet is required; this should be applied for the shortest possible time (e.g. <1 min). The venepuncture must be ‘clean’; blood samples from an indwelling line or catheter should not be used for tests of haemostasis because they are prone to dilution and heparin contamination.

To minimize the effects of contact activation, good-quality plastic or polypropylene syringes should be used. If glass blood containers are used, they should be evenly and adequately coated with silicon.

The blood is thoroughly mixed with the anticoagulant by inverting the container several times. The samples should be brought to the laboratory as soon as possible. If urgent fibrinolysis tests are contemplated, the blood samples should be kept on crushed ice until delivered to the laboratory. Assays of tPA and of PA1-1 antigen are preferably performed on samples taken into trisodium citrate to prevent continued tPA–PA1-1 binding (see p. 621).

If an evacuated tube system is used for collecting samples for different tests, the coagulation sample should be the second or third tube obtained.

Patient identification is of utmost importance. Care must be taken in labelling the patient sample both at the bedside and within the laboratory.

Blood Sample Anticoagulation

The most commonly used anticoagulant for coagulation samples is trisodium citrate. A 32 g/1 (0.109 M) solution (see p. 621) is recommended. Other anticoagulants, including oxalate, heparin and ethylenediaminetetra-acetic acid (EDTA), are unacceptable. The labile factors (factors V and VIII) are unstable in oxalate, whereas heparin and EDTA directly inhibit the coagulation process and interfere with endpoint determinations. Additional benefits of trisodium citrate are that the calcium ion is neutralized more rapidly in citrate and APTT tests are more sensitive to the presence of heparin.

For routine blood coagulation testing, 9 volumes of blood are added to 1 volume of anticoagulant (i.e. 0.5 ml of anticoagulant for a 5 ml specimen). When the haematocrit is abnormal with either severe anaemia or polycythaemia, the blood:citrate ratio should be adjusted.¹⁸ For a 5 ml specimen (total), the amount of citrate should be as follows:

Time of Sample Collection

The time of day when the sample is collected can be an important factor in the interpretation of results. Fibrinolytic activity follows a definite circadian pattern with a trough at around 6 a.m.

The timing of the collection of the blood sample in relation to drug administration should also be taken into consideration (e.g. after subcutaneous heparin therapy).

The timing following administration of factor concentrate samples is very important. The following times are recommended:

Factor VIII: at 15 min

Factor IX: at 30 min

Desmopressin: at 30 min after intravenous infusion, 60 min after intranasal.

Transportation to the Laboratory

An efficient and regular collection service is necessary. It is important that samples are delivered as quickly as possible to prevent deterioration of the labile clotting factors such as factors V and VIII. Automated systems can facilitate rapid delivery, but should be avoided when platelet function tests are to be performed because the associated trauma may affect results. For certain investigations it is necessary for the samples to be placed on ice once taken and delivered immediately to the laboratory.

Centrifugation: Preparation of Platelet-Poor Plasma

Most routine coagulation investigations are performed on platelet-poor plasma (PPP), which is prepared by centrifugation at 2000 g for 15 min at 4°C (approx. 4000 rev/min in a standard bench cooling centrifuge). The sample should be kept at room temperature if it is to be used for PT tests, lupus anticoagulant (LAC) or factor VII assays and it should be kept at 4°C for other assays; the testing should preferably be completed within 2 h of collection. Care must be taken not to disturb the buffy coat layer when removing the PPP.

Samples for platelet function testing, LAC and the activated PC resistance (APCR) test should not be centrifuged at 4°C. These samples should be prepared by centrifugation at room temperature to prevent activation of platelets and release of platelet contents such as phospholipid and factor V. For LAC testing and APCR it is very important that the number of platelets and the amount of platelet debris in the samples are minimized. The platelet count should be below 10 × 10⁹/l. This is best achieved by double centrifugation or filtration of the plasma through a 0.2 μm filter.

Storage of Plasma and Sample Thawing

Some tests such as the PT and APTT are carried out on fresh samples. Certain coagulation assays, unless urgently required, can be performed in batches at a later date on deep frozen plasma. Storage of small aliquots of samples in liquid nitrogen (−196°C) is the optimum, although samples may be frozen at −40°C or −80°C for several weeks without significant loss of most haemostatic activities. Gentle but thorough mixing of samples is essential after thawing and before testing. Once thawed, the sample should never be refrozen.

Some Common ‘Technical’ Errors

A false abnormality of the clotting time may occur in the following situations:

Manual Methods

Detecting clot formation as the endpoint depends to some extent on the rate of its formation: the shorter the clotting time the more opaque is the clot and the easier it is to detect. A slowly forming clot may appear as mere fibrin wisps, which are difficult to detect by eye or machine. In manual work, the observer must try to adopt a uniform convention in selecting the moment in clot formation that will be accepted as the endpoint. It is also important to ensure that the tube can be watched with its lower part under the water or while being quickly dipped in and out so as to avoid cooling and a slowing down of the clot formation. Bubbles also make the determination of the endpoint difficult.

Manual clotting techniques are still used in WHO calibration schemes and therefore should be viewed as an essential skill, despite the ever-increasing reliance on automation. It is worth remembering that not all results produced by an automated analyser are correct; dubious or inconsistent results should be checked manually.

In instrumental work the coagulometer must be shown to detect long clotting times reliably and reproducibly. The various coagulometers available have different means of detecting the endpoint, which may make comparison of results difficult. Some commonly used techniques are as follows.

Electromechanical

Photo-Optical Analysis

Percentage Detection Method

After initiating the clotting reaction, the transmitted light is monitored and a baseline A/D value (bH) is determined for the reaction (bH = 0%) (Fig. 18.2). The reaction is then monitored until the clotting reaction is completed (dH = 100%). The time to an optionally set endpoint, usually 50%, is then determined. At this point the A/D value per unit time shows the greatest change and the fibrin monomer polymerization reaction rate is high. Detection based on this principle enables coagulation analysis to be more accurate at low fibrinogen concentrations in samples with low A/D values and those samples for which the initial amount of A/D value is higher than usual, such as lipaemic and haemolysed samples.

Figure 18.2 Endpoint determination: percentage detection method.

(Reproduced by permission of Sysmex UK Ltd.)

Rate Method

After the start of the reaction, the increase in absorbance per minute is monitored. At the predetermined end time, the final increase in absorbance per minute measurement is made. The rate of the absorbance increase per minute between these two time point measurements is calculated (Fig. 18.3). The calculated change in absorbance (dOD/min) is expressed as the raw data and used to construct a standard curve (i.e. there is no endpoint per se).

Figure 18.3 Analysis of reaction: rate method. This is used for chromogenic assays.

(Reproduced by permission of Sysmex UK Ltd.)

VLin Integral Method

The VLin integral method (Fig. 18.4) evaluates the absorbance per minute of an immunological reaction. This is monitored and mathematical analysis used to determine the peak rate of reaction (maximum velocity). Using this method allows for increase in analytical sensitivity, extended measuring range, reduced measurement time and improved antigen excess reliability when measuring an immunological reaction. The VLin integral evaluation method is used for immunological assays, including D-dimer and VWF antigen.

Figure 18.4 Analysis of reaction: VLin integral method. This is used for immunological assays.

(Reproduced by permission of Sysmex UK Ltd.)

Analysis Time Over

The ‘Analysis Time Over’ check (Fig. 18.5) is used to detect whether the reaction endpoint is correct. If the sample reaction end angle is greater than the permitted angle at maximum detection time, the result will be flagged with an ‘Analysis Time Over’ error. The situation occurs when testing samples with prolonged clotting times and satisfactory endpoint has not been reached by the end of the time allotted for analysis. When this occurs the following checks should be performed:

Figure 18.5 Analyser trace illustrating ‘Analysis Time Over’. The reaction is incomplete at the end of the maximum allotted recording time.

(Reproduced by permission of Sysmex UK Ltd.)

Turbidity Level Over

If the dH exceeds the detection capacity of the A/D converter, the result will not be reported and it may be suspected that sample plasma is turbid or lipaemic (Fig. 18.6). When this occurs, the following checks should be performed:

Figure 18.6 The contributions of sample defects that may contribute to a ‘Turbidity Level Over’ error at different wavelengths.

(Reproduced by permission of Sysmex UK Ltd.)

Clot Signatures: Normal and Abnormal APTT Clot Waveforms

Information on the dynamics of clot formation may also be extracted from the optical profiles generated when performing the PT or APTT tests. It has been demonstrated that such profiles (clot waveforms) show a different pattern in certain clinical conditions compared to normal (Fig. 18.7). Furthermore, the shape of this pattern is predictable for the particular abnormality and the term ‘clot signature’ has been used in this context.

Figure 18.7 Clot signatures: normal and abnormal activated partial thromboplastin time (APTT) clot waveforms. (A) Normal specimen; APTT = 29 s, fibrinogen concentration = 2.89 g/l. (B) Biphasic response in a suspected disseminated intravascular coagulation (DIC) specimen with normal APTT (28.5 s) and elevated fibrinogen (6.74 g/l). (C) Patient treated with heparin (0.52 anti-Xa u/ml); prolonged APTT (81.2 s) and elevated fibrinogen concentration (5.54 g/l). (D) Factor VIII-deficient specimen (<1%), with prolonged APTT (84.1 s) and slightly reduced fibrinogen concentration (1.56 g/l). (E) Factor IX-deficient specimen (<1%) with prolonged APTT (83.4 s) and normal fibrinogen concentration (2.94 g/l).

(Reproduced by permission of Biomerieux.)

The A2 Flag on the MDA system identifies the presence of a biphasic APTT waveform often seen in patients with DIC and a high sensitivity (98%), specificity (98%) and positive predictive value (74%) have been reported.¹⁹

It is important to note that the biphasic APTT waveform has also been observed in samples from patients not diagnosed as having DIC by standard criteria. In this respect, it may indicate an emerging or occult and potentially serious clinical condition associated with the activation of coagulation. Further clinical and laboratory investigation is then warranted.

Molecular Mechanism of the Biphasic Waveform: LC-CRP

The development of the biphasic waveform is a consequence of the formation of a divalent metal ion-dependent complex of C-reactive protein (CRP) and very low density lipoprotein (VLDL) and, to a lesser extent, intermediate density lipoprotein (IDL).

This lipoprotein-complexed CRP has been designated LC-CRP. In the APTT assay, when the citrated plasma is recalcified, the formation of this complex results in a reduction in light transmission, detected by the first slope of the biphasic waveform.

Commonly Used Reagents

Some reagents are common to the majority of first-line tests. They are described here, whereas the reagents specific for one particular test or assay only are described with the details of the relevant test.

Factor-Deficient Plasmas

Plasmas deficient in specific factors are required for many bioassays. They may be obtained from individuals with congenital deficiency of the factor, but frequently these patients will have been treated with plasma concentrates and there is a danger of infection. Many laboratories now use commercial plasmas rendered deficient in the factor by immunodepletion and then lyophilized. However, it is important to establish that these are completely deficient. Once reconstituted, lyophilized plasmas should be gently mixed and left to stand for 20 min before use. If an automated coagulation analyser is used, the factor-deficient plasma should be placed in position 10 min prior to testing.

Principle

The PT test measures the clotting time of recalcified plasma in the presence of an optimal concentration of tissue extract (thromboplastin) and indicates the overall efficiency of the extrinsic clotting system. Although originally thought to measure prothrombin, the test is now known to depend also on reactions with factors V, VII and X and on the fibrinogen concentration of the plasma.

Reagents

Method

Deliver 0.1 ml of plasma into a glass tube placed in a waterbath and add 0.1 ml of thromboplastin. Wait 1–3 min to allow the mixture to warm. Then add 0.1 ml of warmed CaCl₂ and start the stopwatch. Mix the contents of the tube and record the endpoint. Carry out the test in duplicate on the patient’s plasma and the control plasma. When a number of samples are to be tested as a batch, the samples and controls must be suitably staggered to eliminate the time bias. Some thromboplastins contain calcium chloride, in which case 0.2 ml of thromboplastin is added to 0.1 ml plasma and timing is started immediately.

Expression of Results

The results are expressed as the mean of the duplicate readings in seconds or as the ratio of the mean patient’s plasma time to the mean normal control plasma time. The control plasma is obtained from 20 normal men and women (not pregnant and not taking oral contraceptives) and the geometric mean normal PT (MNPT) is calculated. (For further details and a discussion of the importance of the PT in oral anticoagulant control, when results may be reported as an International Normalized Ratio [INR], see Chapter 20.)

Normal Values

Normal values depend on the thromboplastin used, the exact technique and whether visual or instrumental endpoint reading is used. With most rabbit thromboplastins the normal range of the PT is between 11 and 16 s; for recombinant human thromboplastin, it is somewhat shorter (10–12 s). Each laboratory should establish its own normal range.

Interpretation

The common causes of prolonged PTs are as follows:

Principle

The test measures the clotting time of plasma after the activation of contact factors and the addition of phospholipid and CaCl₂′, but without added tissue thromboplastin, and so indicates the overall efficiency of the intrinsic pathway. To standardize the activation of contact factors, the plasma is first preincubated for a set period with a contact activator such as kaolin, silica or ellagic acid. During this phase of the test, factor XIIa is produced, which cleaves factor XI to factor XIa, but coagulation does not proceed beyond this in the absence of calcium. After recalcification, factor XIa activates factor IX and coagulation follows. A standardized phospholipid is provided to allow the test to be performed on PPP. The test depends not only on the contact factors and on factors VIII and IX but also on the reactions with factors X, V, prothrombin and fibrinogen. It is also sensitive to the presence of circulating anticoagulants (inhibitors) and heparin.

Reagents

Method

Mix equal volumes of the phospholipid reagent and the kaolin suspension and leave in a glass tube in the water-bath at 37°C. Place 0.1 ml of plasma into a second glass tube. Add 0.2 ml of the kaolin–phospholipid solution to the plasma, mix the contents and start the stopwatch simultaneously. Leave at 37°C for 10 min with occasional shaking. At exactly 10 min, add 0.1 ml of prewarmed CaCl₂ and start a second stopwatch. Record the time taken for the mixture to clot. Repeat the test at least once on both the patient’s plasma and the control plasma. It is possible to do four tests at 2-min intervals if sufficient stopwatches are available.

Expression of Results

Express the results as the mean of the paired clotting times.

Normal Range

The normal range is typically 26–40 s. The actual times depend on the reagents used and the duration of the preincubation period, which varies in manufacturer’s recommendations for different reagents. These variables also greatly alter the sensitivity of the test to minor or moderate deficiencies of the contact activation system. Laboratories can choose appropriate conditions to achieve the sensitivity they require. Each laboratory should calculate its own normal range.

Interpretation

The common causes of a prolonged APTT are as follows:

The APTT is also moderately prolonged in patients taking oral anticoagulant drugs and in the presence of vitamin K deficiency. Occasionally, a patient with previously undiagnosed haemophilia or another congenital coagulation disorder presents with an isolated prolonged APTT. If the patient’s APTT is abnormally long, the equal mixture test must be set up (see below).

Deficiency or Circulating Anticoagulant?

In cases with a long APTT, a 50:50 mixture of normal and test plasma should be tested to distinguish between factor deficiency and the effect of an inhibitor (see p. 421).

Principle

Thrombin is added to plasma and the clotting time is measured. The TT is affected by the concentration and reaction of fibrinogen and by the presence of inhibitory substances, including fibrinogen/fibrin degradation products (FDPs) and heparin. The clotting time and the appearance of the clot are equally informative.

Reagents

Method

Add 100 μl thrombin solution to 200 μl of control plasma in a glass tube at 37°C and start the stopwatch. Measure the clotting time and observe the nature of the clot (e.g. whether transparent or opaque, firm or wispy). Repeat the procedure with two tubes containing patient’s plasma in duplicate and then with a second sample of control plasma.

Expression of Results

The results are expressed as the mean of the duplicate clotting times in seconds for the control and the test plasma.

Normal Range

A patient’s TT should be within 2 s of the control (i.e. 15–19 s). Times of 20 s and longer are definitely abnormal.

Interpretation of Results

The common causes of prolonged TT are as follows:

Shortening of the TT occurs in conditions of coagulation activation.

A transparent bulky clot is found if fibrin polymerization is abnormal, as is the case in liver disease and some congenital dysfibrinogenaemias.

A gross elevation of the plasma fibrinogen concentration may also prolong the TT. Correction can be obtained by diluting the patient’s plasma with saline (see p. 417).

Fibrinogen Assay (Clauss Technique)

1

If all the first-line investigations are normal in a patient who continues to bleed from the site of an injury or after surgery (or has a history of such bleeding), there are several possible diagnoses:

Second-line investigations required in this situation are specific factor assays for the suspected deficiencies or appropriate screening tests such as the PFA-100 system (see p. 425) or factor XIII assay. Note that some LMWH may be present at therapeutic levels without abnormality of the coagulation screening tests; an anti-Xa assay will reveal their presence.

2

This combination of results is found in the following:

A mixing test should be performed. Factor VII assay is described later. It is usually, but not always, possible to establish from the history whether the patient has received oral anticoagulant drugs. Specific tests for lupus and specific factor assays should be performed, as indicated by the mixing test results. Biochemical measures of liver function should be obtained.

3

An isolated prolonged APTT is found in the following:

The next diagnostic step is to establish whether the patient has a deficiency or an inhibitor by performing the 50:50 mixture test described on p. 421. Mixing tests should be done immediately, followed by the specific assay or tests, as described later.

4

The main causes of a prolonged PT and APTT are as follows:

Mixing experiments using the PT may be useful if there is no history of anticoagulant therapy and no obvious reason for failure of vitamin K absorption (e.g. parenteral feeding, long-term antibiotic treatment). If correction is obtained, specific factor assays should be performed.

5

Abnormalities in all three screening coagulation tests are found in the following:

To distinguish between these conditions, perform a Reptilase or Ancrod time, measure the fibrinogen concentration and measure the level of FDPs or D-dimers in plasma. The pattern may also appear in incipient DIC, but usually the platelet count will also fall in this case.

6

If the only abnormality is a low platelet count, possible causes must be investigated. Premorbid counts and a clinical history should be sought to establish whether the thrombocytopenia is long-standing and possibly constitutional and a blood film should be examined. When it appears to be acquired, the usual approach is to perform a bone marrow aspirate to exclude marrow failure and establish whether megakaryocytes are present. If the number and morphology of megakaryocytes in the marrow is normal, further investigations are undertaken to establish the cause of the presumed peripheral destruction of platelets. Heparin and other drugs are common causes in hospital practice.

7

This pattern of abnormalities of the screening tests is found:

Specific factor assays may be useful if the situation persists. Consider the possibility that the low platelet count has a separate aetiology and that the situation is in fact the same as in Section 4.

8

All the first-line tests are abnormal in the following:

It is only exceptionally necessary to confirm the diagnosis of DIC with additional tests (e.g. by estimating FDP or D-dimer concentration or by carrying out a screening test for the presence of fibrin monomers). Consider the possibility that more than one pathology is present.

Principle

Unexplained prolongation of the PT or APTT can be investigated with simple correction tests by mixing the patient’s plasma with normal plasma. Correction indicates a possible factor deficiency, whereas failure to correct suggests the presence of an inhibitor, but interpretation should initially be cautious; see below.

Reagents

Method

Perform a PT and/or APTT on control, patient’s and a 50:50 (0.05 ml of each) mixture of the control and patient plasma. Perform all the tests in duplicate using a balanced order to avoid time bias. Note that mixing experiments to detect factor VIII inhibitors may require incubation for 2 h (see p. 422).

Interpretation

If the prolongation is the result of a deficiency of a clotting factor, the PT or APTT of the mixture should return to within a few seconds of normal. It is then necessary to identify the specific factor(s) that are deficient.

If the APTT is prolonged and normal plasma fails to correct the APTT, an inhibitor should be suspected. An inhibitor screen and tests for an LAC should be performed.

However, mixing tests may be misleading in two particular circumstances:

Comment

Correction tests are sometimes not as clear-cut as the literature and theory would suggest. Incomplete correction can be difficult to interpret, especially if the initial prolongation is modest. If the correction tests fall into the ‘grey’ area, specific factor levels should be measured and tests for the presence of an LAC should be performed, checking for time-dependent effects and non-linearity.

Principle

The tests use certain physicochemical properties of reagents to bind to inhibitors or abnormal molecules and normalize the prolonged TT. Protamine sulphate has a net electropositive charge and interacts with heparin, as well as binding to FDP, neutralizing the inhibitory effects of both. Toluidine blue is also a charged reagent that will neutralize heparin but has no effect on FDP. It is interesting that toluidine blue normalizes the TT in some dysfibrinogenaemias, probably by interacting with the excess of sialic acid attached to the fibrinogen molecules. Mixing with serum or albumin solution will correct the prolongation of the TT resulting from hypoalbuminaemia.

Reagents

Method

Perform the test as described for TT, adding 0.1 ml of saline to the controls and replacing in the test with protamine sulphate or toluidine blue solution. Also perform a TT on a 50:50 mixture of control and test plasma.

Interpretation

See Table 18.4.

Comment

The endpoint may be difficult to see in samples with a low fibrinogen content in the presence of toluidine blue owing to the dark colour of the reagent. Grossly elevated fibrinogen concentrations or the presence of a paraprotein can cause a prolonged time not corrected by either protamine or toluidine blue. Diluting the test plasma in saline will shorten the TT.

Note on Single Point Factor Assays

Factor assay results are dependent on obtaining parallel lines for the test and reference plasmas. Many automated coagulation analysers will give an assay result obtained from a single dilution assuming that this condition is met. However, this is not always true and if an inhibitor is suspected, results from more than one dilution should be obtained for comparison and to determine parallelism. Similarly, if the result is above or below the linear part of the standard curve, further dilutions should be tested.

One-Stage Assay of Factor VII

One-Stage Assay of Factor VIII

Monitoring Replacement Therapy in Coagulation Factor Defects and Deficiencies

Estimations of factor VIII levels in patients with haemophilia treated with factor VIII concentrates often yield discrepant results. This is primarily because the factor VIII concentrate (diluted in haemophilic plasma) is compared with a plasma standard. In general, two-stage or chromogenic assays reveal greater potency than one-stage assays in this situation. This has been particularly noted in patients who have been treated with B domainless factor VIII (Refacto, Wyeth). In this case a product-specific reference preparation is available from the company. It is recommended that this is used in conjunction with a chromogenic assay, but this may not be necessary.^35–37 In most other cases the clinical experience of using results from one-stage assays remains valid.

Assays of factor VIII concentrates are fraught with difficulty and a detailed discussion is beyond the scope of this chapter.^35,38 The difficulties arise from several problems. First, the concentrate potency may be assigned using either a one-stage assay (as in the USA) or the chromogenic assay (as in Europe). Second, many concentrates, even when diluted in haemophilic plasma, behave differently in one-stage and chromogenic assays. As a result, there are separate WHO standards for factor VIII measurement: a plasma for measurement of factor VIII in plasma samples and a concentrate for measurement of factor VIII in concentrates. This is based on the principle of assaying like against like, although there are so many different concentrates with different characteristics that this is difficult to truly achieve and all must eventually be calibrated against a single plasma pool.³⁷

Principle

Circulating anticoagulants or inhibitors affecting the APTT may act immediately or be time dependent. Normal plasma mixed with a plasma containing an immediately acting inhibitor will have little or no effect on the prolonged clotting time. In contrast, if normal plasma is added to a plasma containing a time-dependent inhibitor, the clotting time of the latter will be substantially shortened. However, after 1–2 h, correction will be abolished and the clotting time will become long again. To detect both types of inhibition, normal plasma and test plasma samples are tested immediately after mixing and also after incubation together at 37°C for 120 min.

Reagents

Method

Prepare three plastic tubes as follows: place 0.5 ml of normal plasma in one tube, 0.5 ml of the patient’s plasma in a second tube and a mixture of 0.25 ml of normal and 0.25 ml of patient’s plasma in a third tube. Incubate the tubes for 120 min at 37°C and then place all three tubes in an icebath or on crushed ice. Next, make a 50:50 mixture of the contents of tubes 1 and 2 into a fourth tube, which serves to check for the presence of an immediate inhibitor. Perform APTTs in duplicate on all four tubes.

Results and Interpretation

See Table 18.5. Note that the incubation period results in a prolongation of the normal plasma APTT.

Method for Detecting Inhibitors in Patients with Haemophilia

A simple inhibitor screen has been reported to be more sensitive than a Bethesda assay in monitoring for the development of inhibitors in haemophilia A and B. Add 0.4 ml patient plasma (or factor-deficient control plasma) to 0.1 ml of 5 iu/ml factor VIII or IX concentrate, giving a final concentration of 1 iu/ml. Mix gently and incubate at 37°C for 60 min for haemophilia A patients and 10 min for haemophilia B patients. Perform appropriate factor assay for the patient and control sample; if the factor level in the patient sample is less than 90% of the control sample the inhibitor screen is positive.⁴⁰

Principle

Factor VIII inhibitors⁴¹ are usually time dependent.³⁸ Thus if factor VIII is added to plasma containing an inhibitor and the mixture is incubated, factor VIII will be progressively neutralized. If the amount of factor VIII added and the duration of incubation are standardized, the strength of the inhibitor may be measured in units according to how much of the added factor VIII is destroyed.

In the Bethesda method, the unit is defined as the amount of inhibitor that will neutralize 50% of 1 unit of factor VIII in normal plasma after 2 h of incubation at 37°C.

Dilutions of test plasma are incubated with an equal volume of the normal plasma pool at 37°C. The normal plasma pool is taken to represent 1 unit of factor VIII. Dilutions of a control normal plasma containing no inhibitor are treated in the same way. An equal volume of normal plasma mixed with buffer is taken to represent the 100% value.

At the end of the incubation period, the residual factor VIII is assayed and the inhibitor strength is calculated from a standard graph of residual factor VIII activity versus inhibitor units.

Inhibitor Assay Modifications

The Bethesda assay and Nijmegen modification give similar results at high levels of factor VIII inhibition. Reports have shown that shifts in pH and protein concentrations will lead to changes in factor VIII stability and inactivation. Factor VIII inactivation increases with pH and reduced protein concentration leads to further inactivation of factor VIII activity. This can result in false-positive results using the unmodified Bethesda method. The Nijmegen modification prevents these discrepancies by buffering normal plasma with 0.1 M imidazole buffer at pH 7.4 and using immunodepleted factor VIII-deficient plasma in the control mixture.⁴² The assay can also be modified to use factor VIII concentrate (Oxford method) or by increasing the incubation time to 4 h (New Oxford method).

Reagents

Method

Pipette into each of a series of plastic tubes 0.2 ml of normal pool plasma. Add 0.2 ml of glyoxaline buffer to the first tube (this tube serves as the 100% value); add 0.2 ml of test plasma dilutions in glyoxaline buffer to each of the other tubes. If the patient’s inhibitor has been assayed previously, this can be used as a guide to the dilutions that should be used. If the patient has not been tested before, a range of dilutions should be set up ranging from undiluted plasma to a 1 in 50 dilution.

Cap, mix and incubate all the tubes for 2 h at 37°C. Then immerse all the tubes in an icebath. Perform factor VIII assays on all the incubation mixtures.

Calculation of Results

Record the residual factor VIII percentage for each mixture assuming the assay value of the control to be 100%. The dilution of test plasma that gives the residual factor VIII percentage nearest to 50% (between 30% and 60%) is chosen for calculating the strength of inhibitor. Results are calculated as shown in Figure 18.9 and in Table 18.6 for three different patients with a mild inhibitor only detected in undiluted plasma, a stronger inhibitor with simple kinetics and an inhibitor with complex kinetics, respectively.

Figure 18.9 Measurement of factor VIII inhibitors. Relationship between the residual factor VIII activity in normal plasma and the inhibitor activity of the test plasma can be read off this plot. At 50% inhibition the test plasma contains, by definition, 1 Bethesda inhibitor unit per ml. Note that the y-axis is a logarithmic scale.

Interpretation

If the residual factor VIII activity is between 80% and 100%, the plasma sample does not contain an inhibitor. If the residual activity is less than 60%, the plasma unequivocally contains an inhibitor. Values between 60% and 80% are borderline and repeated testing on additional samples is needed before the diagnosis can be established.

Tests for Other Inhibitors

Factor IX inhibitors can be measured in a system identical to that described earlier. Because factor IX inhibitors act immediately, there is no need for prolonged incubation; the mixtures can be assayed after 5 min at 37°C. The activity of the inhibitor against porcine factor VIII can be measured by substituting porcine factor VIII concentrate, appropriately diluted in factor VIII-deficient plasma, for normal plasma.

Principle

Fibrinogen in plasma is converted into fibrin by clotting with thrombin and calcium. The resulting clot is weighed and may include other proteins such as some FDPs. It is, however, simpler than the total clottable protein method used for the international standard⁴⁴ and provides a useful comparison for the Clauss.

Reagents

Method

Pipette 1 ml of plasma into a 12 × 75 mm glass tube and warm to 37°C. Place a wooden applicator or swab stick in the tube, add 0.1 ml of CaCl₂ and 0.9 ml of thrombin and mix. Incubate for 15 min at 37°C.

Gently wind the fibrin clot onto the stick, squeezing out the serum. Wash the clot in a tube containing at first 9 g/l NaCl, then water. Blot the clot carefully with filter paper, remove the fibrin from the stick and put into acetone for 5–10 min. Dry the clot in a hot air oven or over a hot lamp for 30 min. Allow it to cool and then weigh it.

Results

The fibrinogen level is expressed as g/l (i.e. the weight of fibrin obtained from 1 ml of plasma ×1000).

Normal Range

Normal range is 1.8–3.6 g/l.

Further Investigations

Whenever a congenital fibrinogen abnormality is suspected, DIC and hyperfibrinolysis must be excluded; FDP should not be in excess and there should be no evidence of the consumption of other coagulation factors and platelets (see p. 440). Immunological or chemical determination of fibrinogen concentration is the next step in investigation. In dysfibrinogenaemias there is often a normal or even raised plasma fibrinogen concentration using these methods, although the functional assays indicate a deficiency. Other tests that may be helpful are the Reptilase time, fibrinopeptide release, factor XIII cross-linking, tests of polymerization, binding to thrombin and lysis by plasmin. DNA analysis to detect the mutation responsible may be useful to allow comparison with other reported phenotypes. Testing the parents or other family members is sometimes a useful means for establishing whether a hereditary fibrinogen abnormality is present.

Bleeding Time

A standard incision is made on the volar surface of the forearm and the time the incision bleeds is measured. Cessation of bleeding is dependent on an adequate number of platelets, the ability of the platelets to adhere to the subendothelium directly and via adhesion molecules such as VWF and fibrinogen. However the test has poor sensitivity for disorders such as VWD, is a poor predictor of bleeding risk and is poorly reproducible. Consequently, it is now rarely performed and readers are referred to previous editions for details.

The PFA-100 System

The in vitro system for measuring platelet–VWF function, PFA-100 (Dade Behring), was introduced as a substitute for the bleeding time. The instrument aspirates a citrated whole-blood sample under constant vacuum from the sample reservoir through a capillary and a microscopic aperture cut into a membrane. The membrane is coated with collagen and either epinephrine or adenosine 5′-diphosphate (ADP). It therefore attempts to reproduce under high shear rates VWF binding, platelet attachment, activation and aggregation, which slowly build a stable platelet plug at the aperture. The time required to obtain full occlusion of the aperture is reported as the ‘closure time’. Collagen/epinephrine is the primary screening cartridge and the collagen/ADP is used to identify possible aspirin use.

Studies have shown this system to be sensitive to platelet adherence and aggregation abnormalities and to be dependent on normal VWF, glycoprotein Ib and glycoprotein IIbIIIa levels but not on plasma fibrinogen or fibrin generation.⁴⁶

The PFA-100 system may reflect VWF function better than the bleeding time, but it is not sensitive to vascular-collagen disorders.^47,48 Studies have shown that many patients with minor platelet disorders such as secretion defects are not detected by the PFA-100.⁴⁹

Principle

ELISA involves coating a special microtitre plate with a primary antibody to von Willebrand factor antigen (VWF:Ag).⁵² A suitable dilution of the test plasma is added to the wells, allowing the VWF:Ag to bind to the primary antibody. After removal of excess antigen by washing the plate, a second antibody, conjugated to an enzyme, usually peroxidase and called the ‘tag’ antibody, is added and this binds to the VWF:Ag already bound to the plate. On addition of a specific substrate, a colour change occurs. After the reaction has been stopped with acid, the optical density (OD) of each well can be measured using an electronic plate reader; the OD is directly proportional to the amount of VWF:Ag present in the test plasma.

Reagents

Method

Dilute the antihuman-VWF:Ag 1:500 in 0.05 M carbonate buffer (i.e. 40 μl antibody in 20 ml buffer) and add 100 μl to each well of the microtitre plate. Incubate for 1 h at room temperature in a moist chamber. Discard antibody and wash three times by immersion in a trough of phosphate buffered saline (PBS) with 0.5 ml/l Tween for 2 min, followed by inversion onto absorbent paper.

Prepare dilutions of the 100% standard 1:10, 1:20, 1:40 and 1:60 in PBS with 1 ml/l Tween. Dilute patient’s and control plasmas 1:10, 1:20 and 1:40 in the same way and add 100 μl of each dilution in duplicate to the wells of the microtitre plate. Incubate for 1 h as before and repeat washing.

Dilute the antihuman-VWF:Ag-peroxidase conjugate 1:500 in 1 ml/l PBS-Tween (i.e. 40 μl antibody in 20 ml buffer) and add 100 μl to each well. Incubate for 1 h. Wash twice in 0.5 ml/l PBS Tween and once in 0.1 M citrate phosphate buffer.

Dissolve 40 mg of substrate (OPD) in 15 ml citrate phosphate buffer. Add 10 μl of 20 volume hydrogen peroxide to the substrate solution immediately before use and then add 100 μl to each well.

When the yellow colour has reached an intensity at which a mid-yellow ring is clearly visible in the bottom of the wells, stop the reaction by the addition of 150 μl of 1 M sulphuric acid. Read the optical density across the plate at 492 nm using a microtitre plate reader. Plot the standard curve on log-linear graph paper. VWF:Ag levels are obtained by reading from the reference curve.

Normal Range

The normal range is approximately 50–200 iu/dl.

Interpretation

The results must be interpreted in conjunction with the results of factor VIII assay and the ristocetin cofactor assay (Table 18.7). VWF:Ag can also be measured by an immunoelectrophoretic assay. (The Laurell rocket method for this is described in the 7th edition of this book.)

Principle

Washed platelets do not ‘agglutinate’ in the presence of ristocetin unless normal plasma is added as a source of VWF. ‘Agglutination’ follows a dose–response curve dependent on the amount of plasma/VWF added. Freshly washed platelets or formalin-fixed platelets can be used in the assay. Fixed platelets take longer to prepare but are not susceptible to aggregation (as distinct from ‘agglutination’) with ristocetin and they can be stored so that they are available for emergency use. Freshly washed platelets are quicker to prepare and retain a functional platelet membrane, but they cannot be retained for later use. Commercial lyophilized, fixed, washed platelet preparations are available. Once reconstituted these preparations are stable for several weeks and should enhance assay standardization.⁵⁴

Reagents

Method

Collect 40–60 ml of normal blood into a one-tenth volume of EDTA–saline in flat-bottom plastic universal containers. Do not use conical bottom containers. Centrifuge at 150–200 g at room temperature (about 20°C) for 15 min.

Pipette, using a plastic pipette, the platelet-rich plasma (PRP) into a plastic container. Mark the level of plasma on the tube. Centrifuge at 1500–2000 g to obtain a platelet button.

Discard the PPP. Resuspend the platelet button in a 2 ml volume of EDTA–citrate–saline by gently squeezing the liquid up and down a pipette until a smooth suspension is formed. Add EDTA–citrate–saline to the 20 ml mark.

Centrifuge at 1500–2000 g for 15 min. Discard the supernatant. Resuspend in EDTA–citrate–saline and leave at room temperature for 20 min to elute the ristocetin cofactor off the platelets.

Centrifuge again, discard the supernatant and resuspend in EDTA–citrate–saline two more times to a total of four washes.

Centrifuge at 1500–2000 g for 15 min. Discard the supernatant and resuspend in citrate–saline using a volume slightly under the original plasma volume (marked on the container). Centrifuge at 800 g for 5 min to remove platelet clumps, white cells and red cells.

Remove the platelet-rich supernatant carefully. Perform a platelet count and dilute the platelet-rich suspension with citrate–saline until the platelet count is about 200 × 10⁹/l.

Leave the platelets at room temperature for 30–45 min to allow the platelets to recover from the trauma of washing and centrifugation.

Reagents for Assay

Standard Curve

A standard curve is obtained by making doubling dilutions, 1 in 2 to 1 in 32 in citrate–saline, of the standard plasma (donor pool, commercial reference plasma or other reference materials). Frozen plasma standards may be preferred because lyophilization can result in pH changes which affect lyophilized platelets. The absorbance resulting from a mixture of 0.4 ml of citrate–saline and 0.1 ml of plasma dilution is taken to represent 100% agglutination and that resulting from the mixture of 0.4 ml of platelet suspension and 0.1 ml of plasma dilution represents zero (0%) agglutination.

Add 5 μl of ristocetin to the cuvette containing the mixture giving zero agglutination and record the agglutination for 2 min. Test each dilution of the standard plasma in a similar way.

The patient’s plasma is tested at two dilutions, depending on the expected concentration of VWF in the plasma. Both dilutions should give agglutination within the range of that of the standard curve.

Reset 100% and zero aggregation for each patient.

A reading of the platelet blank should be repeated at hourly intervals. If the reading differs from the original, the difference must be subtracted from the results of subsequent tests.

Results

Measure ‘agglutination’ at 1 or 2 min depending on the strength of agglutination. All responses must be compared on the same time scale and not read at maximum agglutination.

Plot the standard curve on semi-log paper with agglutination on the linear scale and the concentration of VWF in iu/dl on the log scale (Fig. 18.10). For assay purposes, assign the 1 in 2 dilution of standard plasma a value of 0.50 iu/ml. (Each batch of standard is precalibrated and may not necessarily be 1.0 iu/ml.)

Figure 18.10 Ristocetin cofactor assay. The standard curve is plotted on semi-log paper. Each test plasma is assayed in two dilutions. Plasma 1 (+) produced the following readings: 1 in 4 dilution: 16 divisions of the chart paper = 7 iu (four times [dilution factor] = 28 iu/dl); 1 in 2 dilutions: 22 divisions = (13 iu/dl [twice dilution factor] = 26 iu/dl). The mean of the two readings is 27 iu/dl. Plasma 2 (*) gave the following results: 1 in 2 dilution: 7 divisions = (2.5 iu [twice dilution factor] = 5 iu/dl). 1 in 4 dilution: 5 divisions (not shown). This result was similar to the blank and the plasma was next tested undiluted, giving a reading of 10 divisions = 4 iu/dl. The mean is 4.5 iu/dl (very low).

Read the patient’s VWF concentration directly off the standard curve, correct for the dilution factor and average the two results from the different dilutions.

Normal Range

The normal range is approximately 50–200 iu/dl.

Interpretation

The VWF concentration measured by ristocetin cofactor assay should be interpreted in conjunction with other factor VIII and VWF:Ag assays, as shown in Table 18.7.

Assay Using Formalin-Fixed Platelets

Non-Radioactive Multimer Method

Multimeric analysis of von Willebrand factor antigen is important in the diagnosis and treatment of VWD.⁵⁶ The gold standard method is the autoradiographic method described by Enayat and Hill.⁵⁶ This method uses radioactive iodine (I¹²⁵) and is performed in only a few specialized centres.

The non-radioactive method described here uses a peroxidase-conjugated antibody to VWF:Ag to replace the radioactive antibody described in previous editions.

Plasma samples are diluted in a buffer containing 8 M urea and sodium dodecyl sulphate (SDS) and are heated to ensure mobility of protein is related to size and not molecular charge. Samples are electrophoresed through an agarose stacking gel at pH 6.8 and then through a running gel of higher agarose concentration at pH 8.8. After running overnight on a cooling plate, the protein is fixed in the gel, washed and incubated with a peroxidase-conjugated antibody to VWF:Ag followed by extensive washing. The VWF:Ag multimers are revealed by adding a colour substrate reagent and scanning the gel. The scanned image is modified using photographic editing software (e.g. Microsoft Picture It or Corel Draw Graphics Suite) to produce a black-and-white image suitable for presentation.

The technique described here uses an agarose gel in a discontinuous buffer system. The method appears less prone to technical problems than an acrylamide/agarose system and yet can distinguish clearly the known patterns of VWD subtypes.

Reagents

Preparation of Stock Solutions

These three reagents may be stored at 4°C for up to 3 months.

Preparation of Buffers

Colour Buffer (0.1 M Citrate Phosphate Buffer, pH 5.0)

Preparation of Gels

Electrophoresis (Day 1 Evening)

Set the cooling system used at 8°C to achieve a gel temperature of 13°C. Prepare wicks from J-cloths (Johnson and Johnson) and Whatman No.1 24 cm filter papers. Fold a filter paper in half and mark the folded edge 27 mm from each end. From these marks draw lines at right angles and join the points where they cut the arc. Cut out the rectangle thus drawn and it will act as part of one wick. Also cut two double-thickness J-cloth rectangles 183 × 120 mm. Place 500 ml of cold electrophoresis buffer in each reservoir of the electrophoresis tank.

Once again, carefully disassemble the mould; remove the gel bond, leaving the gel on the glass plate. Using a template, cut 10 wells 10 × 2 mm in the stacking gel 8 mm from the interface of running and stacking gels. Place the gel on the cooling platen. Soak two filter paper wicks in electrophoresis buffer and position over the gel by 5 mm at either end. Soak two J-cloth wicks in electrophoresis buffer, placing one completely over the paper wick at the running gel end and the other over the paper wick at the stacking gel end, leaving a small portion of the paper wick visible.

Pipette 35–40 μl of diluted sample into each well, taking care not to touch the wick. Electrophorese the gel at a constant current of 5 mA per gel (approximately 65 V). Stop the electrophoresis when the blue dye has migrated 1 cm from each well. Carefully remove residual liquid from each well and refill each well with molten stacking gel. Start electrophoresis at the same current. After a total of 18–20 h, the dye will have run off the gel into the wick and electrophoresis is complete.

Gel Fixation (Day 2 Morning)

Remove the gel gently from the glass plate and fix for 1 h using the acid/alcohol fixative solution in a suitable container.

Gel Washing (Day 2)

Once fixed, wash the gel in three changes of distilled water for a total of 3 h. Transfer the gel to a small plastic tray and wash with 1% milk powder for 20 min followed by 10% milk powder for 20 min. Wash the gel extensively in 0.5 ml/l Tween 20 PBS for the remainder of the day.

Addition of the Peroxidase-Conjugated Rabbit Antihuman-VWF:Ag (Day 2 Evening)

Dilute 400 μl of the peroxidase-conjugated rabbit antihuman-VWF in 400 ml of 0.5 ml/l Tween 20 PBS. Then add this to the gel in the tray and mix gently overnight. Place a plastic sheet over the plastic tray to prevent evaporation.

Extensive Washing (Two Days and Nights)

Pour the peroxidase-conjugated rabbit antihuman-VWF mixture to waste. Remove the gel from the plastic tray and place into another flat-bottomed tray. Then wash the gel extensively with frequent changes of 0.5 ml/l Tween 20 PBS for the next 2 days and 2 nights.

Addition of the Colour Substrate (Day 5)

Prepare the colour reagent by adding 6 × 10 mg OPD tablets to 120 ml 0.1 M citrate phosphate buffer. Add 40 μl of hydrogen peroxide just before use. Place the gel into a plastic tray, pour the colour reagent over the gel and mix gently. Ensure the colour reagent gets underneath the gel. When the colour begins to develop, remove the gel from the plastic tray and place between two sheets of gel bond, draining off any excess substrate reagent.

Scanning the Developing Gel

When the multimer patterns become visible, the gel is ready to scan. The gel will continue to develop colour for several minutes and eventually the background gel will become too dark to see the multimer patterns clearly. Scan the gel several times as the colour develops to get the best image of the multimer patterns.

The scanned gel will appear as shown as in Figure 18.11A. The top section is the stacking gel where the samples were added prior to electrophoresis; the main body of the gel is the running gel showing the multimer patterns. The scan is manipulated using photographic editing software (see Fig. 18.11C,D) and the final result shown in Figure 18.11E.

Figure 18.11 von Willebrand factor multimer gel. See text for preparation. (A) Scan of the original stained gel. (B) Adjustment of image tint. (C) Conversion to monochrome image. (D) Adjustment of brightness and contrast. (E) Final gel image.

Interpretation

See Figure 18.12.⁵⁷

Figure 18.12 Autoradiograph of the electrophoretic analysis of von Willebrand factor (VWD) multimer patterns. The largest multimer appear at the top of the gel. The normal pattern with numerous large multimers and a triplet pattern visible in the smaller multimers are shown in lane 7. Lanes 1 and 6 are compatible with type 1 VWD in which there is a generalized decrease in multimer numbers but the normal triplet pattern is retained. In lane 2 there is virtually no VWF detected, indicating type 3 VWD. Lanes 3 and 5 show both abnormality of VWF amount and multimer pattern, indicating type 2A. In lane 4 there is a selective loss of the large multimers typical of type 2B VWD.

Principle

The light absorbance of PRP decreases as platelets aggregate. The amount and the rate of fall are dependent on platelet reactivity to the added agonist provided that other variables, such as temperature, platelet count and mixing speed, are controlled. The absorbance changes are monitored on a chart recorder.

Reagents

Interpretation

Normal and abnormal platelet aggregation curves are shown in Figures 18.14 and 18.15.

Figure 18.14 Traces obtained during the aggregation of platelet-rich plasma. a, Shape change; b, primary wave aggregation; c, secondary wave aggregation; x, angle of the initial aggregation slope; y, height of the aggregation trace; d, lag phase.

(Redrawn from Yardumian DA, Mackie IJ, Machin SJ 1986 Laboratory investigation of platelet function: a review of methodology. Journal of Clinical Pathology 39:701–712.)

Figure 18.15 (A–F) Some examples of platelet aggregation analyses.

(A) Normal and (B) abnormal, responses to adenosine diphosphate.

Blue 0.5 μmol/l

Yellow 1.0 μmol/l

Green 2.0 μmol/l

Black 5.0 μmol/l

(C) Normal and (D) abnormal, responses to epinephrine.

Blue 0.5 μmol/l

Yellow 1.0 μmol/l

Green 2.0 μmol/l

Black 5.0 μmol/l

(E) Responses to collagen and ristocetin.

Blue Low-dose ristocetin 0.75 mg/ml

Yellow High-dose ristocetin 1.5 mg/ml

Green Low-dose collagen 5 μg/ml

Black High-dose collagen 10 μg/ml

(F) Responses to arachidonic acid.

Blue High-dose arachidonic acid 0.5 mg/ml

Yellow Low-dose arachidonic acid 0.25 mg/ml

Calculation of Results

Results can be expressed in one of three ways:^59,60

Normal Range

The platelets of normal subjects usually produce a single reversible primary wave with 1 μmol/l ADP or less, biphasic aggregation with ADP at 2.5 μmol/l and a single irreversible wave at 5 or 10 μmol/l. A single-phase response is observed after a lag phase lasting not more than 1 min with 1 and 4 μg/ml of collagen. A single-phase or biphasic response is seen with 1.2 mg/ml of ristocetin and after 50 and 100 μmol/l of arachidonic acid. Normal ranges have been compiled for the common agonists.⁶² Interpretation can be difficult but reduced maximal aggregation with two or more agonists is highly indicative of a bleeding disorder.⁶¹ Biphasic aggregation is observed with 2–10 μmol/l of adrenaline. A response to a low concentration of ristocetin (0.5 mg/ml) is abnormal and is a feature of type 2B VWD (see below).

Interpretation and Technical Artefacts

The volumes of PRP used will depend on the aggregometer and cuvette used. The smaller the cuvette, the more responses can be tested with a given volume of PRP, but the poorer the optical quality (because of a shorter lightpath) and the more likely the influence of factors such as debris or air bubbles.

Care should be taken to exclude red cells and granulocytes from PRP because these will interfere with the light transmittance and cause reduced response heights, which can be mistaken for abnormal aggregation. In diseases such as thalassaemia, where there may be red cell fragments and membranes, these may be removed by further centrifugation of PRP at 150 g for 2 min or after settling has occurred.

If cryoglobulins are present, they may cause changes in transmittance which resemble the appearance of spontaneous aggregation. Warming the PRP to 37°C for 5 min allows aggregation to be tested in the normal way.

Lipaemic plasma may cause problems in adjusting the aggregometer and the responses may be compressed owing to the small difference in transmitted light between PRP and PPP. Care should be taken in the interpretation of results from such samples.

The pattern of responses in various disorders of platelet function is shown in Table 18.10. For a discussion of hyperaggregability, see p. 461.

Some common technical problems associated with platelet aggregation are described in Table 18.11.

Table 18.11 Technical factors that may influence platelet aggregation tests

PPP, platelet-poor plasma; PRP, platelet-rich plasma.

Platelet Lumiaggregometry

The Chrono-log aggregometers (Chrono-log Corporation) measure platelet function using electrical impedance in whole blood or optical density in platelet-rich plasma; with simultaneous measurement of ATP release by luminescence.

Whole-blood aggregation measures platelet function in anticoagulated blood without the need to isolate them from other blood components. Without the necessity for centrifugation, the entire platelet population is tested and labile factors in the blood (e.g. prostacyclin and thromboxane A₂) that may influence platelet function are preserved.

Results of impedance aggregation tests are quantified by:

The increase in impedance is directly proportional to the mass of the platelet aggregate. Impedance aggregation in blood is not dependent on optical characteristics of the sample, so tests can be performed on lipaemic and thrombocytopenic samples. The method is also useful in situations where sample volume is critical.

ATP secreted by dense granules is measured by a visible light range luminescence technique in either PRP or whole blood. The Lumi-aggregometer measures secretion by a sensitive luminescent (firefly luciferin-luciferase) assay for extracellular ATP in combination with the simultaneous measurement of aggregation. Luminescence measurement of ATP secretion provides unequivocal evidence of normal or impaired dense granule release (as in secretion defects and storage pool deficiency).

Principle

Fibrin clots formed in the presence of factor XIII and thrombin are stable (as a result of crosslinking) for at least 1 h in 5 mol/l urea, whereas clots formed in the absence of factor XIII dissolve rapidly. Quality assurance surveys in the UK have shown that the solubility test for XIII is more sensitive when the sample is clotted with thrombin rather than calcium. Thrombin preparations containing calcium should not be used. The use of 5 M urea as described here will detect factor XIII deficiency of up to 5 iu/dl. One study suggested that deficiency of up to 10% could be detected by using 2% acetic acid as the lysing solution.⁶⁴

Reagents

Method

In duplicate, 0.2 ml patient plasma is mixed with 0.2 ml 10 NIH unit thrombin solution in a glass test tube and incubated at 37°C for 20 min. Set up a normal plasma control in the same way; EDTA-plasma can be included as a negative control. Each tube is filled with approximately 3 ml of urea solution, carefully dislodging the clot, and is left undisturbed at 37°C for 24 h. Inspect each tube for the presence of a clot at regular intervals.

Interpretation

The control clot, if normal, shows no sign of dissolving after 24 h. However, in the absence of factor XIII, the clot will have dissolved. The test is reported as normal if the clot is present and abnormal if the clot is absent. The clot solubility test has poor sensitivity and may only detect levels below approximately 5 iu/dl. The relationship between factor XIII level and adequate haemostasis is uncertain, but there is some evidence that levels of 5–40 iu/dl may also be associated with bleeding. In suspected cases photometric and ELISA assays of factor XIII are available for quantitative measurements.⁶⁵ The introduction of these assays into routine practice may clarify the significance of intermediate levels of factor XIII activity.

Principle

A suspension of latex particles is sensitized with specific antibodies to the purified FDP fragments D and E. The suspension is mixed on a glass slide with a dilution of the serum to be tested. Agglutination indicates the presence of FDP in the sample. By testing different dilutions of the unknown sample, a semiquantitative assay can be performed.⁶⁸

Reagents

Method

Allow the tube with blood to stand at 37°C until clot retraction commences. Then centrifuge the tube and withdraw the serum for testing. It is important that the fibrinogen in the sample is completely clotted or this will be detected by the test. This may be a problem in the presence of heparin or a dysfibrinogenaemia or high levels of FDPs. Addition of a few drops of 100 u (NIH)/ml thrombin will enhance clotting in these cases.

Make 1 in 5 and 1 in 20 dilutions of serum in glycine buffer. Mix 1 drop of each serum dilution with 1 drop of latex suspension on a glass slide. Rock the slide gently for 2 min while looking for macroscopic agglutination. If a positive reaction is observed in the higher dilution, make doubling dilutions from the 1 in 20 dilution until macroscopic agglutination can no longer be seen.

Interpretation

Agglutination with a 1 in 5 dilution of serum indicates a concentration of FDP in excess of 10 μg/ml; agglutination in a 1 in 20 dilution indicates FDP in excess of 40 μg/ml.

Normal Range

Healthy subjects have an FDP concentration of less than 10 μg/ml. Concentrations between 10 and 40 μg/ml are found in a variety of conditions, including acute venous thromboembolism, acute myocardial infarction and severe pneumonia, and after major surgery. High levels are seen in systemic fibrinolysis associated with DIC and thrombolytic therapy with streptokinase.

Principle

When thrombin acts on fibrinogen, some of the monomers do not polymerize but give rise to soluble complexes with plasma fibrinogen and FDP. These complexes can be associated in vitro by ethanol or protamine sulphate. Now rarely used: see previous editions for method.

Principle

The latex agglutination method used to detect crosslinked fibrin D-dimers is identical to the test previously described for FDP, but in this case the latex beads are coated with a monoclonal antibody directed specifically against fibrin D-dimer in human plasma or serum. Because there is no reaction with fibrinogen, the need for serum is eliminated and measurements can be performed on plasma samples.

Reagents

Several manufacturers market kits for the measurement of D-dimers. These usually contain the latex suspension, dilution buffer and positive and negative controls.

Method

The manufacturer’s protocol should be followed. Undiluted plasma is mixed with one drop of latex suspension on a glass slide and the slide is gently rocked for the length of time recommended in the kit. If macroscopic agglutination is observed, dilutions of the plasma are made until agglutination can no longer be seen.

Interpretation

Agglutination with the undiluted plasma indicates a concentration of D-dimers in excess of 200 mg/l. The D-dimer level can be quantified by multiplying the reciprocal of the highest dilution showing a positive result by 200 to give a value in mg/l.

Normal Range

Plasma levels in normal subjects are <200 mg/l. There has been much study of D-dimer assays as a useful way of excluding thrombosis, but there is naturally a compromise between sensitivity and specificity, especially when a rapid turnaround time is required. The lack of an international standard and the poor correlation between kits mean that the use of kits for this purpose should be validated individually. A number of kits using ELISA methods for the detection of D-dimers are now available that have greater sensitivity but are more cumbersome to perform. Latex tests using automated analysers may provide an acceptable compromise.⁶⁹ These tests have now been incorporated into clinical guidelines according to their sensitivity.⁷⁰