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 ‘nonthrombogenic’ (i.e. they promote maintenance of blood in its fluid state), they play an active role in supplying nutrients to the sub-endothelial 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 cell 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, mostly in a truncated form covalently linked to a glycophosphoinositol (GPI) anchor (TFPIbeta) although some full length TFPI (TFPIalpha) is also present and noncovalently bound. 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 diffusing away from the site of injury is rapidly bound to thrombomodulin which can then activate PC bound to the endothelial protein C receptor (EPCR) and a carboxypeptidase which inhibits fibrinolysis (discussed later). Thrombin also stimulates the endothelial cell to produce tissue plasminogen activator (tPA). The endothelium can also synthesise protein S, the cofactor for PC. Finally, endothelium produces von Willebrand factor (VWF) which is essential for platelet adhesion to the subendothelium and stabilises factor VIII (FVIII) 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. 401). Many of the surface proteins are found localised in the specialised lipid rafts and invaginations called caveolae which may provide an important level of regulation.

The endothelial cell participates in vasoregulation by producing and metabolising numerous vaso-active substances. On one hand it metabolises 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 noncollagenous glycoproteins, including fibronectin and VWF. After vessel wall damage has occurred, these components are exposed and are then responsible for platelet adherence. At low shear platelets can bind to collagen but in practice this appears to be largely mediated by VWF binding to collagen. VWF bound to collagen 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.

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 ^, 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 A ₂ (TXA ₂ ), which is a potent vasoconstrictor.

Platelet function in the haemostatic process

The main steps in platelet functions 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 adherent 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 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 IbIX and IIbIIIa complexes are essential at this stage to increase the cell-to-cell contact and facilitate aggregation. Positive feedback is provided by platelet release of ADP, 5-hydroxytryptamine (5HT) and thromboxane. In areas of nonlinear 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 (PLA ₂ ) 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 TXA ₂ . TXA ₂ 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. TXA ₂ is very labile with a half-life of less than 1 min before it is degraded into the inactive thromboxane B ₂ (TXB ₂ ) and malonyldialdehyde.

The second pathway of activation and aggregation can proceed completely independently from the first one: various platelet agonists, including thrombin, ADP, TXA ₂ 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 TXA ₂ .

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.

The resting endothelium helps maintain platelets in a nonactivated state by secreting prostacyclin and NO. 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 several roles in haemostasis:

• 1.
  

  Adhesion and aggregation forming the primary haemostatic plug

  
  
• 2.
  

  Clot retraction

  
  
• 3.
  

  Release of platelet activating and procoagulant molecules

  
  
• 4.
  

  Provision of a procoagulant surface for the reactions of the coagulation system.

The contact activation system

The contact activation system comprises FXII (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. Their 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 FXIIa 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 animal evidence implicates the contact system in platelet-dependent thrombosis although a role in humans is not yet established.

When bound to a negatively charged surface in vitro, FXII 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 FXII 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 (FXI), with which it circulates in a complex, to the negatively charged surface. There are numerous candidates for the physiological surface on which this takes place, including collagen and polyphosphate. The contact system can also activate fibrinolysis by a number of mechanisms: plasminogen cleavage, urokinase plasminogen activator (uPA) activation and tPA release. Most importantly from the laboratory point of view, the contact activation system generates FXIIa, which is able to activate FXI, 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 activated monocytes and, under some conditions, endothelial cells. FVIIa binds to TF in the presence of calcium ions and then becomes enzymatically active. Small amounts of FVIIa are present in the circulation but have virtually no enzymic activity unless bound to TF. The FVIIa-TF complex can activate both FX and FIX and therefore two routes to thrombin production are stimulated. FXa subsequently binds to TFPI and then to FVIIa 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 coagulation factors are the serine proteases factors II, VII, IX and X. However 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 promotes a conformational change in the protein allowing it to bind 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 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 FIXa and FXa, respectively. They both require proteolytic activation by factor IIa or Xa to function. FVIII circulates in combination with VWF, which is present in the form of large multimers of a basic 200 kD monomer. One function of VWF is to stabilise FVIII and protect it from degradation. In the absence of VWF, the survival of FVIII in the circulation is extremely short (< 2 h instead of the normal 8–12 h). VWF may also serve to deliver FVIII to platelets adherent to a site of vascular injury. Once FVIII has been cleaved and activated by thrombin it no longer binds to VWF.

Fibrinogen

Fibrinogen 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%) α chain variant ‘γE’ is also produced by splice variation. Fibrinogen is also found in platelets, but the bulk of this is derived not from synthesis by megakaryocytes but from glycoprotein IIbIIIa-mediated endocytosis of plasma fibrinogen, which is then stored in alpha granules. Fibrin is formed from fibrinogen by thrombin cleavage releasing the A and B peptides. 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 stabilises the primary platelet plug.

Factor XIII

The initial fibrin clot is held together by noncovalent interactions and can be deformed and resolubilised. FXIII, which is also activated by thrombin, is able to covalently crosslink these fibrin monomers. FXIII 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 FXIII deficiency.

Immunological

Immunological tests rely on the recognition of the protein in question by polyclonal or monoclonal antibodies. The antibody is coupled to a system that quantifies the extent of binding. Polyclonal antibodies lack specificity but provide relatively high sensitivity, whereas monoclonal antibodies are highly specific but produce relatively low levels of antigen binding. Immunological assays are often easy to perform, convenient for large batches and can be bought as kits with standardised controls. The obvious drawback of these assays is that they may tell you nothing about the functional capacity of the antigen detected. If possible they should always be carried out in parallel with a functional assay.

The antibody may be bound to a plate but to facilitate automation it is now often bound to microparticles. Antibody binding may then be quantified by some form of luminescence (e.g. chemiluminescence) using a secondary antibody or by changes in turbidity when particles are cross linked (immunoturbidimetric assay). Artefacts in these assays may arise from the presence of rheumatoid factor or other auto-antibodies.

Assays using chromogenic peptide substrates (amidolytic assays)

The serine proteases of the coagulation cascade have narrow substrate specificities and it is possible to synthesise a short peptide specific for each enzyme that has a dye ( p -nitroaniline, p -NA) attached to the terminal amino acid. When the synthetic peptide reacts with the specific enzyme, the dye is released and the rate of its release or the total amount released can be measured photometrically. This gives a measure of the enzyme activity present. Chromogenic substrate assays can be classified into direct and indirect assays. Direct assays can be further subclassified into primary assays, in which a substrate specific for the enzyme to be measured is used, and secondary assays, in which the enzyme or proenzyme measured is used to activate a second protease for which a specific substrate is available. Specific substrates are available for many coagulation enzymes. However, the substrate specificity is not absolute and most kits include inhibitors of other enzymes capable of cleaving the substrate to improve specificity. Indirect assays are used to measure naturally occurring inhibitors and some platelet factors.

The measurement of amidolytic activity is not the same as the measurement of biological activity in a coagulation assay and in some cases may not accurately reflect this. This is particularly important when dealing with the molecular variants of various coagulation factors for which a functional assay may be required. The assays can be automated, carried out in a microtitre plate or in a tube when a spectrophotometer is used to measure the intensity of the colour development.

Coagulation assays

Coagulation assays are functional bioassays and rely on comparison with a control or standard preparation with a known level of activity. In the one-stage system optimal amounts of all the clotting factors are present except the one to be determined, which should be as near to absent as possible. The principles of bioassay, its standardisation and its limitations are considered in detail on page 386.

Coagulation assay techniques are also used in mixing tests to identify a missing factor in an emergency or to identify and quantify an inhibitor or anticoagulant. The advantage of this type of assay is that it most closely approximates the activity in vivo of the factor in question. However, they can be technically difficult to perform and their susceptibility to interference from other plasma components has a detrimental effect on their accuracy and precision.

Fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) describes the distance-dependent transfer of energy from one molecule (the donor) to a second molecule (the acceptor). The transfer of energy leads to a reduction in the donor fluorescence and increase in the acceptor fluorescence. Thus a FRET assay can be used for a function that results in separation of the two molecules. For example, FRET assays for ADAMTS13 involve a chemically modified fragment of the VWF A2 domain spanning the ADAMTS13 cleavage site.

Other assays

Other assays include measurement of coagulation factors using snake venoms, assay of ristocetin cofactor and the amide-release assay for FXIII transglutaminase activity. DNA analysis is sometimes helpful in coagulation and is described in Chapter 8 .

Water baths

A 37 °C water bath is required for manual coagulation tests, incubation steps and the rapid thawing of frozen specimens. Water baths should be set at 37 °C and monitored using a certified thermometer to ensure it varies by no more than ± 0.5 °C. Slight variation in temperature will markedly affect the speed of clotting reactions. A water bath with plastic or glass sides is preferable, and 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 water bath and maintain the water level. Records must be kept.

Refrigerators and freezers

Ensure that the temperature does not move 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. Freezers should maintain a constant temperature and so should not auto-defrost.

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 enzyme-linked immunosorbent assay (ELISA) substrates. Chromogenic substrates should be reconstituted with sterile distilled water; contamination with bacterial enzymes may cause pNA release and yellow discolouration of the reagent. Records of batch numbers, in use dates 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 clocks

Stopclocks are useful for timing incubation periods of several minutes or more, but stopwatches that can 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. Evaluation of laboratory equipment for coagulation testing is detailed in previous editions of this book and now in 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. 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 FVIII, 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. 2). Venous occlusion causes haemoconcentration, increase of fibrinolytic activity, platelet release reaction 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. less than 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. If there is no other source of blood then the line must be flushed thoroughly before sampling and the results scrutinised for possible artefacts.

To minimise 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 silicone.

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. 562).

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

Blood sample anticoagulation

The most commonly used anticoagulant for coagulation samples is trisodium citrate. A 32 g/1 (0.109 M) solution (p. 562) 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 end-point determinations. Additional benefits of trisodium citrate are that the calcium ion is neutralised 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 due to either severe anaemia or polycythaemia, the blood:citrate ratio should be adjusted. 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, direct acting anticoagulants and desmopressin).

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

• 
  

  FVIII: at 15 min

  
  
• 
  

  FIX: at 30 min

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.

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 (approximately 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 is minimised. The platelet count should be below 10 × 10 ⁹ /l. This is best achieved by double centrifugation. Filtration of the plasma through a 0.2 μm filter is not recommended because it may create microparticles.

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. Samples should be thawed at 37 °C to avoid forming cryoprecipitate.

Some common ‘technical’ errors

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

• 1.
  

  Faulty collection of the sample, resulting in it undergoing partial clotting

  
  
• 2.
  

  Underfilling or overfilling of the bottle or high or low haematocrit causing an incorrect volume of citrate in relation to the volume of plasma

  
  
• 3.
  

  An unsuitable anticoagulant, such as EDTA, used in collecting the sample

  
  
• 4.
  

  Collection of blood through a line that has been in contact with heparin or used for concentrate infusion

  
  
• 5.
  

  Contamination of the kaolin/platelet substitute reagent with a trace of thromboplastin

  
  
• 6.
  

  Delay in sample analysis

  
  
• 7.
  

  Use of inaccurate pipettes (documentation of pipette calibration is essential)

  
  
• 8.
  

  Machine malfunction

  
  
• 9.
  

  Incorrect water bath temperature

  
  
• 10.
  

  Calcium chloride at incorrect concentration or not freshly prepared.

Manual methods

Detecting clot formation as the end-point 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 end-point. 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 end-point 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. End-point detection by analysers sometimes fails and dubious or inconsistent results or samples which fail to record a clotting time 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 end-point, which may make comparison of results difficult. Some commonly used techniques follow.

Electromechanical

Photo-optical analysis

Percentage detection method

After initiating the clotting reaction, the transmitted light is monitored and a baseline 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 arbitrarily set end point, usually 50%, is then determined. At this point the H value usually shows the greatest rate of change and the fibrin monomer polymerisation reaction rate is high. Detection based on this principle enables coagulation analysis to be more accurate at low fibrinogen concentrations in samples with low scatter and those samples for which the initial scatter is higher than usual, such as lipaemic and haemolysed samples.

Endpoint determination: percentage detection method.

(Reproduced by permission of Sysmex UK Ltd.)

Rate method

After the start of the reaction the change in absorbance is monitored ( Fig. 18-3 ). After a predetermined time the final absorbance measurement is made. The rate of the absorbance increase per minute between these two time point measurements is calculated. The calculated rate of change in absorbance (Δ OD/min) is used to construct a standard curve (i.e. there is no end-point per se ). This method is used for activity measurement such as in chromogenic substrate assays. It is important that the rate is measured on a linear portion of the curve.

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 evaluates the absorbance per minute of an immunological reaction ( Fig. 18-4 ). This is monitored and mathematical analysis is used to determine the peak rate of change (maximum velocity). The VLin integral evaluation method is used for immunological assays including those for D-dimer and VWF antigen. Using this method allows increased sensitivity, extended measuring range, reduced measurement time and improved antigen excess reliability when measuring an immunological reaction.

Analysis of reaction: VLin integral method.

This is used for immunological assays.

(Reproduced by permission of Sysmex UK Ltd.)

Clot signatures: normal and abnormal activated partial thromboplastin time clot waveforms

On some analysers the clot waveform can be generated from the monitoring of light transmission or absorbance during the PT or APTT ( Fig. 18-7 ). This has been used to assess procoagulant activity and to detect DIC but is not in routine practice.

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, FVIII-deficient specimen (< 1%), with prolonged APTT (84.1 s) and slightly reduced fibrinogen concentration (1.56 g/l). E, FIX-deficient specimen (< 1%) with prolonged APTT (83.4 s) and normal fibrinogen concentration (2.94 g/l).

(Reproduced by permission of bioMérieux.)

CaCl ₂

The working solution is best prepared from a commercial molar solution. Small volumes of 0.025 mol/l concentration should be prepared frequently and stored for short periods to avoid proliferation of microorganisms. Prewarmed CaCl ₂ should always be discarded at the end of the working day. Commercial solutions are available.

Barbitone buffer

• •
  

  50 ml sodium diethyl barbiturate (C ₈ H ₁₁ O ₃ N ₂ Na) 0.2 M (41.2 g/l)

  
  
• •
  

  Add 32.5 ml hydrochloric acid (HCl) 0.2M

  
  
• •
  

  Make up to 200 ml with water (pharmacy grade distilled water www.baxterhealthcare.co.uk ) and correct pH to 7.4 with HCl.

Barbitone buffered saline, pH 7.4

• •
  

  NaCl 5.67 g

  
  
• •
  

  Barbitone buffer, pH 7.4, 1 litre

  
  
• •
  

  Before use, dilute with an equal volume of 9 g/l NaCl.

pH 7.3–7.4 is recommended for most clotting tests.

Glyoxaline buffer.

Dissolve 2.72 g of glyoxaline (imidazole) and 4.68 g of NaCl in 650 ml of water. Add 148.8 ml of 0.1 mol/l HCl and adjust the pH to 7.4. Adjust the volume to 1 litre with water.

• 
  

  Owren veronal buffer

  
  
• •
  

  Sodium acetate: 3.89 g

  
  
• •
  

  Barbitone sodium: 5.89 g

  
  
• •
  

  Sodium chloride: 6.8 g

  
  
• •
  

  Dissolve the salts in 800 ml of water.

  
  
• •
  

  Add 21.5 ml of 1 mol/l HCl, then make up to 1 litre with water, mix and check that the pH is 7.4.

Alternatively the solutions can be purchased commercially ( www.sigmaaldrich.com ).

Principle

The prothrombin time 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 also depends on 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 water bath and add 0.1 ml of thromboplastin. Wait 1–3 min to allow the mixture to warm. Then add 0.1 ml of warmed CaCl ₂ and start the stopwatch. Mix the contents of the tube and record the end-point. 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 time bias. Some thromboplastins contain calcium chloride, in which case 0.2 ml of thromboplastin-Ca 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 (the latter not pregnant and not taking oral contraceptives) and the logarithmic mean normal PT (LMNPT) 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 Normalised Ratio (INR), see Chapter 20 .

Normal values

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

Interpretation

The common causes of a prolonged PT are as follows:

• 1.
  

  Administration of oral anticoagulant drugs (vitamin K antagonists)

  
  
• 2.
  

  The presence of a direct acting inhibitor of factor Xa

  
  
• 3.
  

  Liver disease, particularly obstructive jaundice

  
  
• 4.
  

  Vitamin K deficiency

  
  
• 5.
  

  Disseminated intravascular coagulation

  
  
• 6.
  

  Rarely, a previously undiagnosed factor VII, X, V or prothrombin deficiency or defect.

Note: With prothrombin, FX or factor V deficiency the APTT will also be prolonged.

Principle

The test measures the clotting time of plasma after the activation of contact factors and the addition of phospholipid and CaCl ₂ but without added tissue thromboplastin and so indicates the overall efficiency of the intrinsic pathway. 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, FXIIa is produced, which cleaves FXI to FXIa, but coagulation does not proceed beyond this point in the absence of calcium. After recalcification, FXIa activates FIX and coagulation follows. A standardised 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 CaCl ₂ 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 to 40 s. The actual times depend on the reagents used and the duration of the preincubation period, which varies in manufacturers’ recommendations for different reagents. 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:

• 1.
  

  Disseminated intravascular coagulation

  
  
• 2.
  

  Liver disease

  
  
• 3.
  

  Massive transfusion with plasma-depleted red blood cells

  
  
• 4.
  

  Administration of or contamination with heparin or other anticoagulants

  
  
• 5.
  

  A nonspecific circulating anticoagulant (such as an LAC)

  
  
• 6.
  

  The presence of a direct acting anticoagulant drug (e.g. anti-IIa or anti-Xa agents)

  
  
• 7.
  

  Deficiency of a coagulation factor other than factor VII

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, mixing tests, an inhibitor screen and factor assays should be considered (see below).

Principle

Thrombin is added to plasma and the clotting time is measured. The TT is affected by the concentration and function of fibrinogen and by the presence of inhibitory substances. The clotting time and the appearance of the clot are both 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 the 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 to 19 s). Times of 20 s and longer are definitely abnormal.

Interpretation of results

The common causes of prolonged TT are as follows:

• 1.
  

  Hypofibrinogenaemia as found in DIC and, more rarely, in a congenital deficiency

  
  
• 2.
  

  Dysfibrinogenaemia, either inherited or acquired, in liver disease or in neonates

  
  
• 3.
  

  Extreme prolongation of the TT is nearly always a result of thrombin inhibition; typically unfractionated heparin but also oral or parenteral direct thrombin inhibitors. If a thrombin inhibitor is suspected, a reptilase time test can be carried out (see p. 386) or the test can be repeated after the addition of heparinase. Low molecular weight heparin (LMWH) produces only a slight prolongation at therapeutic levels

  
  
• 4.
  

  Raised concentrations of fibrin degradation products (FDP), as encountered in DIC or liver disease

  
  
• 5.
  

  Hypoalbuminaemia

  
  
• 6.
  

  Paraproteinaemia.

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. 386).

Principle

Diluted plasma is clotted with a strong thrombin solution; the plasma must be diluted to give a low level of any inhibitors (e.g. FDPs and heparin). A strong thrombin solution must be used so that the clotting time over a wide range is independent of the thrombin concentration.

Reagents

• •
  

  Calibration plasma. With a known level of fibrinogen calibrated against an International Reference Standard

  
  
• •
  

  PPP. From the patient and a control

  
  
• •
  

  Thrombin solution. Freshly reconstituted to 100 NIH u per ml in 9 g/l NaCl

  
  
• •
  

  Owren veronal buffer, pH 7.4. See p. 380.

Method

A calibration curve is prepared each time the batch of thrombin reagent is changed or there is a drift in control results; this is used to calculate the results of unknown plasma samples.

Make dilutions of the calibration plasma in veronal buffer to give a range of fibrinogen concentrations (1 in 5, 1 in 10, 1 in 20 and 1 in 40). Part (0.2 ml) of each dilution is warmed to 37 °C, 0.1 ml of thrombin solution is added and the clotting time is measured. Each test should be performed in duplicate. Plot the clotting time in seconds against the fibrinogen concentration in g/l on log/log graph paper. The 1 in 10 concentration is considered to be 100% and there should be a straight line connection between clotting times of 5 and 50 s. Make a 1 in 10 dilution of each patient’s sample and clot 0.2 ml of the dilution with 0.1 ml of thrombin.

The fibrinogen level can be read directly off the graph if the clotting time is between 5 and 50 s. However, outside this time range a different assay dilution and arithmetical correction of the result will be required (i.e. if the fibrinogen level is low and a 1 in 5 dilution is required, divide answer by 2; for a 1 in 20 dilution multiply answer by 2).

The clot formed in this method may be ‘wispy’ as a result of the plasma being diluted and end-point detection may be easier with optical or mechanical automated equipment. These have been assessed with available substrates and give reasonably consistent results. The high concentration of thrombin used raises the risk of carry over into subsequent tests.

Normal range

The normal range is approximately 1.8 to 3.6 g/l.

Interpretation

The Clauss fibrinogen assay is usually low in inherited dysfibrinogenaemia but is insensitive to heparin unless the level is very high (> 0.8 u/ml). High levels of FDPs (> 190 μg/ml), may also interfere with the assay. Because the chronometric Clauss assay is a functional assay it will generally give a relevant indication of fibrinogen function in plasma. When an inherited disorder of fibrinogen is suspected, a physicochemical estimation should be obtained (e.g. clot weight estimate of fibrinogen or total clottable fibrinogen or an immunological assay; see page 392). If a dysfibrinogenaemia is present, it will reveal a discrepancy between the (functional) Clauss assay and the physical amount of fibrinogen present.