The lupus anticoagulant (LAC) is an acquired autoantibody found in various autoimmune disorders and sometimes in otherwise healthy individuals.² LACs are immunoglobulins that bind to certain proteins when bound to phospholipid. The effective sequestration of phospholipid can then cause prolongation of phospholipid-dependent coagulation tests such as the prothrombin time (PT) or activated partial thromboplastin time (APTT). The name ‘anticoagulant’ is misleading because, despite the in vitro effects, patients do not have a bleeding tendency. Instead, there is a clear association with recurrent venous thromboembolism, cerebrovascular accidents and other arterial events and, in women, with recurrent abortions, fetal loss and other complications of pregnancy.³ Therefore, tests for the presence of the LAC should be carried out in all young individuals with unexplained venous or arterial thrombosis and also in women with recurrent-early or late pregnancy loss.⁴ Antibodies of this class are members of a larger group called antiphospholipid or anticardiolipin antibodies. (Although not precisely the same these terms are used interchangeably.) Tests for lupus anticoagulant are usually performed in parallel with tests for the presence of antiphospholipid antibodies, usually by ELISA. In general these tests are not performed by haematology laboratories and are therefore not described here. Serological tests for antiphospholipid antibodies are not standardized and agreement between laboratories is poor. A large number of target proteins have been described but the most important, and the only one for which there is evidence of a pathogenic effect, is β₂-glycoprotein 1.⁵ Increasingly, tests specifically for anti-β₂-glycoprotein 1 antibodies are performed and possible mechanisms for their prothrombotic activity are being elucidated.⁶

The presence of a LAC may be detected by the clotting screen and, depending on the reagents and methods used as well as on the potency and avidity of the antibody, either the PT or APTT may be prolonged. However, the sensitivity of both APTT and PT to LAC varies considerably, so that these tests may well be normal and, if clinically suspected, specific tests should always be performed.⁴ The unmodified test for activated protein C (APC) resistance (see p. 456) is also sensitive to the presence of a LAC.

Patients with a LAC may show other abnormalities, including thrombocytopenia, a positive direct antiglobulin test and a positive antinuclear antibody test. Another frequent target for antiphospholipid antibodies is prothrombin but only rarely are these antibodies sufficient to inhibit or deplete prothrombin activity. Such patients may have a bleeding tendency. A recent international guideline on detection of lupus anticoagulants has been published ⁴ and recommended the following tests:

1. Dilute Russell’s viper venom time (DRVVT) in conjunction with the platelet neutralization test.

2. An APTT test that has a low concentration of phospholipid and uses silica as an activator, thus making it sensitive to the presence of LAC.

There are a large number of additional tests which have in the past been successfully used for the detection of LAC, several of which are no longer recommended due to poor reproducibility, technical problems and lack of standardization.⁴ The kaolin clotting time (KCT) and the dilute thromboplastin inhibition test are retained here because they are still widely used and thought to have some advantages by some authors. Although no single test is sufficiently sensitive to detect all LAC, readers are counselled against performing an excessive (more than two) number of tests because a large number of false positives will be generated. The most recent guidelines for the optimal performance of testing for LAC have been well laid out and are summarized in Box 19.1.⁴

Box 19.1 Recommendations for the optimal laboratory detection of lupus anticoagulant (LAC)⁴

(A) Blood collection

1. Blood collection before the start of any anticoagulant drug or a sufficient period after its discontinuation

2. Fresh venous blood in 0.109 M sodium citrate 9:1

3. Double centrifugation

4. Quickly frozen plasma is required if LAC detection is postponed

5. Frozen plasma must be thawed at 37°C

(B) Choice of the test

1. Two tests based on different principles

2. DRVVT should be the first test considered

3. The second test should be a sensitive APTT (low phospholipids and silica as activator)

4. LAC should be considered as positive if one of the two tests gives a positive result

(C) Mixing test

1. Pooled normal plasma (PNP) for mixing studies should ideally be prepared in-house. Adequate commercial lyophilized or frozen PNP can alternatively be used

2. A 1:1 proportion of patient:PNP should be used, without preincubation within 30 min

3. LAC cannot be conclusively determined if the thrombin time of the test plasma is significantly prolonged

(D) Confirmatory test

1. Confirmatory test(s) must be performed by increasing the concentration of phospholipid content of the screening test(s)

2. Bilayer or hexagonal (II) phase phospholipid (PL) should be used to increase the concentration of PL

(E) Expression of results

Results should be expressed as ratio patient:PNP for all procedures (screening, mixing and confirm)

(F) Transmission of results

A report with an explanation of the results should be given

Sample Preparation

It is essential that all the samples of plasma tested for an LAC should be as free of platelets as possible. This is achieved by further centrifugation of plasma at 2500 g for 10 min. A platelet count of <10 × 10⁹/l should be achieved. The plasma is centrifuged at room temperature to avoid platelet activation because platelet microvesicles may also invalidate the test. After separation, the plasma should be frozen at −70°C as soon as possible to prevent deterioration. Prior to testing, the frozen sample should be rapidly warmed to 37°C in a water bath.

Dilute Russell’s Viper Venom Time

Platelet Neutralization Test

Principle

When an excess of phospholipid, originally in the form of lysed platelets, is added to clotting tests, the tests become insensitive to the presence of a LAC. This appears to be a result of the ability of the platelets/phospholipid to adsorb the LAC. Platelet neutralization reagents are available commercially and are usually provided in DRVVT kits. Commercial reagents are preferred for consistency but a method for preparation is provided below. To utilize this property of platelets, they must be washed to remove contaminating plasma proteins and activated or ‘fractured’ to expose their coagulation factor binding sites.

Reagents for preparation of platelet neutralization reagent

Commercial platelet extract reagent or washed normal platelets

Acid–citrate–dextrose (ACD) anticoagulant solution (see p. 619), pH 5.4, is required for washed platelets. For use, 6 parts of blood is added to 1 part of this anticoagulant

Na₂EDTA. 0.1 mol/l in saline

Calcium-free Tyrode’s buffer. Dissolve 8 g NaCl, 0.2 g KCl, 0.625 g Na₂HPO₄, 0.415 g MgCl₂ and 1.0 g NaHCO₂ in 1 litre of water. Adjust pH if necessary to 6.5 with 1 mol/l HCl.

Method

Collect normal blood into ACD and centrifuge at 270 g for 10 min. Pipette the supernatant platelet-rich plasma (PRP) into a plastic container and centrifuge again to obtain more PRP, which is added to the first lot. Dilute the PRP with an equal volume of the calcium-free buffer and add one-tenth volume of EDTA to give a final concentration of 0.01 mol/l. Centrifuge the mixture in a conical or round-bottom tube at 2000 g for 10 min and discard the supernatant. Gently resuspend the platelet pellet in buffer and 0.01 mol/l EDTA. Centrifuge again, discard the supernatant and resuspend the pellet in buffer alone. Then centrifuge the platelets a third time and resuspend the pellet in buffer without EDTA to give a platelet count of at least 400 × 10⁹/l. The washed platelets may be stored below −20°C in volumes of 1–2 ml. Before use, they must be activated by thawing and refreezing 3–4 times.

Use the washed platelets or the commercial reagent in the dilute RVV test or in the APTT in place of the usual phospholipid reagent. First, determine a suitable dilution by testing a range of doubling dilutions in the test system with control plasma. A suitable dilution gives a similar clotting time to that obtained using control plasma and the phospholipid reagent.

Interpretation

The addition of platelets or a commercial ‘confirm’ phospholipid reagent to the DRVVT system corrects the clotting time when a LAC is present. It does not correct the time when the prolongation is due to a factor deficiency or an inhibitor directed against a specific coagulation factor. However, the ability of different batches of platelets to perform this correction is variable and may vary further with storage. Accordingly, each time the test is performed a plasma sample known to contain a LAC should be tested in parallel to establish the efficacy of the platelets.

Many commercial kits are now available for performing the tests described above. As with all such tests, there is an inevitable trade-off between sensitivity and specificity. This varies with different techniques, kits and coagulometer. One survey of reagents found that the best discriminator of positivity was by using a normalized correction ratio (CR) of DRVVT clotting times as follows:

where P is patient’s clotting time and N is the clotting time of normal plasma and D represents the detection procedure and C represents the confirmation (platelet/phospholipid neutralization) procedure.⁷ A correction of >10% is regarded as positive, but care should be taken to establish a local normal range. Other calculations may also be used such as a simple ratio of P_D/P_C or percentage correction: [(P_D − P_C)/P_D] × 100%.⁴

False-positive results may be obtained in patients receiving intravenous heparin although some reagents contain neutralizing agents. Interpretation may be difficult in patients receiving oral anticoagulants; this can sometimes be overcome by performing the test on a 50:50 mix with normal plasma.

Interpretation of Tests for Lupus Anticoagulant

Detailed instructions for the interpretation of LAC testing have been published.⁴ No single test detects all lupus-like anticoagulants and, if suspected clinically, then two specific tests should be performed before concluding that a LAC is not present.^4,⁸ Conversely, a single positive test should be repeated 12 weeks later because a transient positive may arise as the result of intercurrent illness or medication. It is crucial to distinguish LAC from specific anti-factor VIII antibodies, which are more typically time dependent but may have some immediate effect as well. Specific factor assays can be useful in discrimination but note that a LAC may result in non-parallelism and spuriously low results in these assays. Similarly, some weak LAC are neutralized by 50:50 mixing with normal plasma and sometimes exhibit a time-dependent effect. Some transient non-specific coagulation inhibitors are not detected by tests for LAC. Tests may be falsely negative while taking warfarin.

Kaolin Clotting Time

Principle

When the APTT is performed in the absence of platelet substitute reagent, it is particularly sensitive to a LAC. If the test is performed on a range of mixtures of normal and patient’s plasma, different patterns of response are obtained, indicating the presence of a LAC, deficiency of one or more of the coagulation factors or the ‘lupus cofactor’ effect.⁹

There are commercially available kits based on the KCT, such as Kaoclot (Life Therapeutics USA). This method uses a low-turbidity colloidal kaolin solution, making this reagent slow to settle and therefore suitable for use on automated coagulation analysers. Kaoclot shows high sensitivity to LACs but is not suitable for testing patients undergoing heparin therapy

Reagents

Kaolin. 20 mg/ml in Tris buffer, pH 7.4. This may need to be reduced to 5 mg/ml in some automated analysers (see p. 410)

Normal platelet-poor plasma. Depleted of platelets by second centrifugation

Patient’s plasma. Also platelet depleted

CaCl₂. 0.025 mol/l.

Method

Mix normal and patient plasma in plastic tubes in the following ratios of normal to patient’s plasma: 10:0, 9:1, 8:2, 5:5, 2:8, 1:9 and 0:10. Pipette 0.2 ml of each mixture into a glass tube at 37°C. Add 0.1 ml of kaolin and incubate for 3 min, then add 0.2 ml of CaCl₂ and record the clotting time.

Results

Plot the clotting times against the proportion of normal to patient’s plasma on linear graph paper as shown in Figure 19.1.

Figure 19.1 Curves obtained using the kaolin clotting time (KCT) to test for the presence of a lupus anticoagulant (see text).

Interpretation

The pattern obtained for each patient must be critically assessed. A convex pattern (Pattern 1) indicates a positive result, whereas a concave pattern (Pattern 4) indicates a negative result. Pattern 2 indicates a coagulation factor deficiency and a LAC. Pattern 3 is found in plasma that contains a LAC but is also deficient in a cofactor necessary for the full inhibitory effect. The initial rate of slope is important because a steep slope indicates a positive result. This allows the test to be simplified so that only the tests of 100% normal and of 80% normal/20% test plasmas need be performed. The slope can be calculated using the ratio of KCT at 20% test plasma and KCT at 100% normal control plasma (N). For a positive result the ratio at this point should be ≥1.2.

Thus,

A control KCT of <60 s may indicate contamination of the control plasma with phospholipid.

Dilute Thromboplastin Inhibition Test

Principle

When the thromboplastin used for the PT is diluted, the PT becomes prolonged. At a certain point (usually 1:50–1:500 dilution) the concentration of phospholipid is low enough for the test to become sensitive to phospholipid binding antibodies and when a LAC is present the ratio of the test plasma to normal plasma clotting time increases. This test is now considered more useful because some thromboplastin reagents (e.g. Innovin) are more sensitive to LACs. However, it should be noted that diluting thromboplastin makes the system sensitive to low levels of factor VIII as are encountered in mild haemophilia, acquired haemophilia and low levels of factor V or factor VII. Care should be taken that these disorders are not confused. In one study, the test was determined to be positive when the dilute PT ratio (test/mean normal) using Innovin at 1:200 dilution was >1.15.¹⁰

Other Acquired Thrombophilic States

There are numerous other disorders that are associated with an increased risk of thrombosis but are not usually diagnosed using coagulation-based tests. Appropriate tests for some of these such as myeloproliferative neoplasms and paroxysmal nocturnal haemoglobinuria are found elsewhere in this book. One of the most important factors precipitating thrombosis is malignancy. However, the value of extensive testing for possible malignancy in patients with thrombosis remains contentious; some studies have shown that a history and examination combined with a few simple tests detect the large majority of malignancy and other systemic disorders. Other studies suggest that more intensive screening including abdominal and pelvic scans are required to achieve this. Large numbers of ‘tumour marker’ analyses result in numerous false positives. Even when tests have been effective in detecting occult malignancy it is not clear there is any improvement in outcome.^11,¹²

Investigation of inherited thrombotic states

Testing for thrombotic syndromes remains frequent, despite doubts about its clinical utility.¹ Patients with disorders of pregnancy and those with thrombotic disorders are often referred for investigation. Prior to testing for thrombophilia consideration should be given to the likely benefits including alteration in management that can be achieved. This requires a carefully taken history, noting in particular the circumstances of any previous thrombotic event, a family history of thrombosis and identification of any coexisting disorders. The relevant tests are described below.

Antithrombin (AT)

AT ¹³ (previously known as antithrombin III) is the major physiological inhibitor of thrombin and factors IXa, Xa and XIa. AT deficiency is found in approximately 2% of cases of thrombosis and may be acquired or congenital. Various methods are available for measuring either functional activity or antigenic quantity of AT. The functional methods are based on the reaction with thrombin or factor Xa and can be coagulation based or chromogenic assays. A chromogenic assay is described below.

Antithrombin (AT) Measurement Using a Chromogenic Assay

Principle

In the presence of heparin, AT reacts rapidly to inactivate thrombin by forming a 1:1 complex. The chromogenic AT assay is a two-step procedure. In the first step, the plasma sample is incubated with a fixed quantity of thrombin and heparin. In the second step the residual thrombin is measured spectrometrically by its action on a synthetic chromogenic substrate, which results in the release of p-nitro-aniline (pNA) dye. The use of bovine thrombin avoids interference in the assay by heparin cofactor II; this can also be achieved by measuring the Xa-neutralizing capacity of the AT and an appropriate chromogenic substrate. The assay thus measures heparin cofactor activity rather than progressive AT activity and may therefore also detect AT variants with altered heparin binding.

Method

Carry out the procedure on dilutions of a standard plasma to construct a standard graph. Then test dilutions of the test plasma in an identical manner and read the results directly from the standard graph.

The reagents provided and details of the method vary among manufacturers and should be closely followed. There may also be variation between different batches of the same reagent.

Normal range

The normal range is generally between 0.75 and 1.25 iu/ml. Some manufacturers suggest a slightly narrower range (i.e. 0.8–1.20 iu/ml), but it is preferable for each laboratory to establish its own normal range. Repeated freezing and thawing of samples, as well as storage at or above −20°C, results in a reduction in AT concentration.

Interpretation

In an inherited deficiency, the AT concentration is usually <0.7 iu/ml. Most cases are heterozygotes for null mutations (type I deficiency) and have levels of approximately 50% of normal. Be aware that numerous type 2 variants have been described affecting the reactive site, the heparin binding site or having pleiotropic effects, sometimes resulting in assay results that are close to normal. The clinical significance of heterozygous heparin-binding site mutations is probably low. Further tests such as AT antigen, crossed immunoelectrophoresis or mutation analysis may be required to identify variant molecules.^14,¹⁵ A low level of AT may be acquired as a result of active thrombosis, liver disease, heparin therapy, nephrotic syndrome or asparaginase therapy; very low values are sometimes encountered in fulminant disseminated intravascular coagulation (DIC) or liver failure. Normal newborns have a lower AT concentration (0.60–0.80 iu/ml) than adults. In neonates who are congenitally deficient, very low values (0.30 iu/ml and lower) may be found. It is also important to remember that oral anticoagulant therapy may increase the AT concentration by approximately 0.1 iu/ml in cases of congenital deficiency.

Antithrombin Antigen Determination

AT antigen can be assessed using various methods such as enzyme-linked immunosorbent assay (ELISA), immuno-electrophoresis assays and latex agglutination (nephelometry).

Principle

Latex agglutination assays are based on the agglutination of a suspension of antibody-coated, micro-latex particles in the presence of plasma containing AT antigen (the antibody is attached by covalent bonding). The wavelength is such that light can pass through the latex suspension unabsorbed. However, in the presence of AT antigen, the antibody-coated latex particles agglutinate to form aggregates of diameter greater than the wavelength of the light; the latter is then absorbed. There is a direct relationship between the observed absorbance value and the concentration of the antigen being measured. The convenience of this form of test is that it can be performed on automated analysers.

Protein C (PC)

PC ^16,¹⁷ is a vitamin K-dependent protein. After activation by thrombin, which is accelerated in the presence of thrombomodulin on the vascular endothelium, PC complexes with phospholipids and protein S (PS) to degrade factors Va and VIIIa. Inherited heterozygous PC deficiency is found in 2–4% of first-episode thromboses and 5–7% of all recurrent thromboembolic episodes in young adults.¹⁸^–²⁰ The importance of the PC–PS system is evidenced by the catastrophic syndrome of purpura fulminans in neonates with homozygous PC or PS deficiency.²¹ Acquired PC deficiency is found in all conditions associated with vitamin K deficiency or defect, including oral anticoagulant therapy. A low plasma concentration is also found in DIC, sepsis (especially meningococcal septicaemia), in liver disease, sickle cell disease and in the early postoperative period.

PC can be measured using a chromogenic assay, a coagulation assay or an antigenic method.²²

Measurement of Functional Protein C by the Protac Method

Principle

In the presence of a specific snake venom activator, PC is converted into its active form. This allows the activation to be carried out in whole plasma without separation of PC. Activated PC is measured by its action on one of the specific synthetic substrates (e.g. S-2366, CBS 65.25). The reaction is stopped by the addition of 50% acetic acid and the p-nitro-alanine produced is measured in a spectrometer at 405 nm.

Reagents

Platelet-poor plasma. Standard and test samples are centrifuged at 1500–2000 g for 15 min. After centrifugation, plasma can be stored indefinitely at −40°C or below

Protac. This is an activator derived from the venom of Agkistrodon contortrix contortrix (Southern copperhead snake). It is obtained commercially; each vial contains lyophilized powder, which is reconstituted and stored according to the manufacturer’s instructions

Specific chromogenic substrate. Reconstituted and stored according to the manufacturer’s instructions

Barbitone buffered saline. See p. 409

Acetic acid. 50%.

Method

Construct the standard curve according to the instructions using a calibrated reference plasma.

The assay is carried out by a two-step method. In the first step, plasma and activator are incubated for an exact period of time. In the second step, the specific chromogenic substrate is added and the reaction is stopped with acetic acid, again at a precise point in time. Read the amount of the dye produced at 405 nm against a blank obtained as follows: acetic acid, activator and chromogenic substrate are first mixed, then activated standard or patient’s plasma is added to the mixture and the absorbance is measured at 405 nm. The manufacturer’s instructions must be closely followed. Plot the PC activity against the corresponding absorbance reading on linear graph paper.

Normal range

The normal range is 0.70–1.40 iu/ml. Preferably, each laboratory should establish its own normal range.

Further Investigation for Protein C Deficiency

If inherited PC deficiency is suspected, an immunological assay may also be carried out with an ELISA-based kit, which will distinguish a type 1 or type 2 deficiency. The amidolytic assay described here does not detect the rare type 2 PC deficiency due to mutations in the Gla domain, although they can be detected by a coagulation-based assay.²² The specificity of the chromogenic substrate is limited and is augmented by the inclusion of substances that inhibit other enzymes capable of cleaving the substrate. In some circumstances, this can fail and spuriously high PC activities can be obtained, which may obscure PC deficiency.²³ PC activity and antigen are reduced in patients taking oral vitamin K antagonists, although it is sometimes possible to make a provisional diagnosis of PC deficiency by using a PC:VIIc ratio.²⁴ It is also important to exclude vitamin K deficiency and/or liver disease by assaying other vitamin K-dependent factors. Family studies should be carried out whenever possible.

Clotting-Based Protein C Assay

Principle

PC clotting assays use an APTT reagent incorporating a PC activator derived from the Southern copperhead snake venom (Agkistrodon contortrix contortrix), PC-deficient plasma and calcium chloride. The APTT reagent activates both PC and the factors of the intrinsic pathway. The clotting time of normal plasma is long (>100 s), whereas that of PC-deficient plasma is normal (30 s). The degree of prolongation of the clotting time when patient plasma is mixed with PC-deficient plasma is proportional to the concentration of PC in the patient plasma.

Unlike chromogenic PC assays, PC clotting assays are sensitive to functional PC defects such as phospholipid binding (mutations in the Gla domain) and calcium binding. However, they are also sensitive to anticoagulants, Factor V Leiden, LACs and raised factor VIII levels. A functional protein C activity assay can also be performed using the DRVVT which may be less sensitive to these effects.²⁵

Protein C Antigen

PC antigen can be measured using a conventional ELISA. Commercial kits are available.

Protein S (PS)

PS is also a vitamin K-dependent protein that acts as a cofactor for activated PC. It is similar to the serine proteases of the coagulation system in having a Gla domain and four EGF domains; however, instead of a protease domain it has a large terminal domain closely homologous to sex hormone-binding globulin (SHBG). In plasma, 60% of PS is bound to C4b-binding protein (C4bBP) via the SHBG ²⁶ and does not possess any APC cofactor activity; the remaining 40% is free and available to interact with APC. Functional assays of PS are based on the capacity of PS to augment the prolongation of a clotting test time by APC. However, PS has some APC-independent anticoagulant activity that can also be measured in coagulation assays and it can also act as a cofactor for TFPI.^27,²⁸ Measurement of the total and free PS antigen is possible using enzyme-linked immuno-assays. All three measurements are considered together here but usually measurement of free PS is adequate.

Enzyme-Linked Immunosorbent Assay of Free and Total Protein S

Principle

The total PS in plasma is detected by a standard ELISA using polyclonal antibodies.²⁹ The analysis is then repeated using plasma in which C4bBP-bound PS has been removed by polyethylene glycol (PEG) precipitation. This gives a measure of free PS.

Reagents

Polytheneglycol (PEG) precipitation solution. Dissolve 100 g of PEG 8000 in 200 ml of sterile water. Prepare approximately 50 ml of working PEG by diluting the stock solution to exactly 18.75% with sterile water. Store in 2-ml aliquots at −20°C.

Coating buffer (phosphate buffered saline, pH 7.2). 0.39 g Na₂HPO₄.2H₂O, 2.68 g Na₂HPO₄.12H₂O, 8.474 g NaCl. Make up to 1 litre and adjust to pH 7.2; store at 4°C.

Wash buffer. This is the same as the coating buffer, but it contains 0.5 M NaCl and 0.2% v/v Tween 20. Add 10.37 g of NaCl to 1 litre of coating buffer and 0.2% Tween 20. (Mix well.) Store at 4°C.

Dilution buffer. This is the washing buffer with 30 g/1 PEG 8000. Store at 4°C.

Substrate buffer Na₂HPO₄ (citrate phosphate buffer, pH 5.0). 7.3 g citric acid, 23.87 g. Na₂HPO₄.12H₂O. Make up to 1 litre with water. Adjust pH to 5.0.

o-phenylenediamine

Anti-PS and anti-PS peroxidase conjugated. (Dako Ltd)

Sulphuric acid, 1 M

Microtitre plates. (Greiner Labortechnick Ltd)

Standards and controls

Hydrogen peroxide. 30% w/v.

Methods

Dilute the antihuman PS immunoglobulin 1:1000 in coating buffer (i.e. 20 μl in 20 ml of buffer). Add 0.1 ml to each well of a microtitre plate, cover with Parafilm and leave overnight in a moist chamber at 4°C. On the day the assay is to be performed, warm an aliquot of working PEG solution to 30°C. Accurately pipette 200 μl of standard, patient’s and control plasma samples into conical Eppendorf tubes; warm for 5 min at 37°C. Add exactly 50 μl of warmed PEG, immediately cap and vortex mix twice for exactly 5 s each time. Place in a water/crushed ice mixture. In turn, treat all the samples identically. Leave for 30 min on the melted ice. Centrifuge for 30 s in the Eppendorf centrifuge. Then return to ice and remove 100 μl into a labelled tube (taking care not to remove any precipitate).

Prepare dilutions of control and patient’s samples in PEG dilution buffer as follows. For total PS, dilute 0.05 ml of reference plasma in 8 ml of diluent. Use the PEG precipitated reference plasma for measuring free PS; add 0.1 ml to 4 ml of dilution buffer.

Prepare a range of standards from these stock solutions using the same dilution schedule for free and total PS.

A. Stock solution = 1.25 iu/ml.

B. 0.8 ml stock + 0.2 ml buffer = 1.0 iu/ml.

C. 0.6 ml stock + 0.4 ml buffer = 0.75 iu/ml.

D. 0.4 ml stock + 0.6 ml buffer = 0.5 iu/ml.

E. 0.2 ml stock + 0.8 ml buffer = 0.25 iu/ml.

F. 0.1 ml stock + 0.9 ml buffer = 0.125 iu/ml.

G. 0.05 ml stock + 0.95 ml buffer = 0.0625 iu/ml.

Control and patient’s samples are tested at two dilutions – total PS plasma: 1:200 and 1:400 and free PS PEG supernatants: 1:50 and 1:100. Shake out the contents of the previously prepared plate and blot on tissue. Wash the plate three times in wash buffer by filling all the wells, leaving for 2 min, shaking out the contents, blotting and repeating. Add 100 μl of each dilution of standard, control or patient’s plasma in duplicate across the plate. Cover and incubate for 3 h in a wet box at room temperature. Wash the plate as described earlier. Dilute 2 μl of peroxidase-labelled antibody in 24 ml of dilution buffer. Add 100 μl of diluted tag (peroxidase-conjugated) antibody to each well and leave in a wet box for 2–3 h at room temperature. Wash the plate as described earlier. Make up the substrate solution by adding 8 mg of o-phenylenediamine to 12 ml of citrate phosphate buffer. Immediately before use add 10 μl of hydrogen peroxide. Add 100 μl of substrate solution to each well. When the weakest standard has a visible yellow colour, add 150 μl of 1 M sulphuric acid to each well. Read the optical densities on a plate reader at 492 nm. Plot the optical densities against plasma dilutions on double-log graph paper and read the patient’s values from the corresponding calibration curve (i.e. total against total and free against free).

The polyclonal antibody should have similar affinities for free and bound PS; high plasma dilutions and long incubation times help to avoid differential affinity leading to error. Alternatively, two monoclonal antibodies (capture and tag) with the same affinity for free and bound PS can be used (Asserachrom).

Automated assays using antibodies to distinguish free and total PS are now available.³⁰^–³²

Protein S Functional Assay

Principle

Functional PS can be assessed using coagulation-based assays activated by different means. In one commercial assay (American Diagnostica Inc) dilutions of normal and test plasmas are mixed with PS-deficient plasma. Activation of these mixtures is achieved by a reagent containing factor Xa, activated PC and phospholipid. After a 5 min activation time, clot formation is initiated by the addition of calcium chloride. Under these conditions, the prolongation of the clotting time is directly proportional to the concentration of PS in the patient plasma. The use of factor Xa as the activator minimizes the potential interference by high levels of factor VIII.

A PS function assay may also be based on the PT, in which case the effect of factor VIII is again bypassed. The PT-based PS assay uses PS-depleted plasma activated by Protac, thus providing activated PC. The PT is increased by the APC–PS-mediated destruction of factor Va, which occurs in the presence of PS from the test and control plasmas. The PT is measured using bovine thromboplastin and prolongation is proportional to PS activity.

These tests are performed according to the manufacturer’s instructions and many tests can be automated.

Because the assays are subject to interference by other plasma factors, it is recommended that the test plasma is assayed at two different dilutions to ensure parallelism with the standard curve.

PS functional assays are designed to measure the PC cofactor activity of PS, but as discussed earlier, this is not its only anticoagulant activity. PS that is bound to C4bBP, is inadequately γ-carboxylated or has been cleaved by thrombin does not have PC cofactor activity but its effect on the assays is unknown.

Interpretation of Protein S Functional and Antigenic Assays

PS deficiency has been classified into three subtypes according to the pattern of results obtained in functional and antigenic assays (Table 19.1).

Table 19.1 Classification of protein S deficiency

Studies have suggested that the type I and type III patterns are both the result of the same genetic defect and that the difference may be the result of an age-related increase in C4bBP.^33,³⁴

Although an estimate of PS functional activity would be ideal for diagnosing PS deficiency, the functional PS assays available are problematic. Like other functional assays they are prone to external influences: factor V Leiden (FVL), LAC and levels of other coagulation factors. Fortunately type II PS defects appear to be extremely rare; many previously diagnosed cases proved to be due to FVL. Thus measurement of free PS is the preferred method for detecting PS deficiency.^35,³⁶ Low levels of PS may be an acquired phenomenon during pregnancy and with oral anticoagulation, nephrotic syndrome, use of oral contraceptives, systemic lupus erythematosus, HIV infection and liver disease. Catastrophically low levels have been reported in children after varicella infection owing to autoantibody production.³⁷ It is important to note that the normal range for premenopausal women is significantly lower than in other groups and local normal ranges should be determined to avoid misinterpretation, paying attention to the additional effects of hormonal therapy and artefactual reduction in PS as described earlier.^38,³⁹ Although C4bBP is elevated during an acute-phase reaction, the PS-binding β chain does not increase and as a result free PS does not decrease.⁴⁰

Activated Protein C Resistance

In 1993, Dahlback et al.⁴¹ described an inherited tendency to thrombosis characterized by a defective plasma response to activated PC. This became known as activated PC resistance (APCR) and was subsequently shown in >90% of cases to result from a polymorphism encoding the amino acid change Arg506Glu subsequently named factor V Leiden.⁴² This mutation destroys a cleavage site for APC, which greatly slows APC inactivation of factor Va. It also blocks the conversion by APC of factor V into factor Vi, which acts as a cofactor for APC degradation of factor VIIIa. APCR is found in approximately 20% of patients with a first episode of venous thrombosis.

Principle ⁴³

When activated PC (APC) is added to plasma and an APTT is performed, there is normally a prolongation of the clotting time as a result of factor V and factor VIII degradation. The original detection of this phenomenon was by means of a modified APTT, but it can also be detected using modifications of the PT, DRVVT and factor Xa clotting time. These tests all vary somewhat in their sensitivity and specificity for the FVL mutation, which is generally improved by mixing the test plasma with factor V-deficient plasma. This reduces the effect of other factors such as factor VIII and prothrombin, which can alter estimation of APCR ⁴⁴ and restores the sensitivity of the test in patients who are taking oral anticoagulants. However, the test remains sensitive to interference by LACs. Numerous commercial kits are available for these tests.

Expression of Results

APCR was originally reported as a simple ratio of clotting times with and without APC. The result can be normalized by expressing this as a ratio of the same result obtained with normal plasma, i.e.

when T = test and N = normal.

The use of a normalized ratio improves day-to-day precision and may also improve accuracy. However, it is extremely important that the pooled normal plasma does not contain FVL because very small amounts (2.5%) markedly affect the response to APC. A normal range should be established locally and its relationship to the presence of FVL should be determined.

Interpretation

The Leiden thrombophilia survey estimated the relative risk of thrombosis for APCR to be approximately 7.⁴⁵ Studies using DNA analysis alone have generally found slightly lower relative risks.⁴⁶ Most testing strategies have been directed toward producing tests that have a high sensitivity and specificity for FVL to avoid the need for DNA analysis. It seems that ‘acquired APCR’ or APCR resulting from other causes represents a prothrombotic state even in the absence of FVL,⁴⁷ as does the presence of acquired APCR in prothrombotic states such as pregnancy. These are not (except LACs) detected after mixing with factor V-deficient plasma. Some laboratories use a combination of plasma and DNA testing to assess patients’ status but increasingly DNA analysis alone is performed and this can be combined with analysis of the prothrombin gene (below).

Increased Prothrombin, Factor VIII and Other Factors

A later finding from the Leiden thrombophilia survey was that elevated levels of prothrombin were significantly associated with thrombosis.⁴⁸ Most elevated levels were associated with a mutation in the 3′ untranslated region of the gene (G20210A). The mutation is detected by a simple polymerase chain reaction-based test (see p. 148). Subsequently, other factors, including factor VIII, factor IX and factor XI, have been shown to have an association with thrombosis when elevated.⁴⁹^–⁵¹

Heparin Cofactor II

There is no clear evidence that heparin cofactor II (HCII) deficiency is more prevalent in patients with thrombosis than in the normal population; consequently, testing is not recommended as part of thrombophilia investigation.⁵² (A method for measuring HCII is described in previous editions of this book.)

Fibrinolytic system

Investigation of Suspected Dysfibrinogenaemia

Congenital dysfibrinogenaemia, which may be associated with thrombosis, should be suspected in individuals with a prolonged thrombin time and a slightly or moderately reduced fibrinogen concentration in plasma. The presence of a dysfibrinogen is proved when a significant (usually two-fold) discrepancy is found between the Clauss and clot weight assays. (For details of investigation see p. 412.)

Investigation of the Fibrinolytic System: General Considerations

The investigation of fibrinolysis has an uncertain place in haemostasis. It seems well-established that uncontrolled fibrinolytic capacity as a result of plasmin inhibitor or plasminogen activator inhibitor (PAI-1) deficiency can lead to a haemorrhagic tendency, although these are rare.^53,⁵⁴ Conversely it has been difficult to demonstrate that an impaired fibrinolytic capacity results in a tendency to venous thrombosis. This may be attributed in part to the poor reproducibility of the global tests such as euglobulin clot lysis or fibrin plate lysis but it has not been resolved by use of either specific assays or genetic polymorphic markers.⁵⁵ More recently a plasma clot lysis time has been developed which has been shown to detect a reduced fibrinolytic potential associated with an increased risk of first and recurrent thrombosis.^56,⁵⁷ Moreover, this defect was associated with levels of thrombin-activatable fibrinolysis inhibitor (TAFI), PAI-1, plasminogen and tissue plasminogen activator (tPA), although for the latter two the association was lost after adjusting for other variables. This test is not yet in routine clinical use. High levels of tPA were shown to be predictive of myocardial infarction in the ECAT (European Concerted Action on Thrombosis and Disabilities) study, but it is possible that this unexpected association can be interpreted as demonstrating an abnormality of endothelial function rather than a problem with fibrinolysis per se.⁵⁸^–⁶¹

Fibrinolysis shows considerable diurnal variation as well as interference from plasma lipids and stress. It is therefore generally recommended that these tests be performed in the morning after an overnight fast, after a period of no smoking and after the subject has lain resting for ≥15 min (the plasma half-life of tPA is approximately 5 min). Great care is required in obtaining and handling samples for the assays described later.⁶² Tests for fibrin and fibrinogen degradation products are described in Chapter 18.

Investigation of ‘Fibrinolytic Potential’

The ‘fibrinolytic potential’ is measured as the combined effect of plasminogen activators and inhibitors. The concentration of activators may be increased by venous occlusion or by the administration of desmopressin (1-deamino-8-D-arginine vasopressin). The global tests, euglobulin lysis time and fibrin plate lysis are described first followed by assays for specific components of the fibrinolytic system.

Euglobulin Clot Lysis Time

Principle

When plasma is diluted and acidified, the precipitate (euglobulin) that forms contains plasminogen activator (mostly tPA), plasminogen and fibrinogen. Most of the plasmin inhibitors are left in the solution. The precipitate is redissolved, the fibrinogen is clotted with thrombin and the time for clot lysis is measured.

Reagents

Acetic acid. 0.01%

Bovine thrombin. 10 NIH u/ml

Fresh platelet-poor plasma from the patient and control. Because tPA is very labile, blood must be collected into cooled sample tubes, placed on ice and processed immediately

Glyoxaline buffer. pH 7.4. See p. 409.

Method

Place venous blood in a plastic tube containing citrate; after mixing, keep the tube in an icebath. Centrifuge the sample as soon as possible (never later than 30 min after collection) at 4°C at 1200–1500 g. Pipette 1.0 ml of plasma into 9 ml of acetic acid. Mix well and keep on ice for 15 min. Centrifuge at 4°C for 15 min, at 1500 g, to deposit the white euglobulin precipitate. Discard the supernatant, invert the tubes, then wipe the walls with cotton wool on an applicator stick until completely dry inside. Add 0.5 ml of glyoxaline buffer and dissolve the precipitate. Place duplicate 0.3 ml volumes of the dissolved euglobulin fractions from the patient and control in glass tubes and obtain clotting by adding 0.1 ml of thrombin. Leave undisturbed at 37°C and inspect for clot lysis at 15 min intervals.

Normal range

The normal range is 90–240 min.

Interpretation

A technical problem leading to an artefactually long lysis time is the failure to maintain a low temperature throughout all the stages of the test. Furthermore, the fact that a variable amount of PAI-1 precipitates in the euglobulin fraction makes it essential to analyse a normal control on each occasion the test is performed. Exercise and prolonged venous stasis shorten the lysis times. There is also a significant diurnal variation; lysis time is longer in the morning than at noon or in the afternoon. Prolonged fibrinolysis (as found during fibrinolytic therapy) may result in plasminogen depletion and give rise to a falsely long lysis time. In DIC, a low fibrinogen concentration in the patient’s plasma gives a wispy clot, which dissolves rapidly and results in a falsely short lysis time. Conversely, high levels of fibrinogen result in a prolonged lysis time.

Long lysis times are found in the last trimester of pregnancy, in the postoperative period, after myocardial infarction, in individuals who are obese and in many cases of recurrent venous thrombosis. Very short lysis times are seen in some haematological or disseminated malignancies and in cirrhosis. A short lysis time is also seen in factor XIII deficiency.

Lysis of Fibrin Plates

Principle

Most commercially available fibrinogen preparations are contaminated with plasminogen. If a standard fibrinogen solution is poured into a Petri dish and clotted with CaCl₂ and thrombin, a solid fibrin plate is obtained. If the euglobulin fraction under test is placed on the plate, the plasminogen in the plate is converted into plasmin and a zone of lysis appears around the sample. The area of lysis is proportionate to the concentration of plasminogen activator in the euglobulin fraction.⁶³

Reagents

Bovine fibrinogen

Bovine thrombin. 50 NIH u/ml

Calcium chloride. 0.025 mol/l

Barbitone buffered saline. (see p. 409)

Platelet-poor plasma. From the patient and a control; collected as described for euglobulin lysis time.

Equipment

Equipment includes plastic Petri dishes.

Method

To prepare the fibrin plate, dilute the fibrinogen in buffered saline to obtain a final concentration of 1.5 g/l. Pipette 10 ml of diluted fibrinogen into a Petri dish. Place it on a level tray. Add 0.5 ml of CaCl₂ and 0.2 ml of thrombin solution. Mix the contents by swirling quickly. The plate clots within 10 to 20 s; it must clot evenly to be suitable for the test. Leave the plate undisturbed for 20 min. The prepared plates can then be kept for 3–4 days at 4°C.

Carefully apply 30 μl of the euglobulin fraction, prepared as described in the previous test, to the surface of the plate. There is no need to cut a well. Place in an incubator at 37°C for 24 h. This preparation time can be shortened by the addition of exogenous plasminogen.⁶³

Perform all tests (patient and control) in duplicate.

Results

Calculate the zone of lysis by measuring two diameters in mm at right angles to each other. Multiply the two values to obtain the approximate area of lysis in mm².

Normal range

The normal range is variable but is usually between 40 and 60 mm².

Interpretation

The area of lysis may be difficult to define because of incomplete lysis. Only areas of complete, clear lysis should be measured. In other respects the interpretation is as for the euglobulin clot lysis time except that the levels of plasminogen and fibrinogen in the test plasma do not affect the result. The same problems in preparing the euglobulin fraction apply as does the necessity for a normal control.

Venous Occlusion Test

Principle

Localized venous occlusion ⁶⁴ of an arm for a standardized period is used as a stimulus for release of tPA from the vessel wall. The original intention was that this would be a better measure of functional defects in fibrinolysis than a resting sample. Preocclusion and postocclusion lysis times, using the previously described euglobulin lysis or the fibrin plate lysis tests, are measured. In normal subjects fibrinolysis is greatly enhanced by occlusion. However, given the problems associated with global assays of fibrinolysis, it seems preferable to perform specific measurements of tPA before and after occlusion.

Method

Withdraw blood from the arm to be tested without stasis, place it in a citrate-containing tube and keep in an icebath. Inflate the sphygmomanometer cuff to a pressure midway between the systolic and diastolic pressure. Leave the inflated cuff on for 10 min. Take a sample of venous blood from below the cuff immediately before deflation and place on ice. Measure the lysis in both samples, as described previously. This test is uncomfortable and some patients may not be able to tolerate as much as 10 min of occlusion. Petechiae are commonly seen after the test is completed.

Results

The postocclusion lysis times should be shorter than the preocclusion times. Shortening by at least 30 min is found in most normal subjects.

Interpretation

Failure to enhance lysis is found in some cases of recurrent venous thrombosis, in people who are obese, after surgery, trauma or severe illness and in Behçet’s syndrome. It may also result from a failure to release the activator because insufficient pressure was applied or the occlusion time was too short. Normal people vary in the degree of response: ‘good’ responders increase the concentration of tPA by three-fold to four-fold, whereas ‘poor’ responders may consistently show only a very slight enhancement of fibrinolysis even with longer occlusion times. When comparing plasma levels of proteins preocclusion and postocclusion, an adjustment for changes in haematocrit may be required.⁶⁵ The effect of the venous occlusion test and the levels of tPA are very variable over time.^66,⁶⁷

Investigation of Suspected Plasminogen Defect or Deficiency

Inherited plasminogen deficiency or defect may be found in 2–3% of unexplained thromboses in young people.^68,⁶⁹ However, there is no good evidence that deficiency is associated with an increase risk of thrombosis. The only consistent clinical finding appears to be ligneous conjunctivitis.⁷⁰ The laboratory screening should be carried out using a functional assay based on full transformation of plasminogen into plasmin by activators. Such assays can be caseinolytic, fibrin substrate or chromogenic.

Chromogenic Assay for Plasminogen

Principle

In this two-step amidolytic assay, plasminogen is first complexed with excess streptokinase. In the second step, the plasmin-like activity of the streptokinase–plasminogen complex is measured by its effect on a plasmin-specific peptide (e.g. S-2251). The amount of the dye released is proportional to the amount of plasminogen available in the sample for complexing with streptokinase. The streptokinase–plasminogen complex is not significantly inhibited by the plasma plasmin inhibitors. An excess of plasminogen-free fibrinogen can be added to maximize the activity and avoid confounding by the presence of fibrin degradation products (FDPs).

Reagents and method

Details can be found in the manufacturer’s instructions.

Normal range

The normal range is usually approximately 0.75–1.60 iu/ml.

Interpretation

Plasminogen concentration is reduced in the newborn, in patients with cirrhosis, in DIC and during and after thrombolytic therapy, but the assay is less reliable in these circumstances when fibrinogen/fibrin degradation products may augment plasmin activity. A high concentration of fibrinogen can also augment plasmin activity and make the assay less reliable. Plasminogen is an acute-phase reactant and an increased concentration is found in infection, trauma, myocardial infarction and malignant disease. Hereditary plasminogen deficiency is most commonly due to a type I deficiency and so an antigenic test is usually sufficient. If suspicion remains high then a functional assay will be required.

Tissue Plasminogen Activator Amidolytic Assay

Principle

Different amidolytic assays for tPA have been described.^71,⁷² One relies on the activation of purified plasminogen to plasmin in the presence of fibrinogen fragments, which stimulate the tPA activity in the test plasma. The plasmin is measured using a specific chromogenic substrate. In the second method, tPA is captured on specific antibodies bound to a solid-phase matrix such as a microtitre plate; the various plasma inhibitors of tPA and plasmin are washed away, plasminogen is added together with a stimulator of tPA activity and the plasmin produced is measured with chromogenic substrates. Alternatively, chromogenic substrates specific for tPA may be used, but there are specificity problems, especially in the plasma assays.

Interpretation

tPA is secreted into plasma in its active form but rapidly complexes with its principal inhibitor PAI-1. The amount of active tPA in the plasma is a result of this equilibrium and represents only a small fraction of the total (antigenic) tPA. This process continues after blood sampling unless blood is taken into an appropriate acidic anticoagulant (see above).

tPA can also be measured by ELISA using monoclonal antibodies on microtitre plates, although concentration closely parallels the PAI-1 concentration and says little about the proportion of free, active tPA.

Plasminogen Activator Inhibitor Activity Assay

Principle

Plasma anti-plasminogen activator activity is almost entirely due to PAI-1. A fixed amount of tPA is added in excess to undiluted plasma and part of it rapidly complexes with PAI-1. Plasminogen in plasma is then activated into plasmin by the residual, uncomplexed tPA. The amount of plasmin formed is directly proportional to the residual tPA activity and inversely proportional to the PAI activity of the sample. The amount of plasmin generated is measured using a plasmin-specific substrate.

Reagents are available in kit form and the manufacturer’s instructions must be closely followed. The normal range, in particular the lower limit, is not clearly defined and many normal subjects have levels below the assay’s lower limit of detection.^73,⁷⁴ Each laboratory should establish its own range until reliable normal values become available.

The time of sampling must be standardized. Early morning (7 a.m.) samples have much greater levels of activity than those done later in the day. Rapid sample processing is extremely important because PAI leaks from platelets in sampled blood and PAI-1 in plasma rapidly converts to a latent (inactive) form. An ELISA assay is also available to measure the total PAI-1 present.

Plasminogen Activator Inhibitor Antigen Assay

Principle

Microplate wells coated with an anti-PAI-1 monoclonal antibody are incubated with samples and standards. PAI-1 present in the samples and standards is bound to the solid phase during this incubation. Unbound substances are then removed by washing. An enzyme-labelled anti-PAI-1 monoclonal antibody (conjugate) is added. The conjugate binds to the antibody–antigen complexes formed in the previous incubation. Unbound conjugate is then removed by washing. Finally, enzyme substrate is added. The action of the bound enzyme on the substrate produces a blue colour, which turns yellow after stopping the reaction with acid. The absorbances are read in a microplate reader at 450 nm. The amount of colour is proportional to the concentration of PAI-1. The assay is specific for total PAI-1, including both free and complexed forms.

Specimen Collection

Blood should be collected into CTAD tubes or Diatube H (Stago) and immediately cooled on ice; CTAD is a buffered tri-sodium citrate solution with theophylline, adenosine and dipyridamole. Vacutainer coagulation tubes with CTAD (Becton-Dickinson) samples can be stored on ice for up to 7 h in the collection tubes. If the sample is not tested immediately, it should be separated and frozen as soon as possible.

Reagents and Method

This is available in a kit based on an ELISA (Chromogenix).

Normal Range

The normal range is usually 11–69 ng/ml. However, each laboratory should establish its own normal range.

Plasmin Inhibitor (α₂ Antiplasmin) Amidolytic Assay

Principle

Plasma dilutions are incubated with excess plasmin, a proportion of which are inhibited by antiplasmins. The residual, uninhibited plasmin is measured using a specific chromogenic substrate. Plasmin inhibitor is the major circulating inhibitor of plasmin and forms complexes much faster than other inhibitors; if the reaction times are short, the assay effectively measures plasmin inhibitor only.

Different commercial kits are available containing all the necessary reagents. The manufacturer’s instructions should be carefully followed. Care is required when aliquoting the plasmin solution, which has a high viscosity because of its glycerol content. Note that plasmin bound to α₂ macroglobulin may escape inhibition, thus under-estimating inhibitor activity.

The usual normal range is between 0.80 and 1.20 iu/ml. Congenital plasmin inhibitor deficiency is associated with a severe bleeding tendency. A reduced concentration is also found in liver disease, in DIC and during thrombolytic therapy. Plasmin inhibitor increases with age and is higher in Caucasians than in Africans.

Platelet ‘hyperreactivity’ and activation

Platelets may be more reactive than normal as a consequence of in vivo activation by thrombin or non-endothelial surfaces, such as prosthetic valves or Dacron grafts. This can sometimes be detected by a lowered threshold (increased sensitivity) for aggregating agents. Because there is considerable variation in response to aggregating agents in normal people, the attempts to show platelet hyperaggregability are rarely successful and the results are frequently inconsistent. Spontaneous aggregation of platelets in the blood can also be demonstrated.⁷⁵

Platelets that have formed a part of a platelet thrombus and have been released into the circulation may show a measurable decrease in their ability to aggregate because of a loss of some granular content. The released contents can be measured in plasma; the α-granule proteins, β-thromboglobulin and platelet factor 4 are the constituents most commonly measured. Overall the problems with these tests make them of doubtful utility;⁵⁹ they are described in previous editions of this book.

Several genetic polymorphisms have been reported to affect the reactivity of platelet glycoproteins and P selectin. Although they may be important in population studies, their clinical significance for individual patients remains unclear.^76,⁷⁷

Platelet Activation: Flow Cytometry

The problems associated with previous tests of platelet activation have been circumvented to some extent by the application of flow cytometric analysis of platelets in whole-blood samples.

Principle

The activation of platelets is associated with the appearance of new antigenic determinants on the platelet surface. Some of these are molecules present in platelet granules brought to the surface during degranulation (e.g. CD62P, CD63, LAMP-1 and CD40L) and others are new conformations of existing molecules (e.g. the ligand-induced binding site on GpIIbIIIa). These can be detected using fluorescein-conjugated antibodies and the degree of expression can be quantified by flow cytometry. This gives a measure of platelet activation with a much greater degree of sensitivity than platelet factor 4 or β-thromboglobulin estimation and may still be successful in the presence of thrombocytopenia. Samples may need to be collected into inhibitors of platelet activation such as PGE₁. Numerous alternative surface molecules are available (Table 19.2). These tests have not yet entered routine laboratory practice but are proving increasingly useful in research.⁷⁸ An alternative approach is offered by the PFA-100 (see p. 425) in which short closure times may be indicative of platelet hyperreactivity and/or hyperreactive von Willebrand factor species.

Table 19.2 Indicators of platelet activation detectable by flow cytometry

Name	CD Designation	Comment
GpIb, IX, V	CD42	Decreases
GpIIbIIIa	CD41	Increases
Phosphatidyl serine	–	Increases Detected by Annexin V binding
Lysosomal Integral membrane protein (gp53, granulophysin)	CD63	Indicates lysosomal degranulation
P selectin	CD62P	Indicates α granule release, subsequently cleaved and measurable in plasma
Fibrinogen	–	Surface-bound fibrinogen increases
IIbIIIa activation	–	Conformation change in IIbIIIa produced by activation, detected by PAC-1 antibody

Homocysteine

Following the observation that patients with homocystinuria have venous and arterial thromboses with accelerated vascular damage, there has been considerable interest in patients with less marked elevation of plasma homocysteine (hyperhomocysteinaemia). This has been shown to have an association with arterial and venous thrombosis but the assay has little clinical utility and dietary interventions have been ineffective.^79,⁸⁰ Until recently, homocysteine has been measured by high-performance liquid chromatography or mass spectroscopy, but an ELISA-based assay is now available that allows it to fit more easily into coagulation laboratory practice. To standardize study results, homocysteine is measured either while fasting or after a methionine load. Rapid processing of samples is required because homocysteine quickly leaches out of red blood cells.

Markers of coagulation activation

Numerous commercial kits are available for measuring molecules produced by coagulation activation.

Principle

The activation of many proteins active in coagulation is mediated by proteolytic cleavage with the release of small peptides: activation peptides. The most frequently measured of these is prothrombin fragment 1 + 2, which is released when prothrombin is converted to thrombin. It has an appreciable half-life of approximately 45 min, which allows a measurable concentration to accumulate in plasma and provides an indication of the rate at which thrombin is being generated.

An alternative is to measure the concentration of thrombin–antithrombin complexes (TAT), which provides similar information. Plasmin–antiplasmin complexes provide corresponding information about fibrinolysis. These can all be measured using commercially available ELISA kits but are not used routinely and are not required for normal diagnostic work.⁸¹ Other tests such as fibrinopeptide A require exceptional care and the use of special anticoagulants to prevent in vitro activation of the sample.

A plasmin cleavage product of crosslinked fibrin, D-dimer (see p. 441) is another measure of activity in the coagulation system. Several studies have shown that elevated levels of D-dimer are an indicator of future risk of thrombosis. It is not yet certain whether they have on their own, or in conjunction with other factors, sufficient predictive value to alter management but they are currently being incorporated into management protocols. The test is usually performed after oral anticoagulants have been discontinued.