NEW DEVELOPMENTS IN NEAR PATIENT (POC) TESTING

Dr Ian Mackie, Haemostasis Research Unit, Haematology Dept., University College London, 3^rd Floor, Jules Thorn Bldg., 48 Riding House St., London W1W 7EY

Point of care (POC) testing is used variably amongst different NHS Trusts and their Departments. The main clinical uses are in the areas of: oral anticoagulant control (OAC), coagulopathy screening (ie. FFP requirement), heparin management (mainly cardiac theatre & ITU), assessment of transfusion triggers during major surgery, and platelet function testing. In theory, all of this should be under the control of Trust POC committees and covered by the rules of CPA Accreditation (Haematology departments are responsible for POC haematology devices used within their Trust). This presentation will focus on developments in POC for OAC and the use of thrombelastography.

There are 3 main semi-automated instruments available in the UK for OAC: the Thrombotrak, KC1 and Thrombi-Stat. These instruments are reliable and reasonably portable, but require some technical skills to pipette the blood sample and reagents into the reaction cups. The growth area of POC for OAC is therefore with small devices having no requirement for pipetting due to the use of closed reagent strips or cuvettes. The devices currently available have different methods of end-point detection: CoaguCheck S (ferromagnetic particles and light reflectance), Protime 3 (capillary blood movement and optical detection of cessation of flow), INRatio (electrochemical sensor) and Hemochron Junior Signature (capillary blood movement and optical detection of blood flow). These devices require a small volume of whole blood and are normally used with fingerprick (capillary) samples. The Rapidpoint Coag Monitor and the Avosure PT devices are not currently available in the UK, while the CoaguCheck XS (Roche), SmartCheck (UniPath) and Lifescan (Johnson & Johnson) devices may appear in the near future. There has been considerable improvement in the technology of POC devices, but some problems still exist, such as how to correct the PT of non-anticoagulated whole blood into the equivalent citrated plasma INR value. It is difficult for manufacturers to accurately assign ISI values to test strips and errors may be introduced by using house reference materials in between the test strip and the International Reference Preparations. However, some devices now show consistently good performance against traditional PT methods. The biggest area of concern centres on internal QC and external QA. Commercial IQC plasma samples tend to have wide target ranges that greatly limit their utility. Some devices rely on built in IQC and it is not possible to use QC plasmas, meaning that independent QC is not possible and the IQC system may suffer from any inherent flaws in the device. EQA is essential to ensure that results are accurate and in agreement with those from other patients and healthcare centres; sometimes this can only be provided by periodic parallel testing with venous samples or against a clinic POC device that is itself controlled in an EQA scheme.  A further problem is that while some test strips are available on the drug tariff and may therefore be prescribed, QC and EQA samples are not freely available in this way, so that patients are effectively discouraged from ensuring the validity of their INR results.

The MHRA evaluates coagulation devices and produces informative reports, which are now freely available through the internet (www.mhra.gov.uk). One way forward for the future of OAC appears to be patient self-testing (PST) or self management (PSM), to limit the size of hospital clinics, reduce transport costs and improve the quality of life for patients. The MHRA has evaluated the CoaguCheck S in both PST and PSM settings. PSM and PST were both successful and implemented without problems, giving an overall time within target therapeutic range of 70.0% compared to 62.5% in the previous 6 months by traditional clinic care (p=0.03). However, PST and PSM are not always desired by patients and only 13% of the clinic patients at UCLH enrolled in the trial. Whether this was due to anxiety about signing informed consent forms or concern about performing a self-test is unclear. A further extension of PST is Telemedicine and we have recently been involved in a feasibility study where patients self-tested and INR results were automatically transferred from their POC device, via the internet, to the anticoagulant nurse, who could then advise on the date of next test and warfarin dose. After initial teething troubles with hardware configuration and server performance, the Telemedicine system worked well, with data being consistently, securely and accurately transferred. This system also transferred QC data, so that performance and compliance could be monitored by the hospital clinic.

The final area of development is in thrombelastography using the TEG and ROTEM devices. There has been considerable improvement in the technology of these devices, the software used and the reagent systems. Present applications include: cardiac surgery, liver transplant surgery, assessment of hypercoagulability and evaluation of haemostatic agents and inhibitors. The impact of platelet function and fibrinogen can be differentiated on the devices using platelet agonists or inhibitors. Anaesthetists at the Royal free Hospital have been assessing the use of the TEG and ROTEM to indicate transfusion requirements during liver transplant surgery. Thrombelastographic methods showed good agreement with each other as triggers for platelet concentrate transfusion, but weak agreement with platelet count. There was weak agreement with PT as a trigger for FFP infusion, but good agreement with Clauss fibrinogen assay for cryoprecipitate infusion. In the absence of a randomised clinical trial, it is unclear which trigger algorithm has the best clinical application. The current thrombelastographic triggers would lead to at least twice as many units of platelets being infused compared to platelet count assessment, but perhaps they are required clinically, since the platelet count is not a measure of platelet function. The use of TEG and ROTEM devices appears to be growing, since they not only provide rapid results close to the patient, but also give dynamic information about the rate and strength of clot formation in a whole blood milieu. In particular, they may be expected to give useful data on some aspects of the contribution of platelet function and fibrin tensile strength to the forming clot, which cannot be assessed by traditional methods.

ANTIPHOSPHOLIPID ANTIBODIES:  WHICH TESTS ARE CLINICALLY RELEVANT?

Professor Mike Greaves, Department of Medicine and Therapeutics, University of Aberdeen

Antiphospholipid syndrome is an important condition because the major clinical manifestations are life or limb threatening thrombosis and pregnancy failure.  Diagnosis depends upon the demonstration of persistently positive tests for antiphospholipid antibody, typically lupus anticoagulant and/or anticardiolipin. However these supporting laboratory investigations for the confident diagnosis of the syndrome suffer from serious limitations.  These include suboptimal standardisation and reproducibility, and lack of specificity.

Whilst diagnosis of the fully developed syndrome may be straightforward, for example when there is a typical clinical history in combination with persistently abnormal coagulation tests which have the characteristics of lupus inhibitor, or anticardiolipin antibodies in moderate or high titre, diagnostic uncertainty is common in other cases.  This is principally because of the high prevalence of alternative causes of thrombosis in the population and the frequency of low titre antiphospholipid antibodies of unknown significance, especially in older subjects.

From the accumulated published evidence it can be reasonably concluded that:

Lupus anticoagulant is more specific than anticardiolipin antibody

IgG anticardiolipin is more specific than IgM anticardiolipin

Higher titre anticardiolipin antibodies are more specific than low titre antibodies

Numerous avenues have been explored to improve the approach to laboratory diagnosis.  The standardisation of anticardiolipin assays remains problematic^1,2, although better standardisation of lupus anticoagulant tests and adherence to evidence-based guidelines may have had some impact ³.  The introduction of alternative tests for supposedly more specific antiphospholipid antibodies, especially anti-beta2 glycoprotein I, offered hope but in practice this has not been realised to any great degree ⁴.  The recent description of a lupus anticoagulant assay which discriminates beta2 glycoprotein I-dependent antibodies from other lupus inhibitors is of potential importance as it would appear that it is those antibodies which are most closely linked to thrombosis ⁵.

There is much still to learn about antiphospholipid antibodies.  For example, although recurrent thrombosis is a major feature of the classical syndrome, there is some evidence that even non-persistent antibodies may predict future first thrombotic events in some populations ^6,7.  There is also debate over the best approach to treatment in various clinical situations ^8,9.  Large clinical studies with close attention to laboratory diagnostic parameters will be necessary to address these issues.

References:

1 Reber et al,  Thrombos Haemostas 1995 73:444

2 Favoloro & Silvestrini, Am J Clin Pathol 2002 118:548

3 Jennings et al, BJH 2002 119:364

4 Reber et al, Thrombos Haemostas 2002 88:66

5 de Laat et al, Blood 2004 104:3598

6 Ginsburg et al, Arch Int Med 1992 117:997

7 Vaarala et al, Circulation 1995 91:23

8 Farquharson et al, Obstet Gynecol 2002 100:408

9 Levine et al, JAMA 2004 291 :576

LUPUS ANTICOAGULANT TESTING:  ANY NEARER STANDARDISATION?

Dr I Jennings, UK NEQAS for Blood Coagulation, Sheffield. UK.

Unlike many other tests included in a thrombophilia screening profile, there is consensus over the importance of screening for lupus anticoagulant, as risks of recurrence are sufficiently high to influence clinical judgement on duration, and possibly even intensity, of treatment (1,2).

However, there remains no correlation between the titre or potency of antiphospholipid antibodies, and the level of thrombotic risk.  It is therefore equally important to identify the presence of “weak” as well as “strong” lupus anticoagulants.  Successive UK NEQAS for Blood Coagulation exercises have identified poor accuracy when samples from subjects with weak LA or factor deficiencies have been distributed (3), and similar findings have also been reported by others (4).

Improvement in discrimination between weak LA positive and LA negative plasmas may be achieved through standardization of the many methods and algorithms in use for LA screening, and availability of reliable, well-characterised reference plasmas may be of value in this respect.  However, sub-optimal results have been observed with a reference plasmas derived from patient and normal plasmas (5,6).  Further investigation of a reference plasma panel revealed a pH-dependent sensitivity of some DRVVT methods. 

The use of plasma spiked with monoclonal antibodies or purified IgG as an alternative source of control plasma has been described (7,8), and the former approach was also investigated in a UK NEQAS supplementary exercise.  Although the majority of centres reported the correct interpretation for all samples distributed in this exercise, variation between methods and reagents was observed (9).  At identical antibody concentrations, strength of response was greater with anti-prothrombin than anti-β2GP1 antibodies, and responsiveness of APTT reagents showed greatest agreement between patient and anti-prothrombin samples.  However, a high proportion of negative interpretations were reported with one DRVVT kit with a sample spiked with anti-prothrombin antibodies.

Measurement of the phospholipid content of DRVVT reagents indicated significant correlation (r>0.85, P<0.03) between normalized test/confirm ratios and the phospholipid concentration in confirm reagents for 2 out of 3 monoclonal antibody-spiked samples and a LA positive patient sample, with higher ratios obtained using reagents with higher phospholipid concentrations.  However, the pattern of results obtained with different sources and concentrations of platelet-based confirmatory reagents indicate an unpredictable response to plasma spiked with monoclonal antibodies.

Artificially prepared reference plasmas will be a useful tool in improving standardisation between LA screening methods; it is important however to recognize that these plasmas may not always behave in the same way as plasma from all patients with LA, and standardization may be best achieved through careful adherence to published guidelines on LA testing (10,11) together with use of reference plasma panels prepared from a mixture of sources and potencies.

References: 

1.    Meroni PL, Moia M, Derksen RH, Tincani A, McIntyre JA, Arnout JM, Koike T, Piette JC, Khamashta MA, Shoenfeld Y (2003).  Venous thromboembolism in the antiphospholipid syndrome: management guidelines for secondary prophylaxis.  Lupus; 12: 504-7.

2.    Bauer KA (2003).  Management of thrombophilia.  J Thromb Haemost; 1: 1429-34.

3.    Jennings I, Kitchen S, Woods TAL, Preston FE, Greaves M. (1997) Clinically important inaccuracies in testing for the lupus anticoagulant: An analysis of results from three surveys of the UK NEQAS for Blood Coagulation.  Thrombosis & Haemostasis, 77 (5), 934-7.

4.    Roussi J, Roisin JP, Goguel A (1996).  Lupus anticoagulants.  First French Interlaboratory Etalonorme Survey.  Am. J. Clin. Pathol; 105: 788-793.

5.    Mackie IJ, Gardiner C, Machin SJ, Greaves M, Malia R, Jones DW, Winter M, Taberner D, Leeming D.  (1998)  Medical Devices Agency evaluation of lupus anticoagulant kits.  Report MDA/98/43.  Medical Devices Agency.  London: HMSO

6.    Jennings I.  pH and buffering – effects on DRVVT results.  Presentation to Lupus subcommittee, ISTH SSC meeting, Venice 2004.

7.    Arnout J, Meijer P, Vermylen J. (1999)  Lupus anticoagulant testing in Europe: an analysis of results from the first European Concerted Action on Thrombophilia (ECAT) survey using plasmas spiked with monoclonal antibodies against human β2-glycoprotein 1.  Thrombosis & Haemostasis, 81, 929-34.

8.    Tripodi A, Biasiolo A, Chantarangkul V, Pengo V (2003).  Lupus anticoagulant (LA) testing: performance of clinical laboratories assessed by a national survey using lyophilized affinity-purified immunoglobulin with LA activity.  Clin Chem; 49: 1608-14.

9.    Jennings I, Mackie I, Arnout J, Preston FE (2004).  Lupus anticoagulant testing using plasma spiked with monoclonal antibodies: performance in the UK NEQAS proficiency testing programme. J Thromb Haemost. 2004 Dec;2(12):2178-84.

10.  Greaves M, Cohen H, Machin SJ, Mackie I (2000).  Guidelines on the investigation and management of the antiphospholipid syndrome. Br J Haematol; 109: 704-15.

11.  Jennings I, Kitchen S, Woods TAL, Greaves M, Preston FE. (2002) Lupus anticoagulant (LA) screening; Impact of national testing guidelines and reference plasmas on diagnostic accuracy.  Br J Haematol, 117, 84.

REGULATION OF BLOOD COAGULATION BY THE PROTEIN C ANTICOAGULANT PATHWAY, NOVEL INSIGHTS INTO STRUCTURE FUNCTION RELATIONSHIPS AND MOLECULAR RECOGNITION

Björn Dahlbäck, Department of Laboratory Medicine, Clinical Chemistry, Lund University, The Wallenberg laboratory, University Hospital, Malmö, SE-205 02 Malmö, Sweden

The protein C system provides important control of blood coagulation by regulating the activities of factor VIIIa (FVIIIa) and factor Va (FVa), cofactors in the activation of factor X and prothrombin, respectively.  The system comprises membrane-bound and circulating proteins that assemble into multi-molecular complexes on cell surfaces. Vitamin K-dependent protein C, the key component of the system, circulates in blood as zymogen to an anticoagulant serine protease.  It is efficiently activated on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin.  The endothelial protein C receptor (EPCR) further stimulates the protein C activation.  Activated protein C (APC) together with its cofactor protein S inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged phospholipid membranes.  Efficient FVIIIa degradation by APC requires not only protein S but also intact FV, which like thrombin is a Janus-faced protein with both pro- and anticoagulant potential. In addition to its anticoagulant properties, APC has anti-inflammatory and anti-apoptotic functions, which are exerted when APC binds to EPCR and proteolytic cleaves protease activated receptor 1 (PAR-1).  The protein C system is physiologically important and genetic defects affecting the system are the most common risk factors of venous thrombosis.  The proteins of the protein C system are composed of multiple domains and the three-dimensional structures of several of the proteins are known.  The molecular recognition of the protein C system is progressively being unraveled, giving us new insights into this fascinating and intricate molecular scenario at the atomic level.  These new insights are the focus of this presentation.

GLOBAL vs. SPECIFIC COAGULATION ASSAYS – THE WHEEL TURNS FULL CIRCLE

Dr T W Barrowcliffe, NIBSC, Potters Bar, UK

In the early days of therapy of bleeding disorders, global tests such as whole blood clotting time and thrombin generation were used as a guide to diagnosis and treatment.  However, these were largely abandoned when assays for specific clotting factors such as FVIII and FIX were developed.  More recently, with the development of knowledge about the complex interactions of clotting factors it has become recognised that a patient’s haemostatic state may not be solely dependent on the level of the particular deficient factor, and some of the old global tests, particularly the thrombin generation test, have enjoyed a resurgence of interest.

One aspect of the thrombin generation test which has boosted its renaissance is the development of improved methodology, allowing increased precision and automation.  However, unlike the specific assays, where a well developed system of standardisation has developed over the last 20 years, standardisation of this test is only at an early stage, and reproducibility between laboratories is poor. Recent studies in our laboratory have investigated various technical modifications to the assay in the context of diagnosis and treatment of haemophilia.  A sensitive method has been developed which can detect FVIII and FIX activity down to 0.1% of normal.  It has also been found that the thrombin generation test in haemophilic plasma can be normalised by the addition of concentrates to as little as 0.3 IU/mL, suggesting that investigation of lower therapeutic doses of concentrates may be warranted.

HAEMATOLOGY IN THE UK – PAST, PRESENT AND FUTURE

 Sir John Lilleyman, Medical Director, National Patient Safety Agency, 4-8 Maple Street, London W1T 5HD

There are two stories of haematology, the first is the unravelling of the mysteries of blood and its diseases which probably began when Hippocrates was a lad, and the second is the evolution of the clinical specialty in the UK, which began in 1948.  I aim to tell the second story, with only brief reference to the first.

The National Health Service allowed laboratories in hospitals to differentiate luxuriously into the 4 classical disciplines, and each supported a consultant in charge. Haematologists mostly evolved from clinical pathologists, but there was a fair number in teaching hospitals who were academics or ‘physicians with an interest’.  Developing the clinical specialty was spurred by its recognition by the new RCPath in 1964 and by the RCP in 1968, and the MCPath was the gateway.

Gradually the shift for consultants has been out of the routine laboratory to clinics and research laboratories, though more so (as ever was) in teaching hospitals.  Technological developments in therapy for previously untreatable diseases have made pressures grow for larger units for tertiary care.  Routine laboratory tests are becoming more sophisticated and portable, moving in the opposite direction from secondary to primary care.

My guess is that the haematologists of the mid 21^st century will be aggregated into larger but fewer groups offering mostly on line or outreach support to secondary care and on-site care chiefly in specialised units splintering into subspecialities such as stem cell therapies, haemostasis, malignant disease and gene therapy. But wherever diagnostic laboratory work is carried out in future, quality assurance will become no less important. Long live NEQAS.