A 9-year retrospective assessment of laboratory testing for activated protein C resistance: evolution of a novel approach to thrombophilia investigations.
OBJECTIVE
To assess international and local trends in laboratory testing for activated protein C (APC) resistance (APCR) and factor V Leiden (FVL). Also, to compare local results of FVL testing with a variety of different clot-based APCR assays to assess utility for detection of APCR both related and unrelated to FVL.


METHODS
Local test statistics and test result patterns were evaluated and international literature was reviewed over the past 9 years. Direct comparisons of FVL testing by DNA analysis against (a) the standard APTT-based APCR assay, with and without pre-dilution with factor V deficient (FVD) plasma, or with and without normalisation, and (b) three alternative RVVT-based procedures (most recently using a commercial RVVT-based procedure called GradiLeiden V; GLV). In total, data obtained over the past 7 years, using referred samples from over 1,000 patients, have been assessed.


RESULTS
The 9-year retrospective assessment has seen many changes in test-based processes. Locally, test requests for both APCR and FVL have consistently increased. We suspect this has been fuelled in part by media reports of 'economy class syndrome' (ECS) and associated general public and clinical concern. Current request patterns number around 800 APCR and 1,600 FVL per year. Interestingly, most requests are for one or either test, with joint requests comprising less than 20% of those overall. Although test requests are increasing, detection of the FVL defect as a proportion of test requests is actually falling (from a high of over 25% in 1996 to around 14% currently). Whether this suggests an increasing tendency for clinical ordering in the absence of appropriate clinical histories is a matter of concern. Consistent with previous findings, the original and commonly used APTT-based procedure was found to show the least correlation with DNA findings, with a large overlap between FVL and non-FVL individuals. The alternate-RVVT-based procedures showed much better differentiation. Thus, for the APTT-based method, in order to ensure 100% sensitivity, an APC ratio cut-off value of 3.1 was required, and this yielded only 49.1% specificity. In contrast, for the GLV RVVT-based method, in order to ensure 100% sensitivity, an APC ratio cut-off value of 1.65 was required, and this yielded 96.6% specificity.


CONCLUSIONS
It is important to recognise the limitation of APTT-based assays to discriminate FVL. However, a combination of RVVT- and APTT-based testing is still recommended, as this will provide excellent discriminatory power for the FVL defect, particularly negative prediction, in addition to detection of potential APCR unrelated to FVL, as well as detection of other potential haemostatic disturbances. Accordingly, we detail strategies, including a test algorithm that we are currently using to improve our detection of APCR and prediction of FVL, and use of clotting-based procedures as the first-line approach.
Favaloro
Emmanuel J
EJ
Department of Haematology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Western Sydney Area Health Service, NSW, Australia. emmanuel@icpmr.wsahs.nsw.gov.au
Orsag
Ina
I
Bukuya
Melinda
M
McDonald
David
D
D020016:Activated Protein C Resistance / Q000175:diagnosis*; D004247:DNA / Q000032:analysis; D005165:Factor V / Q000032:analysis / Q000235:genetics; D006801:Humans; D013677:Medical Laboratory Science / Q000592:standards* / Q000639:trends*; D010314:Partial Thromboplastin Time; D011517:Prothrombin Time; D012189:Retrospective Studies; D019851:Thrombophilia / Q000175:diagnosis*
C095381:factor V Leiden; D005165:Factor V; D004247:DNA