David McDonald

Laboratory investigation of thrombophilia: the good, the bad, and the ugly.

Thrombophilia can be broadly defined as an increased tendency toward hypercoagulability and venous thrombosis. There are several defined risk factors for thrombosis, and these are generally distinguished as either acquired or congenital, although sometimes this distinction is blurred because of interrelationships. Congenital risk factors include deficiencies or defects in natural anticoagulants, such as antithrombin, Protein C and Protein S, and genetic polymorphisms such as prothrombin G20210A and cleavage-resistant forms of factor V (in particular factor V Leiden), that lead to a condition commonly known as activated protein C resistance. Acquired risk factors include antiphospholipid antibodies, detected as lupus anticoagulants and/or anticardiolipin antibodies and/or anti-beta-2-glycoprotein-I antibodies. High levels of clotting factors, dysfibrinogenemia, hyperhomocysteinemia, prolonged immobilization, increasing age, surgery, trauma, cancer, obesity, poor nutrition, pregnancy, oral contraceptives, and hormone replacement therapy comprise just some of the other risk factors. Each of these elements constitutes a component of increased risk, which is compounded when concomitant. There is ongoing debate regarding relative and compound risks, the value of laboratory screening, whom and when to screen for these markers, which tests and methodologies to use, and the form and duration of therapeutic management. The current article explores several important issues primarily from a scientific perspective and predominantly related to laboratory testing. Many of these issues appear to be simply overlooked by some clinicians managing patients with thromboses. In brief, although there is potential significance in testing for various thrombophilia-associated markers, this value is limited and greatly diminishes when inappropriately applied. The application of excessive or inappropriate thrombophilia testing is of particular concern, and the net effect of current worldwide testing trends is likely to be more detrimental than beneficial. In short, it is likely that current generalized testing is simply doing more harm than good, and thus that ordering practice requires scrutiny.

External Quality Assurance of DNA Testing for Thrombophilia Mutations

Because of the potential implications of results of genetic analyses of thrombophilic mutations, laboratories must undertake stringent internal quality control measures and participate in external quality assurance (QA) programs. A small number of external QA surveys of thrombophilic defects have been conducted across a large number of molecular laboratories and generally have indicated favorable levels of correct responses. The Royal College of Pathologists of Australasia QA program has conducted external QA testing of factor V Leiden G1691 A, prothrombin G20210A, and MTHFR C677T gene mutations for the past 5 years, including 133 DNA samples in 10 multilaboratory surveys. Of 3,799 responses, the overall success rate was 98.63%; the poorest individual sample result was 15% incorrect for a homozygous factor V Leiden sample. Success rates in identifying specific mutations were 98.13% for factor V Leiden, 98.84% for prothrombin G20210A, and 99.3% for the MTHFR C677T mutation. Among responding laboratories, 51% (20/39) made at least 1 error; 3 of 39 laboratories were responsible for 46% of all errors (24/52). Although encouraging, these data underscore the need for ongoing participation of molecular diagnostic laboratories in external QA programs to ensure the provision of quality genetic testing services.

A 9-year retrospective assessment of laboratory testing for activated protein C resistance: evolution of a novel approach to thrombophilia investigations

Aims: 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 1000 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 1600 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‐RVVTbased 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.Abbreviations: APC, activated protein C; APCR, activated protein C resistance; APTT, activated partial thromboplastin time (test); FV, factor V; FVIII, factor VIII; FVD, factor V deficient (plasma pre‐dilution); FVL, factor V Leiden (mutation); GLV, GradiLeiden V (RVVT‐based APCR; Gradipore Ltd, Australia); PCA, protein C activator; PCI, protein C impedance (RVVT‐based APCR; Gradipore Ltd); PCP, protein C pathway (RVVT‐based APCR; Gradipore Ltd); RVVT, Russell Viper venom time (test).

Histiocytic necrotizing lymphadenitis (Kikuchi's disease): cytologic diagnosis by fine-needle biopsy.

A cytologic diagnosis of histiocytic nerotizing lymphadenitis (Kikuchis lymphadenitis) was made in a 14-yr-old female with cervical lymphadenopathy, Fever, neutropenia, and hepatosplenomegaly. A predominance of reticulum cells, foamy macrophages, and karyorrhectic debirs are cluse to the diagnosis in the fine-needle biopsy smears. Subsequent histology confirmed the diagnosis of Kikuchis lymphadenitis. The differential diagnoses are discussed including malignant lymphoma, which was excluded by morphology as well as flow cytometry and polymerase chain reaction (PCR) studies.

External quality assurance of molecular analysis of haemochromatosis gene mutations

Background: The Royal College of Pathologists of Australasia Quality Assurance Programs has conducted an external quality assurance programme for the testing of the haemochromatosis gene (HFE) mutations C282Y and H63D. Methods: A total of 10 surveys have been undertaken over a period of 6 years from 2000 to 2005. Results: Of the 3016 responses received, the overall success rate was found to be 99.47% (3000/3016). A total of 16 errors were found, 6 for C282Y and 10 for H63D. Only one sample was associated with more than one error, in which 2 of 23 respondents classified a normal sample as heterozygotic for H63D. Overall performance was observed to vary minimally between surveys, from a low of 91.3% correct (21/23 responses) for a normal sample to 100% correct in most (85/100) samples. Of the 10 complete surveys, four returned a 0% error rate. In one survey in 2004, seven incorrect responses were returned by one laboratory, all of which were secondary to transcriptional errors. Overall success rates per assay were 99.61% (1532/1538) for C282Y and 99.32% (1468/1478) for H63D. Over a period of 6 years from 2000 to 2005, the proportion of respondents using polymerase chain reaction (PCR) and restriction enzyme analysis fell from 85% to around 30%, whereas the proportion of laboratories using real-time PCR rose from 5% to around 55%, as indicated by the questionnaire surveys of methods used by participants. Discussion: Encouraging levels of testing proficiency for two common genetic mutations are indicated by these data, but they also confirm the need for participation of molecular diagnostic laboratories in external quality assurance programmes to ensure the ongoing provision of high-quality genetic testing services.

A clinical audit of congenital thrombophilia investigation in tertiary practice

Background: The presumed cause of congenital thrombophilia can now be explained in ∼50% of familial thrombosis cases following evaluation of a range of markers, primarily comprising factor V Leiden (FVL), activated protein C resistance (APCR), protein C (PC), protein S (PS) and antithrombin (AT). However, the effectiveness of such evaluations is largely determined by limiting improper investigations, either in inappropriate patients or at unsuitable timepoints. Aim: To evaluate clinical ordering patterns for a range of thrombophilia associated tests at a tertiary level public facility. Methods: Several independent audits into clinical requests for FVL, APCR, PC, PS, and AT testing were performed at our institution. Results: We identified a wide variety of clinical ordering background, although most requests related to ‘thrombosis’ or ‘obstetric’ indications. For FVL, the detection rate of heterozygotes continues to decline and is currently ∼10% of investigations. For APCR, review of clinical requests and clinical notes indicated that around 36% of investigations occurred whilst patients were on anticoagulant therapy. For PC, PS and AT investigations, additional testing of samples that yielded low test results for PC, PS and/or AT indicated that an alarming 80% of these cases likely derived from patients on anticoagulant therapy. Conclusion: These results continue to reflect on poor patient or timing selection for congenital thrombophilia investigations that compromises the utility of these tests. In total, this would yield a very high rate of false positive identification for disorders that patients do not have, raising the question: are broadly based congenital thrombophilia investigations doing more harm than good?

Chronic gingivitis in a new BTK mutation

Abstract:u2002 A 5‐yr‐old Caucasian boy with a new mutation in Brutons tyrosine kinase (BTK) is described. Full sequencing of the BTK gene revealed a point mutation in exon 17 resulting in an amino acid change from tryptophan to serine at location 581 of the tyrosine kinase domain. Clinically the child presented with chronic gingivitis and had no prior history of bacterial infections. Whereas serum immunoglobulin M (IgM) levels were undetectable, IgG levels were in the low normal range. The gingivitis completely resolved after intravenous immunoglobulin therapy. Lymphocyte phenotyping revealed 0.05% B cells in his peripheral blood, which were IgG−, IgM+, IgD+, CD38+, CD20+, CD27−. However, 40% of the B cells also expressed CD5. This subpopulation of B cells has not previously been described in X‐linked agammaglobulinaemia (XLA) patients. We suggest that the occurrence of CD5+ B cells could correlate with a late onset and mild clinical presentations of XLA.

The diagnostic dilemma: dual presentations of clinical mucosal bleeding and venous thrombosis associated with the presence of thrombophilia markers and mild reduction in von Willebrand factor.

A prothrombotic and hemorrhagic state can separately manifest in one patient and can potentially cause several diagnostic problems. We report an intriguing case as an example of a potential hemostasis-based diagnostic dilemma. A 29-year-old female patient presented with a personal history of menorrhagia and other mucosal bleeding and renal ovarian thrombosis. Previous investigations had uncovered several diagnostic anomalies, including von Willebrand disease (VWD), factor V Leiden (FVL), antiphospholipid syndrome, and thrombocytopaenia. Previous therapy in this patient included heparin and warfarin for the thrombosis and desmopressin acetate (DDAVP) and antifibrinolytic therapy for surgical management. Subsequent laboratory testing with fresh samples consistently confirmed an equivocal (borderline normal/abnormal) level of von Willebrand factor (VWF) and FVL with activated protein C resistance (APCR). A patient sample, differentially labeled according to the tests being performed, was later distributed for blind testing to participants within several modules of the RCPA Quality Assurance Program (QAP). Most participants reported a low level of VWF consistent with possible mild Type 1 VWD, and most (but not all) reported a positive finding for APCR. All participants correctly reported the sample as heterozygous for the FVL mutation, negative for the Prothrombin gene mutation G20210A, and heterozygous for the methylenetetrahydrofolate reductase (MTHFR) mutation C677T. Interestingly, a significant number of laboratories performing Protein S testing using clot-based procedures also identified a false Protein S deficiency. In conclusion, this exercise showed how, either depending on the clinical review and specific laboratory investigation and tests performed, a pro-bleeding diagnosis (of either VWD or thrombocytopenia) or pro-thrombophilia risk (Antiphospholipid Syndrome or FVL/APCR or false Protein S deficiency) could potentially and differentially arise in the one patient.