A consensus review on the development of palliative care for patients with chronic and progressive neurological disease.

BACKGROUND AND PURPOSE: The European Association of Palliative Care Taskforce, in collaboration with the Scientific Panel on Palliative Care in Neurology of the European Federation of Neurological Societies (now the European Academy of Neurology), aimed to undertake a review of the literature to establish an evidence-based consensus for palliative and end of life care for patients with progressive neurological disease, and their families. METHODS: A search of the literature yielded 942 articles on this area. These were reviewed by two investigators to determine the main areas and the subsections. A draft list of papers supporting the evidence for each area was circulated to the other authors in an iterative process leading to the agreed recommendations. RESULTS: Overall there is limited evidence to support the recommendations but there is increasing evidence that palliative care and a multidisciplinary approach to care do lead to improved symptoms (Level B) and quality of life of patients and their families (Level C). The main areas in which consensus was found and recommendations could be made are in the early integration of palliative care (Level C), involvement of the wider multidisciplinary team (Level B), communication with patients and families including advance care planning (Level C), symptom management (Level B), end of life care (Level C), carer support and training (Level C), and education for all professionals involved in the care of these patients and families (Good Practice Point). CONCLUSIONS: The care of patients with progressive neurological disease and their families continues to improve and develop. There is a pressing need for increased collaboration between neurology and palliative care.

Prevalence of brain and spinal cord inclusions, including dipeptide repeat proteins, in patients with the C9ORF72 hexanucleotide repeat expansion: a systematic neuropathological review.

AIM: The current literature shows no consensus on the localization and number of characteristic neuronal inclusions [p62 and dipeptide repeat proteins (DRPs) positive, TDP-43-negative and TDP-43 positive] in the brain and spinal cord of patients with the hexanucleotide repeat expansion on chromosome 9 (C9ORF72-positive patients). This may be due to small sample sizes. A valid brain map of the inclusions in C9ORF72-positive patients may improve clinicopathological correlations and may serve as a reference for neuropathologists. METHODS: We performed a systematic review on 42 pathological studies to assess the pooled prevalence rates and density (a measure of the number of inclusions per brain region) of (phosphorylated)-TDP-43, p62 and DRP neuronal inclusions in seven brain regions and the spinal cord of 261 C9ORF72-positive patients with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and ALS-FTD. RESULTS: In the cerebellum and hippocampus, the pooled prevalence rates of TDP-43 neuronal cytoplasmic inclusions (NCIs; cerebellum: 3.9%; hippocampus: 68.3%) were lower than those of DRP (cerebellum: 97.2%; hippocampus 97.1%). Moreover, TDP-43 inclusion density was lower compared with p62 inclusion density in these regions. The pooled prevalence rate of TDP-43 NCI in the substantia nigra was high (94.4%). DISCUSSION: The findings of this systematic review largely confirm findings of previous smaller studies on the localization and prevalence of inclusions in the central nervous system of C9ORF72-positive patients. The high prevalence of TDP-43 inclusions in the substantia nigra is a relatively new finding and is probably related to the relatively high prevalence of parkinsonism in C9ORF72-positive patients.

RYR1-related myopathies: a wide spectrum of phenotypes throughout life.

BACKGROUND AND PURPOSE: Although several recent studies have implicated RYR1 mutations as a common cause of various myopathies and the malignant hyperthermia susceptibility (MHS) trait, many of these studies have been limited to certain age groups, confined geographical regions or specific conditions. The aim of the present study was to investigate the full spectrum of RYR1-related disorders throughout life and to use this knowledge to increase vigilance concerning malignant hyperthermia. METHODS: A retrospective cohort study was performed on the clinical, genetic and histopathological features of all paediatric and adult patients in whom an RYR1 mutation was detected in a national referral centre for both malignant hyperthermia and inherited myopathies (2008-2012). RESULTS: The cohort of 77 non-related patients (detection rate 28%) included both congenital myopathies with permanent weakness and 'induced' myopathies such as MHS and non-anaesthesia-related episodes of rhabdomyolysis or hyperCKemia, manifested throughout life and triggered by various stimuli. Sixty-one different mutations were detected, of which 24 were novel. Some mutations are present in both dominant (MHS) and recessive modes (congenital myopathy) of inheritance, even within families. Histopathological features included an equally wide spectrum, ranging from only subtle abnormalities to prominent cores. CONCLUSIONS: This broad range of RYR1-related disorders often presents to the general paediatric and adult neurologist. Its recognition is essential for genetic counselling and improving patients' safety during anaesthesia. Future research should focus on in vitro testing by the in vitro contracture test and functional characterization of the large number of RYR1 variants whose precise effects currently remain uncertain.

Myositis with endomysial cell invasion indicates inclusion body myositis even if other criteria are not fulfilled.

The objective of this study was to investigate if patients with endomysial mononuclear cell infiltrates invading non-necrotic fibers have a disease course consistent with inclusion body myositis (IBM), irrespective of other histopathological and clinical characteristics. All patients with a muscle biopsy showing endomysial inflammation with invasion of non-necrotic muscle fibers during the period 1979-2006 in two tertiary neuromuscular referral centers were classified into three groups: 1) patients whose biopsies also showed rimmed vacuoles; 2) patients whose biopsies showed no vacuoles but fulfilled clinical criteria for IBM, and 3) patients whose biopsies showed no vacuoles, and also did not fulfill clinical criteria for IBM (unclassified patients). These groups were compared with regard to age, gender, clinical features, and disease course including response to immunosuppressive treatment. Eighty-one individuals (41 men) were included. Rimmed vacuoles were found in 49 patients (60.5%). Fourteen patients (17.3%) fulfilled clinical criteria for IBM and 18 patients (22.2%) were unclassified at presentation. At follow up (mean duration 9 years) three women remained unclassified (4%). There were no differences in disease course or effect of treatment between the three groups. Men had more often rimmed vacuoles than women (73% vs 48%; p = 0.018), and women more often than men were unclassified. Women tended to show more often temporary improvement if treated (p = 0.07), but none had sustained improvement. In conclusion, patients with a muscle biopsy showing endomysial cell infiltration with invasion of non-necrotic muscle fibers most probably have IBM, regardless of clinical and other pathological features. Women lack typical features more often than men.

Clinical characterisation of Becker muscular dystrophy patients predicts favourable outcome in exon-skipping therapy.

OBJECTIVE: Duchenne and Becker muscular dystrophy (DMD/BMD) are both caused by mutations in the DMD gene. Out-of-frame mutations in DMD lead to absence of the dystrophin protein, while in-frame BMD mutations cause production of internally deleted dystrophin. Clinically, patients with DMD loose ambulance around the age of 12, need ventilatory support at their late teens and die in their third or fourth decade due to pulmonary or cardiac failure. BMD has a more variable disease course. The disease course of patients with BMD with specific mutations could be very informative to predict the outcome of the exon-skipping therapy, aiming to restore the reading-frame in patients with DMD. METHODS: Patients with BMD with a mutation equalling a DMD mutation after successful exon skipping were selected from the Dutch Dystrophinopathy Database. Information about disease course was gathered through a standardised questionnaire. Cardiac data were collected from medical correspondence and a previous study on cardiac function in BMD. RESULTS: Forty-eight patients were included, representing 11 different mutations. Median age of patients was 43 years (range 6-67). Nine patients were wheelchair users (26-56 years). Dilated cardiomyopathy was present in 7/36 patients. Only one patient used ventilatory support. Three patients had died at the age of 45, 50 and 76 years, respectively. CONCLUSIONS: This study provides mutation specific data on the course of disease in patients with BMD. It shows that the disease course of patients with BMD, with a mutation equalling a 'skipped' DMD mutation is relatively mild. This finding strongly supports the potential benefit of exon skipping in patients with DMD.

Idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIMs), except for sporadic inclusion body myositis (sIBM), present with subacute symmetrical weakness of the limb girdle muscles, an elevated serum creatine kinase activity, and inflammatory cells in the muscle biopsy (necrotizing autoimmune myopathy being an exception). In dermatomyositis, additional skin abnormalities are found. IIMs are nowadays subclassified into the following categories: (1) dermatomyositis (DM), including (1a) classic dermatomyositis, which may be associated with connective tissue disorders (CTDs) and malignancy, (1b) juvenile dermatomyositis, and (1c) clinical amyopathic dermatomyositis; (2) polymyositis (PM) encompassing (2a) classical PM and (2b) nonspecific or overlap myositis, associated with CTD; (3) autoimmune necrotizing myopathy, associated with malignancy, statin use and CTD; and (4) sporadic IBM, sometimes associated with CTDs. These conditions result from chronic immune activation after exposure to environmental risk factors in individuals with a predisposing genetic background. A strong association of autoantibodies with distinct clinical phenotypes and prognosis is found in patients with myositis. Inflammatory myopathies, sporadic IBM excluded, are amenable to immunosuppressive and immunomodulation therapies. The prognosis of IIM is not well known since long-term outcome and prognostic factors vary widely. Disease-related mortality rates in PM and DM are at least 10%. In DM mortality is attributed to cancer and pulmonary complications. Juvenile dermatomyositis has a low mortality rate. Because chronic immunosuppressive therapy is associated with significant side-effects, and many patients remain (partially) refractory to treatment, novel therapeutic agents that are safe and effective are needed.

Teaching adjuvant endocrine breast cancer treatment to medical students.

BACKGROUND: In undergraduate medical education, students are supposed to acquire knowledge and understanding about the basic principles of adjuvant breast cancer treatment. The best education method in this context is unknown. In this randomised study we assessed the effect of designing a patient education poster on knowledge, perceived participation and students' satisfaction compared with case-oriented education concerning endocrine therapy for breast cancer patients. METHODS: This study was conducted in the Bachelor Oncology Course for undergraduate students in Medical Science of the Radboud University Nijmegen Medical Centre. In the experimental group, students designed and created a patient education poster in small groups. In the control group, students answered case-based questions in small groups. Knowledge was tested at different moments using multiple-choice questions. To assess perceived participation and satisfaction, students filled out questionnaires. RESULTS: 329 students participated in the study. No difference in knowledge was observed between the experimental and control group. However, students in the control group reported a higher perceived participation and satisfaction compared with the students in the experimental group (p<0.05). CONCLUSION: In this study, working on case-based questions was preferred compared with designing a patient education poster in terms of students' perceived participation and satisfaction. Working on case-based questions may be appreciated by medical students as most relevant for their future profession. We advocate more attention to the importance of patient education in the medical curriculum, to help students realise the relevance of this aspect of medical profession.

EFNS review on the role of muscle biopsy in the investigation of myalgia.

BACKGROUND: Myalgia, defined as any pain perceived in muscle, is very common in the general population and a frequent cause for referral to neurologists, rheumatologists and internists in general. It is however only rarely due to primary muscle disease and often referred from ligaments, joints, bones, the peripheral and central nervous system. A muscle biopsy should only be performed if this is likely to be diagnostically useful. At present no 'guidelines' exist. METHODS: An EFNS panel of muscle specialists was set to review relevant studies from PubMed dating as far back as 1/1/1990. Only Class IV studies were available and therefore the recommendations arrived at are 'best practice recommendations' based on information harvested from the literature search and expert opinion. RESULTS: Muscle cramps should be recognized while drugs, infections, metabolic/ endocrinological and rheumatological causes of myalgia should be identified from the history and examination and pertinent laboratory tests. A muscle biopsy is more likely to be diagnostically useful if myalgia is exertional and if one or more of the following apply: i) there is myoglobinuria, (ii) there is a second wind phenomenon, (iii) there is muscle weakness, (iv) there is muscle hypertrophy /atrophy, (v) there is hyperCKemia (>2-3× normal), and (vi) there is a myopathic EMG. CONCLUSIONS: Patients presenting with myalgia can be recommended to have a biopsy based on careful history and examination and on simple laboratory screening.

Congenital myopathy with focal loss of cross-striations revisited.

In 1977 Wijngaarden et al. reported a Dutch family with a congenital myopathy characterized by external ophthalmoplegia and a remarkable histological feature, focal loss of cross-striations. A small number of other families with similar clinical and pathological features led to the consideration of this congenital myopathy as a distinct entity. Here we present more than 30years of follow-up from the Dutch family and report recently identified compound heterozygous mutations in the skeletal muscle ryanodine receptor (RYR1) gene, c.10627-2A>G and p.Arg3539His (c.10616G>A). Focal loss of cross-striations on muscle biopsy is another histopathological feature that should raise the possibility of RYR1 involvement.

Dermatomyositis and polymyositis: new treatment targets on the horizon.

Polymyositis (PM) and dermatomyositis (DM) are rare idiopathic inflammatory myopathies (IIM) with a presumed autoimmune pathogenesis. Typical features are subacute onset, proximal, symmetric muscle weakness, elevated serum creatine kinase, and mononuclear cell infiltrates in the muscle biopsy. Strong support for an autoimmune pathogenesis comes from histopathological findings in biopsies of affected muscles. Furthermore, the association with autoantibodies supports the notion that immune-mediated inflammation is involved. PM and DM may occur in isolation or in connection with a connective tissue disease or cancer. The current treatment for IIM consists of first-line high-dose steroids and various conventional second-line treatments. Improvements in treatment for IIM are hampered by difficulties in the design of trials and the low incidence and prevalence of the disease. Cytokines and chemokines are factors involved in the inflammatory process in IIM, and are candidates for future therapeutic targets. Preliminary data with anti-tumour necrosis factor therapy are not very promising, but results of blockers of the lymphotoxin signalling pathway are to be awaited. Anti-B cell therapy may be a valuable therapeutic option for treatment of refractory IIM. The effects of anti-interferon-alpha in IIM are to be awaited, as are results of other anti-cytokine therapies and anti-chemokine therapy. Outcome measures to be used in clinical trials in II M include at present the core sets of outcome proposed by the International Myositis Assessment Clinical Study Group (IMACS).

The Lambert-Eaton myasthenic syndrome 1988-2008: a clinical picture in 97 patients.

BACKGROUND: Neuromuscular symptoms in patients with Lambert-Eaton myasthenic syndrome (LEMS) and a small cell lung cancer (SCLC) develop more rapidly than in LEMS patients without a SCLC. We studied how this clinical information, which is readily available at the first consultation, can be used to predict the presence of SCLC. PATIENTS AND METHODS: In our study we included 52 LEMS patients with SCLC and 45 non-tumor patients (NT-LEMS). We interviewed patients using a structured checklist and reviewed their clinical records. We compared frequency and onset of symptoms during the course of LEMS. RESULTS: In the first six months, over half the SCLC-LEMS patients had developed seven separate symptoms, while NT-LEMS patients developed only two symptoms. Proximal leg weakness and dry mouth were early symptoms in both groups. Rapid involvement of proximal arm muscles (p=0.0001), distal arm muscles (p=0.0037), distal leg muscles (p=0.0002), dysartria (p=0.0091) and the presence of erectile dysfunction (p=0.007) were found significantly more often in SCLC-LEMS patients in both cohorts. Cerebellar symptoms, although present in 9% of LEMS patients, were almost exclusively related to SCLC-LEMS. CONCLUSION: A rapidly progressive course of disease from onset in LEMS patients should raise a high suspicion of SCLC. Special attention should be paid to involvement of upper extremities, involvement of distal arm and distal leg muscles, to erectile dysfunction and probably ataxia in order to discriminate between SCLC-LEMS and NT-LEMS.

Spontaneous recovery of dermatomyositis and unspecified myositis in three adult patients.

Dermatomyositis (DM), polymyositis and unspecified myositis are idiopathic inflammatory myopathies in which prednisone is usually started as soon as the diagnosis has been established. Therefore, little is known about the natural history of these diseases and spontaneous recovery may escape attention. Here, we present three patients who achieved remission without administration of immunosuppressants. In these three patients, treatment was not started because of spontaneously improving symptoms and signs during the diagnostic process. After 3-5 years, all patients are still free of muscle weakness. These case reports demonstrate that spontaneous long lasting remission can occur in a small proportion of patients with subacute onset idiopathic inflammatory myopathies. In some patients, immunosuppressive treatment with the risk of serious side effects can perhaps be omitted. However, close and frequent monitoring is required in these instances.

EFNS guideline on diagnosis and management of limb girdle muscular dystrophies.

The limb girdle muscular dystrophies (LGMD) are termed as such as they share the characteristic feature of muscle weakness predominantly affecting the shoulder and pelvic girdles; their classification has been completely revised in recent years because of elucidation of many of the underlying genetic and protein alterations in the various subtypes. An array of diagnostic measures is possible but with varying ease of use and availability. Several aspects of muscle cell function appear to be involved in the causation of muscle pathology. These cellular variations may confer some specific clinical features thus permitting recognition of the LGMD subtype and hence directing appropriate levels of monitoring and intervention. Despite an extensive literature on the individual limb girdle dystrophies, these publications may be impenetrable for the general neurologist in this increasingly complex field. The proposed guidelines suggest an approach to the diagnosis and monitoring of the limb girdle dystrophies in a manner accessible to general neurologists.

Novel 111In-labelled bombesin analogues for molecular imaging of prostate tumours.

PURPOSE: It has been shown that some primary human tumours and their metastases, including prostate and breast tumours, overexpress gastrin-releasing peptide (GRP) receptors. Bombesin (BN) is a neuropeptide with a high affinity for these GRP receptors. We demonstrated successful scintigraphic visualisation of BN receptor-positive tumours in preclinical studies using the radiolabelled BN analogue [(111)In-DTPA-Pro(1),Tyr(4)]BN. However, the receptor affinity as well as the serum stability of this analogue leave room for improvement. Therefore new (111)In-labelled BN analogues were synthesised and evaluated in vitro and in vivo. METHODS AND RESULTS: The receptor affinity of the new BN analogues was tested on human GRP receptor-expressing prostate tumour xenografts and rat colon sections. Analogues with high receptor affinity (low nM range) were selected for further evaluation. Incubation in vitro of GRP receptor-expressing rat CA20948 and human PC3 tumour cells with the (111)In-labelled analogues resulted in rapid receptor-mediated uptake and internalisation. The BN analogue with the best receptor affinity and in vitro internalisation characteristics, Cmp 3 ([(111)In-DTPA-ACMpip(5),Tha(6),betaAla(11),Tha(13),Nle(14)]BN(5-14)), was tested in vivo in biodistribution studies using rats bearing GRP receptor-expressing CA20948 tumours, and nude mice bearing human PC3 xenografts. Injection of (111)In-labelled Cmp 3 in these animals showed high, receptor-mediated uptake in receptor-positive organs and tumours which could be visualised using planar gamma camera and microSPECT/CT imaging. CONCLUSION: With their enhanced receptor affinity and their rapid receptor-mediated internalisation in vitro and in vivo, the new BN analogues, and especially Cmp 3, are promising candidates for use in diagnostic molecular imaging and targeted radionuclide therapy of GRP receptor-expressing cancers.

Long-term outcome in polymyositis and dermatomyositis.

BACKGROUND: Although polymyositis and dermatomyositis are regarded as treatable disorders, prognosis is not well known, as in the literature long-term outcome and prognostic factors vary widely. AIM: To analyse the prognostic outcome factors in polymyositis and adult dermatomyositis. METHODS: We determined mortality, clinical outcome (muscle strength, disability, persistent use of drugs and quality of life) and disease course and analysed prognostic outcome factors. RESULTS: Disease-related death occurred in at least 10% of the patients, mainly because of associated cancer and pulmonary complications. Re-examination of 110 patients after a median follow-up of 5 years showed that 20% remained in remission and were off drugs, whereas 80% had a polycyclic or chronic continuous course. The cumulative risk of incident connective tissue disorder in patients with myositis was significantly increased. 65% of the patients had normal strength at follow-up, 34% had no or slight disability, and 16% had normal physical sickness impact profile scores. Muscle weakness was associated with higher age (odds ratio (OR) 3.6; 95% confidence interval (CI) 1.3 to 10.3). Disability was associated with male sex (OR 3.1; 95% CI 1.2 to 7.9). 41% of the patients with a favourable clinical outcome were still using drugs. Jo-1 antibodies predicted the persistent use of drugs (OR 4.4, 95% CI 1.3 to 15.0). CONCLUSIONS: Dermatomyositis and polymyositis are serious diseases with a disease-related mortality of at least 10%. In the long term, myositis has a major effect on perceived disability and quality of life, despite the regained muscle strength.

Late onset axonal Charcot-Marie-Tooth phenotype caused by a novel myelin protein zero mutation.

A late onset axonal Charcot-Marie-Tooth phenotype is described, resulting from a novel mutation in the myelin protein zero (MPZ) gene. Comparative computer modelling of the three dimensional structure of the MPZ protein predicts that this mutation does not cause a significant structural change. The primary axonal disease process in these patients points to a function of MPZ in maintenance of the myelinated axons, apart from securing stability of the myelin layer.

Laing early onset distal myopathy: slow myosin defect with variable abnormalities on muscle biopsy.

BACKGROUND: Laing early onset distal myopathy (MPD1) is an autosomal dominant myopathy caused by mutations within the slow skeletal muscle fibre myosin heavy chain gene, MYH7. It is allelic with myosin storage myopathy, with the commonest form of familial hypertrophic cardiomyopathy, and with one form of dilated cardiomyopathy. However, the clinical picture of MPD1 is distinct from these three conditions. OBJECTIVE: To collate and discuss the histological features reported in the muscle biopsies of MPD1 patients and to outline the clinical features. RESULTS: The phenotype of MPD1 was consistent, with initial weakness of great toe/ankle dorsiflexion, and later development of weakness of finger extension and neck flexion. Age of onset was the only variable, being from birth up to the 20 s, but progression was always very slow. The pathological features were variable. In this retrospective series, there were no pathognomonic diagnostic features, although atrophic type I fibres were found in half the families. Rimmed vacuoles are consistently seen in all other distal myopathies with the exception of Myoshi distal myopathy. However, they were found in a minority of patients with MPD1, and were not prominent when present. Immunohistochemical staining for slow and fast myosin showed co-expression of slow and fast myosin in some type I fibres, possibly indicating a switch to type II status. This may be a useful aid to diagnosis. CONCLUSIONS: The pathological findings in MPD1 are variable and appear to be affected by factors such as the specific muscle biopsied, the age of the patient at biopsy, and the duration of disease manifestations.

[The practice guideline 'Dermatomyositis, polymyositis and sporadic inclusion body myositis']. / Richtlijn 'dermatomyositis, polymyositis en sporadische "inclusion body"-myositis'.

This guideline presents recommendations for the diagnosis and treatment of dermatomyositis, polymyositis and sporadic inclusion body myositis (sIBM) according to the best available evidence. Characteristic skin abnormalities can be sufficient for the diagnosis of dermatomyositis. In case of doubt, a skin biopsy is advisable. A muscle biopsy is indicated when other examinations are inconclusive and the musculature is involved. The working group considers screening for cancer to be required in adults with dermatomyositis and presents recommendations for the way that this should be done. At least one-third of all patients with polymyositis has, or will develop, an associated inflammatory connective tissue disease. If a patient with a connective tissue disease develops symmetrical, proximal muscle weakness in the course of weeks or months, this may be assumed to be due to polymyositis. In the absence ofpre-existing connective tissue disease, demonstration of a mononuclear cell infiltrate in muscle tissue is a prerequisite for the diagnosis ofpolymyositis. The histopathology of muscle tissue is used as the gold standard for the diagnosis of sIBM. The practice guideline presents criteria for the concept 'activity' of myositis. Disease activity serves as a guideline for the treatment of polymyositis and dermatomyositis. The treatment of choice for dermatomyositis and polymyositis is high-dose prednisone. Physical activity does not have a negative effect on the course of these diseases. The long-term prognosis ofdermatomyositis and polymyositis is not well known. The clinical course of sIBM is slowly progressive.

Determinants of the APTT- and ETP-based APC sensitivity tests.

BACKGROUND: A reduced sensitivity for activated protein C (APC) is associated with an increased risk of venous thrombosis even in the absence of the factor (F)V Leiden mutation. This risk has been demonstrated with two APC sensitivity tests, which quantify the effects of APC on the activated partial thromboplastin time (APTT) and the endogenous thrombin potential (ETP), respectively. OBJECTIVES: We examined determinants of both APC sensitivity tests in the control group of the Leiden Thrombophilia Study (LETS). METHODS: Multiple linear regression analysis was performed with normalized APC-SR(APTT) or APC-SR(ETP) as dependent variable and putative determinants [levels of FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII A subunit, FXIII B subunit, protein S total, protein S free, protein C, tissue factor pathway inhibitor (TFPI) total, TFPI free, antithrombin and fibrinogen] as independent variables. RESULTS AND CONCLUSIONS: The major determinant of the APTT-based test was FVIII level, followed by FII level. The ETP-based test was influenced most by free protein S and free TFPI levels. In both tests FXa formation plays a major role, as the effect of FVIII and TFPI on the tests seems to be executed via FXa. The ETP-based test was also strongly influenced by oral contraceptive use, even when we adjusted for all the clotting factors listed above. This means that the effect of oral contraceptives on the ETP-based test is not fully explained by the changes of coagulation factor levels investigated in this study, and that the molecular basis of acquired APC resistance during use of oral contraceptives remains to be established.