This study aims to assess the diagnostic value of a rapid bedside test for H-FABP, in addition to history taking and physical examination in primary care patients suspected of ACS. In addition, the balance between costs and effects of applying the H-FABP bedside test in primary care will be evaluated.

The study design is depicted in figure 1. Patients are primarily recruited by GPs working at one of three participating out-of-hours GP services in the region of Utrecht, The Netherlands (1 urban and 2 semi-urban). Additionally, 25 GPs from group practices will recruit patients during daytime hours. Diagnostic assessment during the initial GP consultation includes standardised history taking and physical examination and rapid H-FABP testing (see below). To allow for a definitive decision whether ACS is present ("gold" or reference standard) an ECG is recorded and a venous blood sample is collected in all patients (for measurement of currently preferred biomarkers including troponin, creatinin kinase (CK) and creatinin kinase-myocardial band (CK-MB)), irrespective of whether or not they are referred to hospital. In patients who are referred to hospital these measurements are performed as part of routine care. Patients who are not referred to hospital are visited at home by qualified GP laboratory service personnel to perform the above mentioned tests. Blood samples are obtained between 12 to 36 hours after onset of complaints in order to allow for a definitive diagnosis of AMI. Using this time interval we adopt a safe margin for a troponin rise to become detectable in the blood.

The study protocol was approved by the Medical Ethics Committee of the University Medical Center Utrecht, The Netherlands.

All patients with symptoms suggestive of ACS who present themselves to a GP are eligible for inclusion in the study. Presenting symptoms will typically include chest pain or discomfort at rest, but also atypical "vague" complaints such as abdominal discomfort, dizziness or sudden onset of dyspnoea.

Excluded are patients with complaints lasting more than 24 hours, as H-FABP levels usually return to normal 24–36 hours after onset of myocardial ischemia 911. Also excluded are patients who require instant hospital referral and those patients in whom no written informed consent is obtained.

The H-FABP bedside test (Cardiodetect^® Rennesens GmbH, Berlin) can easily be performed by the GP by drawing four drops of capillary whole blood from the patient's finger and applying them onto the test-strip. Within 15 minutes the H-FABP test result (elevated or non-elevated plasma FABP) is available. For study purposes the result is concealed by a blinding-strip. The test is de-blinded by the GP after he/she has made the referral decision. Test results are documented on a standardized case record form, together with findings from history taking and physical examination. Other items on the form include age, gender, prior AMI and treatment for AMI (bypass surgery or percutaneous coronary intervention). Also recorded are patient delay and doctor delay and a probability estimate by the GPs (prior to the H-FABP test result) that a patient has ACS, their decision about referral to hospital and the result of the H-FABP test.

A previous study by Grijseels et al provided a pre-hospital decision rule based on items from history taking and physical examination for patients suspected of ACS 13. We will validate the diagnostic accuracy of this clinical score and estimate the added value of the H-FABP bedside test.

An expert panel consisting of two cardiologists and one GP will establish the final diagnosis using all available patient information, including signs and symptoms, ECG and biomarker levels (troponin-T or -I, CK and CK-MB). These results are available for all patients, including patients that are not referred to hospital, as they are visited at home by qualified GP laboratory personnel for performance of ECG and laboratory tests.

AMI will be defined in accordance with guidelines from the European Society of Cardiology and the American College of Cardiology 15 The diagnosis of AMI is established when patients have suggestive symptoms (such as chest pain) and a maximal concentration of troponin T or I exceeding the decision limit (99^th percentile of the values for a reference control group) within the first 36 hours after the onset of complaints and/or CK-MB values greater than two times the upper reference limit on at least one occasion during the same time frame. The presence of ST- and T-wave changes on the ECG, notably ST elevations and Q-waves, can further confirm AMI. Unstable angina (UA) is defined as symptoms of chest pain and ST- and/or T-wave changes on the ECG suggestive of ischemia, without elevation of troponin or CK-MB above the decision limits 15.

As already mentioned, every GP will decide about referral to hospital in accordance with daily practice, using only history taking and physical examination and, when available, ECG. This decision is made without using the H-FABP test result. For safety reasons an exception is made for patients with a positive H-FABP test result in whom the GP initially decided not to refer. In these cases, the GP is instructed to change his initial management decision in favor of hospital referral.

Using 2 by 2 tables the diagnostic value of the H-FABP test alone and in combination with the clinical score will be assessed, using AMI as the outcome, and positive and negative predictive values, sensitivity and specificity will be calculated with 95% confidence interval.

Multivariable regression analysis with receiver operating characteristic (ROC) curves will be used to determine whether the H-FABP test provides added diagnostic value beyond history taking and physical examination (summarized in a clinical score). Two diagnostic models will be tested: one using only the clinical score, the other one consisting of the clinical score together with the H-FABP test result. This will lead to 2 different areas under the ROC-curve (AUC), where the difference in AUC presents the added value of the H-FABP test.

As AMI is notoriously difficult to diagnose in women and the elderly we will perform subgroup analyses in these specific patient categories 1.

A frequently used 'rule of thumb' recommends that for each diagnostic determinant included in a multivariable logistic regression analysis at least 10 events (in this case AMI) are necessary 1516. Our study includes 2 diagnostic determinants (clinical score and H-FABP test result). Thus, a population in which at least 20 patients with AMI is required. Although available estimates vary, Dutch studies show that in more than 10% of patients suspected of AMI by the GP the diagnosis is confirmed 213. Therefore, 200 patients with suspected AMI need to be included in our study. We will include 300 patients to allow for subgroup analyses.

In diagnostic prediction research the physician ideally should be blinded to the results of the test under study in order to prevent bias in the ascertainment of the disease. In this study, the H-FABP test result may influence the inferences drawn from medical history and physical examination, and thereby influence the referral decision of the GP, especially since it is notoriously difficult to decide about the presence or absence of an ACS based on clinical assessment only.

There are however two important reasons why a fully blinded H-FABP test is not feasible in our study. Firstly, previous hospital based studies showed that the H-FABP bedside test has a positive predictive value of well above 80% 17181920. We therefore instructed the GPs to decide on the referral before de-blinding the test result, but for safety reasons we also instructed them to refer patients with a positive H-FABP test result to hospital irrespective of their initial referral decision. Secondly, in the hours following application of the test, discoloration of the test-strip occurs which negatively influences the interpretation and thereby the accuracy of the test results. Therefore the test has to be read shortly after its performance.

It is neither very realistic nor feasible to ask written informed consent to study participation from a patient in an acute life threatening situation, such as with symptoms suggestive for ACS. Therefore the Medical Ethics Committee agreed to ask verbal consent from patients for taking the H-FABP test by the GP. Subsequently patients are given the opportunity for written informed consent after having read an information letter at a more convenient moment. Patients may also decide to withdraw their consent then or at any time thereafter. Only patients who return a written informed consent are included in our study.

We have chosen for a phased introduction of our study in 3 different GP out-of-hour services (weekdays from 5 pm-8 am and weekends). Participating centers are notified of the study progress by a monthly overview of the number of participants and a 2-monthly newsletter with background information on the study, frequently asked questions and tips and tricks, for instance on how to draw the required amount of capillary blood. We anticipate including 300 patients within 36 months.