Portal de Periódicos da CAPES

Prognostic value of serial platelet reactivity measurements on long‐term clinical outcome in patients with ST‐elevation myocardial infarction undergoing primary PCI

2008; Elsevier BV; Volume: 6; Issue: 10 Linguagem: Inglês

10.1111/j.1538-7836.2008.03112.x

1538-7933

Gianluca Campo, Marco Valgimigli, Alice Frangione, Matteo Tebaldi, Roberto Ferrari,

Coronary Interventions and Diagnostics

Several studies have documented that platelet reactivity (PR) plays a critical role in determining ischemic complications after ST‐segment elevation myocardial infarction (STEMI) and percutaneous coronary intervention (PCI) [1Huczek Z. Filipiak K.J. Kochman J. Piatkowski R. Grabowski M. Roik M. Malek L.A. Jaworski P. Opolski G. Baseline platelet reactivity in acute myocardial infarction treated with primary angioplasty influence on myocardial reperfusion, left ventricular performance, and clinical events.Am Heart J. 2007; 154: 62-70Crossref PubMed Scopus (0) Google Scholar, 2Campo G. Valgimigli M. Gemmati D. Percoco G. Tognazzo S. Cicchitelli G. Catozzi L. Malagutti P. Anselmi M. Vassanelli C. Scapoli G. Ferrari R. Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY study.J Am Coll Cardiol. 2006; 48: 2178-85Crossref PubMed Scopus (143) Google Scholar, 3Angiolillo D.J. Fernandez‐Ortiz A. Bernardo E. Alfonso F. Macaya C. Bass T.A. Costa M.A. Variability in individual responsiveness to clopidogrel: clinical implications, management, and future perspectives.J Am Coll Cardiol. 2007; 49: 1505-16Crossref PubMed Scopus (855) Google Scholar, 4Angiolillo D.J. Bernardo E. Sabate M. Jimenez‐Quevedo P. Costa M.A. Palazuelos J. Hernández‐Antolin R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Guzman L.A. Bass T.A. Macaya C. Fernandez‐Ortiz A. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease.J Am Coll Cardiol. 2007; 50: 1541-7Crossref PubMed Scopus (320) Google Scholar, 5Fuchs I. Frossard M. Spiel A. Riedmüller E. Laggner A.N. Jilma B. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS.J Thromb Haemost. 2006; 4: 2547-52Crossref PubMed Scopus (119) Google Scholar]. Moreover, baseline PR influences the responsiveness to antiplatelet therapy [2Campo G. Valgimigli M. Gemmati D. Percoco G. Tognazzo S. Cicchitelli G. Catozzi L. Malagutti P. Anselmi M. Vassanelli C. Scapoli G. Ferrari R. Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY study.J Am Coll Cardiol. 2006; 48: 2178-85Crossref PubMed Scopus (143) Google Scholar, 6Gurbel P.A. Bliden K.P. Hiatt B.L. O’Connor C.M. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity.Circulation. 2003; 107: 2908-13Crossref PubMed Scopus (1443) Google Scholar, 7Angiolillo D.J. Fernandez‐Ortiz A. Bernardo E. Ramírez C. Barrera‐Ramirez C. Sabaté M. Hernández R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Costa M.A. Bass T.A. Macaya C. Identification of low responders to a 300‐mg clopidogrel loading dose in patients undergoing coronary stenting.Thromb Res. 2005; 115: 101-8Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. All together, these studies carry two important limitations. First, PR was determined with a single assessment and whether patients with high PR at the time of the first event maintain high PR also during the follow‐up is unknown. Second, the long‐term clinical implications (>1 year) of high PR has been less investigated [4Angiolillo D.J. Bernardo E. Sabate M. Jimenez‐Quevedo P. Costa M.A. Palazuelos J. Hernández‐Antolin R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Guzman L.A. Bass T.A. Macaya C. Fernandez‐Ortiz A. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease.J Am Coll Cardiol. 2007; 50: 1541-7Crossref PubMed Scopus (320) Google Scholar, 5Fuchs I. Frossard M. Spiel A. Riedmüller E. Laggner A.N. Jilma B. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS.J Thromb Haemost. 2006; 4: 2547-52Crossref PubMed Scopus (119) Google Scholar]. In the present study, we evaluated the PR variations during a 2‐year follow‐up and we assessed their association with outcome. For this purpose, 135 STEMI patients (65 ± 12 years, male 73%) treated with primary PCI were recruited from the cath‐lab of the University Hospital of Ferrara. As control population, 25 volunteers without previous cardiovascular events, matched for cardiovascular risk factors, were enrolled. This study was approved by the local ethics committee and all patients gave written informed consent. Four blood samples were collected: visit T0, at entry before any therapy; visit T1, at discharge; visit T2, at 1 year; visit T3, at 2 years. All patients, at T2 and T3, were clinically stable. A single blood sample from volunteers was collected. PR was measured with Platelet Function Analyzer 100 (PFA‐100). In the PFA‐100 system, blood is aspirated through a capillary into a membrane coated with collagen and adenosine diphosphate (CADP) or epinephrine (CEPI). PR is reported as the closure time (seconds, CADP‐CT or CEPI‐CT) needed to form a platelet plug and occlude the membrane. CADP‐CT inversely reflects PR [2Campo G. Valgimigli M. Gemmati D. Percoco G. Tognazzo S. Cicchitelli G. Catozzi L. Malagutti P. Anselmi M. Vassanelli C. Scapoli G. Ferrari R. Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY study.J Am Coll Cardiol. 2006; 48: 2178-85Crossref PubMed Scopus (143) Google Scholar, 5Fuchs I. Frossard M. Spiel A. Riedmüller E. Laggner A.N. Jilma B. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS.J Thromb Haemost. 2006; 4: 2547-52Crossref PubMed Scopus (119) Google Scholar]. The clinical end‐points were death, reinfarction, and target vessel revascularization (TVR) (major adverse cardiac event, MACE). Continuous data are presented as means ± SD, categorical variables as number (percentages). Comparisons were performed byt‐test and two‐sided Fisher’s test, respectively. Correlations were tested by Pearson analysis. Results of PFA‐100 were normally distributed. PR temporal variations are estimated by linear mixed model, with an autocorrelation structure of order 1. We defined patients with CADP‐CT under median value as high PR group and those with CADP‐CT above median value as low PR group. The prognostic value of variables was examined using a Cox‐proportional hazards model. Receiver‐operating characteristics (ROC) analysis was used for an exploratory evaluation of the best cut‐off point of CADP‐CT to predict MACE. Probability was significant at 1. Before admission, 10 (7.4%) patients were being treated with aspirin and one (0.7%) with thienopyridines. At T2 and T3, 116 (91%) and 112 (90%) patients were still taking aspirin, 19 (15%) and 16 (13%) thienopyridines. There was a progressive CADP‐CT increase from baseline to 2 years (P< 0.01) (Fig. 1). As compared with controls, at T0 and T1 CADP‐CT was lower in STEMI patients (P< 0.01), whereas no differences were observed at T2 and T3 (P= 0.4). In STEMI patients, CADP‐CT values throughout the different blood samples were highly correlated (Fig. 1). In spite of the CADP‐CT increase, the difference between high and low PR groups remains unchanged (Fig. 1). Patients with high PR at T0 showed lower CADP‐CT values even at T1, T2 and T3 (Fig. 1). The same pattern was observed also using the PR value at T2 to stratify the population in the high vs. low group. Of note, 85% (95% CI, 74–91%), 81% (95% CI, 68–89%) and 77% (95% CI, 64–86%) of patients in the high PR group at T0 remained in the high PR group at T1, T2 and T3, respectively. No differences in baseline characteristics and antiplatelet therapy were observed stratifying the study population in the high vs. low PR group. Only diabetes mellitus tended to be more frequent in the high PR group (P= 0.09). CEPI‐CT increases from T0 to T1 (151 ± 43 vs. 232 ± 83,P< 0.01). No variations were observed from T1 to T3 (232 ± 83 vs. 240 ± 80 vs. 236 ± 81,P= 0.5). CEPI‐CT shows a weak correlation with CADP‐CT (r= 0.35 at T0,r= 0.3 at T1,r= 0.32 at T2,r= 0.38 at T3). Of note, high PR patients (according CADP‐CT at T0) showed lower CEPI‐CT values as compared with the low PR group (138 ± 45 vs. 165 ± 40 at T0,P< 0.01; 215 ± 73 vs. 259 ± 50 at T1,P< 0.01; 217 ± 84 vs. 258 ± 77 at T2,P< 0.01; 216 ± 86 vs. 255 ± 73 at T3,P< 0.01). We observed 11 (8%) deaths, eight (5%) re‐infarctions and nine (6%) TVR for restenosis. Patients with MACE, as compared with those without, showed lower CADP‐CT throughout the follow‐up (67 ± 11 vs. 82 ± 9 at T0,P< 0.01; 93 ± 17 vs. 105 ± 11 at T1,P< 0.01; 97 ± 22 vs. 113 ± 19 at T2,P< 0.01; 100 ± 15 vs. 114 ± 18 at T3,P< 0.01). The increase in the risk of composite end‐point was 6 (95% CI: 2.3–15,P< 0.01) in the high PR group at T0 and 5 (95% CI: 1.2–22,P= 0.04) in that group at T2. Diabetes mellitus and Killip class at entry were predictors of MACE. At multivariate analysis PR remained an independent predictor for MACE (HR 4.5; 95% CI, 2–12;P< 0.01). No association between CEPI‐CT and clinical outcome was found. Figure 1 shows ROC analysis; 74% and 78% of patients (95% CI 58–85% and 95% CI 61–89%, respectively) under the T0 cut‐off are again under the cut‐off at T1 and T2. The present study shows that PR decreases significantly after STEMI, but patients with high PR at baseline tend to maintain a high PR also during a 2‐year follow‐up and they have an over 4‐fold increase in 2‐year cardiovascular event rates compared with those with low PR. This is the first study that includes several PR evaluations across a 2‐year period, two during the acute phase and two during the chronic phase, many months after the index event. As previously reported [8Linden M.D. Furman M.I. Frelinger A.L. Fox M.L. Barnard M.R. Li Y. Przyklenk K. Michelson A.D. Indices of platelet activation and the stability of coronary artery disease.J Thromb Haemost. 2007; 5: 761-5Crossref PubMed Scopus (93) Google Scholar], our study confirms that PR is higher during the hospitalization for STEMI and that it returns to normal values during the follow‐up. Interestingly, this PR decrease is observed, in absolute terms, in all patients, but those with high PR at entry consistently remained in the high PR group. We found that, in patients with STEMI, the cut‐off to distinguish high vs. low PR group changes during the follow‐up, being lower in the acute phase and higher in the chronic phase, but a high number of patients (≈75%; 95% CI, 60–88%) under the cut‐off at the baseline continue to remain under the cut‐off also in the following determinations, showing a worse clinical outcome. Several studies reported PR as independent predictor of myocardial necrosis, myocardial reperfusion and clinical outcome [1Huczek Z. Filipiak K.J. Kochman J. Piatkowski R. Grabowski M. Roik M. Malek L.A. Jaworski P. Opolski G. Baseline platelet reactivity in acute myocardial infarction treated with primary angioplasty influence on myocardial reperfusion, left ventricular performance, and clinical events.Am Heart J. 2007; 154: 62-70Crossref PubMed Scopus (0) Google Scholar, 2Campo G. Valgimigli M. Gemmati D. Percoco G. Tognazzo S. Cicchitelli G. Catozzi L. Malagutti P. Anselmi M. Vassanelli C. Scapoli G. Ferrari R. Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY study.J Am Coll Cardiol. 2006; 48: 2178-85Crossref PubMed Scopus (143) Google Scholar, 3Angiolillo D.J. Fernandez‐Ortiz A. Bernardo E. Alfonso F. Macaya C. Bass T.A. Costa M.A. Variability in individual responsiveness to clopidogrel: clinical implications, management, and future perspectives.J Am Coll Cardiol. 2007; 49: 1505-16Crossref PubMed Scopus (855) Google Scholar, 4Angiolillo D.J. Bernardo E. Sabate M. Jimenez‐Quevedo P. Costa M.A. Palazuelos J. Hernández‐Antolin R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Guzman L.A. Bass T.A. Macaya C. Fernandez‐Ortiz A. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease.J Am Coll Cardiol. 2007; 50: 1541-7Crossref PubMed Scopus (320) Google Scholar, 5Fuchs I. Frossard M. Spiel A. Riedmüller E. Laggner A.N. Jilma B. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS.J Thromb Haemost. 2006; 4: 2547-52Crossref PubMed Scopus (119) Google Scholar]. Our study supports these data and adds two key points: (i) high PR predicts worse 2‐year outcome and its estimation may be performed both at entry and at discharge or later; (ii) the cut‐off value changes according to the timing of PR measurement. In particular, our findings confirm the data reported by Angiolilloet al., where PR measured in diabetic patients during the chronic phase predicts 2‐year adverse events [4Angiolillo D.J. Bernardo E. Sabate M. Jimenez‐Quevedo P. Costa M.A. Palazuelos J. Hernández‐Antolin R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Guzman L.A. Bass T.A. Macaya C. Fernandez‐Ortiz A. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease.J Am Coll Cardiol. 2007; 50: 1541-7Crossref PubMed Scopus (320) Google Scholar]. We may speculate that high PR patients have an interplay between genetic factors (e.g. platelet receptor polymorphisms), concomitant diseases (e.g. diabetes mellitus [4Angiolillo D.J. Bernardo E. Sabate M. Jimenez‐Quevedo P. Costa M.A. Palazuelos J. Hernández‐Antolin R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Guzman L.A. Bass T.A. Macaya C. Fernandez‐Ortiz A. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease.J Am Coll Cardiol. 2007; 50: 1541-7Crossref PubMed Scopus (320) Google Scholar]), or altered drug metabolism (e.g. aspirin, clopidogrel, statins), which all together may determine permanently hyperactive platelets and worse outcome. Recently, prasugrel demonstrated to be more effective at preventing ischemic events than clopidogrel [9Wiviott S.D. Braunwald E. McCabe C.H. Horvath I. Keltai M. Herrman J.P. Van De Werf F. Downey W.E. Scirica B.M. Murphy S.A. Antman E.M. Prasugrel versus clopidogrel in patients with acute coronary syndromes.N Engl J Med. 2007; 357: 2001-15Crossref PubMed Scopus (5507) Google Scholar]. Unfortunately, this beneficial effect is accompanied by an increase in the rate of bleeding [9Wiviott S.D. Braunwald E. McCabe C.H. Horvath I. Keltai M. Herrman J.P. Van De Werf F. Downey W.E. Scirica B.M. Murphy S.A. Antman E.M. Prasugrel versus clopidogrel in patients with acute coronary syndromes.N Engl J Med. 2007; 357: 2001-15Crossref PubMed Scopus (5507) Google Scholar]. In order to preserve the ischemic advantage and minimize the bleeding complications, a more aggressive antiplatelet treatment could be reserved during the acute phase, whereas tailoring the intensity of treatment based on serial PR measurements may optimize the long‐term clinical outcome after STEMI. Our study presents some limitations. Several techniques may be used to measure PR [1Huczek Z. Filipiak K.J. Kochman J. Piatkowski R. Grabowski M. Roik M. Malek L.A. Jaworski P. Opolski G. Baseline platelet reactivity in acute myocardial infarction treated with primary angioplasty influence on myocardial reperfusion, left ventricular performance, and clinical events.Am Heart J. 2007; 154: 62-70Crossref PubMed Scopus (0) Google Scholar, 2Campo G. Valgimigli M. Gemmati D. Percoco G. Tognazzo S. Cicchitelli G. Catozzi L. Malagutti P. Anselmi M. Vassanelli C. Scapoli G. Ferrari R. Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY study.J Am Coll Cardiol. 2006; 48: 2178-85Crossref PubMed Scopus (143) Google Scholar, 3Angiolillo D.J. Fernandez‐Ortiz A. Bernardo E. Alfonso F. Macaya C. Bass T.A. Costa M.A. Variability in individual responsiveness to clopidogrel: clinical implications, management, and future perspectives.J Am Coll Cardiol. 2007; 49: 1505-16Crossref PubMed Scopus (855) Google Scholar, 4Angiolillo D.J. Bernardo E. Sabate M. Jimenez‐Quevedo P. Costa M.A. Palazuelos J. Hernández‐Antolin R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Guzman L.A. Bass T.A. Macaya C. Fernandez‐Ortiz A. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease.J Am Coll Cardiol. 2007; 50: 1541-7Crossref PubMed Scopus (320) Google Scholar, 5Fuchs I. Frossard M. Spiel A. Riedmüller E. Laggner A.N. Jilma B. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS.J Thromb Haemost. 2006; 4: 2547-52Crossref PubMed Scopus (119) Google Scholar, 6Gurbel P.A. Bliden K.P. Hiatt B.L. O’Connor C.M. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity.Circulation. 2003; 107: 2908-13Crossref PubMed Scopus (1443) Google Scholar, 7Angiolillo D.J. Fernandez‐Ortiz A. Bernardo E. Ramírez C. Barrera‐Ramirez C. Sabaté M. Hernández R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Costa M.A. Bass T.A. Macaya C. Identification of low responders to a 300‐mg clopidogrel loading dose in patients undergoing coronary stenting.Thromb Res. 2005; 115: 101-8Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Linden M.D. Furman M.I. Frelinger A.L. Fox M.L. Barnard M.R. Li Y. Przyklenk K. Michelson A.D. Indices of platelet activation and the stability of coronary artery disease.J Thromb Haemost. 2007; 5: 761-5Crossref PubMed Scopus (93) Google Scholar]. Each technique shows advantages and drawbacks, and their knowledge is crucial in order to analyze and understand the data. We used a rapid point‐of‐care test as PFA‐100 because previous studies demonstrated that CADP cartridges are fairly insensitive to the effects of aspirin and thienopyridines [5Fuchs I. Frossard M. Spiel A. Riedmüller E. Laggner A.N. Jilma B. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS.J Thromb Haemost. 2006; 4: 2547-52Crossref PubMed Scopus (119) Google Scholar, 6Gurbel P.A. Bliden K.P. Hiatt B.L. O’Connor C.M. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity.Circulation. 2003; 107: 2908-13Crossref PubMed Scopus (1443) Google Scholar, 7Angiolillo D.J. Fernandez‐Ortiz A. Bernardo E. Ramírez C. Barrera‐Ramirez C. Sabaté M. Hernández R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Costa M.A. Bass T.A. Macaya C. Identification of low responders to a 300‐mg clopidogrel loading dose in patients undergoing coronary stenting.Thromb Res. 2005; 115: 101-8Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Linden M.D. Furman M.I. Frelinger A.L. Fox M.L. Barnard M.R. Li Y. Przyklenk K. Michelson A.D. Indices of platelet activation and the stability of coronary artery disease.J Thromb Haemost. 2007; 5: 761-5Crossref PubMed Scopus (93) Google Scholar, 9Wiviott S.D. Braunwald E. McCabe C.H. Horvath I. Keltai M. Herrman J.P. Van De Werf F. Downey W.E. Scirica B.M. Murphy S.A. Antman E.M. Prasugrel versus clopidogrel in patients with acute coronary syndromes.N Engl J Med. 2007; 357: 2001-15Crossref PubMed Scopus (5507) Google Scholar, 10Geiger J. Teichmann L. Grossmann R. Aktas B. Steigerwald U. Walter U. Schinzel R. Monitoring of clopidogrel action: comparison of methods.Clin Chem. 2005; 51: 957-65Crossref PubMed Scopus (165) Google Scholar] and give an accurate PR evaluation correlated with the clinical outcome [1Huczek Z. Filipiak K.J. Kochman J. Piatkowski R. Grabowski M. Roik M. Malek L.A. Jaworski P. Opolski G. Baseline platelet reactivity in acute myocardial infarction treated with primary angioplasty influence on myocardial reperfusion, left ventricular performance, and clinical events.Am Heart J. 2007; 154: 62-70Crossref PubMed Scopus (0) Google Scholar, 2Campo G. Valgimigli M. Gemmati D. Percoco G. Tognazzo S. Cicchitelli G. Catozzi L. Malagutti P. Anselmi M. Vassanelli C. Scapoli G. Ferrari R. Value of platelet reactivity in predicting response to treatment and clinical outcome in patients undergoing primary coronary intervention: insights into the STRATEGY study.J Am Coll Cardiol. 2006; 48: 2178-85Crossref PubMed Scopus (143) Google Scholar, 3Angiolillo D.J. Fernandez‐Ortiz A. Bernardo E. Alfonso F. Macaya C. Bass T.A. Costa M.A. Variability in individual responsiveness to clopidogrel: clinical implications, management, and future perspectives.J Am Coll Cardiol. 2007; 49: 1505-16Crossref PubMed Scopus (855) Google Scholar, 4Angiolillo D.J. Bernardo E. Sabate M. Jimenez‐Quevedo P. Costa M.A. Palazuelos J. Hernández‐Antolin R. Moreno R. Escaned J. Alfonso F. Bañuelos C. Guzman L.A. Bass T.A. Macaya C. Fernandez‐Ortiz A. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease.J Am Coll Cardiol. 2007; 50: 1541-7Crossref PubMed Scopus (320) Google Scholar, 5Fuchs I. Frossard M. Spiel A. Riedmüller E. Laggner A.N. Jilma B. Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS.J Thromb Haemost. 2006; 4: 2547-52Crossref PubMed Scopus (119) Google Scholar]. Our sample size was adequate to evaluate the PR variations during the follow‐up, but it is too small to validate the long‐term PR predictive role. Our analysis may be considered exploratory. Moreover, our data are obtained in a STEMI population and our cut‐offs are specific for this subset. Our cut‐offs should not apply to stable patients, and future studies are clearly needed. In conclusion, patients with high PR at baseline tended to maintain a high PR also during the follow‐up and had an over 4‐fold increase in 2‐year cardiovascular event rates. The authors state that they have no conflict of interest. The authors thank G. Parrinello (Medical Statistics Unit, University of Brescia, Italy) for his assistance in the statistical analysis.