Colonoscopy findings after increasing two-stool faecal immunochemical test (FIT) cut-off: Cross-sectional analysis of the SCREESCO randomized trial.

BACKGROUND: We determined the impact of an increased two-stool faecal immunochemical test (FIT) cut-off on colonoscopy positivity and relative sensitivity and specificity in the randomized controlled screening trial screening of Swedish colons conducted in Sweden. METHODS: We performed a cross-sectional analysis of participants in the FIT arm that performed FIT between March 2014 and 2020 within the study registered with ClinicalTrials.gov, NCT02078804, who had a faecal haemoglobin concentration of at least 10 µg/g in at least one of two stool samples and who underwent a colonoscopy (n = 3841). For each increase in cut-off, we computed the positive predictive value (PPV), numbers needed to scope (NNS), sensitivity and specificity for finding colorectal cancer (CRC) and advanced neoplasia (AN; advanced adenoma or CRC) relative to cut-off 10 µg/g. RESULTS: The PPV for AN increased from 23.0% (95% confidence intervals [CI]: 22.3%-23.6%) at cut-off 10 µg/g to 28.8% (95% CI: 27.8%-29.7%) and 33.1% (95% CI: 31.9%-34.4%) at cut-offs 20 and 40 µg/g, respectively, whereas the NNS to find a CRC correspondingly decreased from 41 to 27 and 19. The PPV for AN was higher in men than women at each cut-off, for example 31.5% (95% CI: 30.1%-32.8%) in men and 25.6% (95% CI: 24.3%-27.0%) in women at 20 µg/g. The relative sensitivity and relative specificity were similar in men and women at each cut-off. CONCLUSION: A low cut-off of around 20-40 µg/g allows detection and removal of many AN compared to 10 µg/g while reducing the number of colonoscopies in both men and women.

A wide scan of plasma proteins demonstrates thioredoxin reductase 1 as a potential new diagnostic biomarker for hepatocellular carcinoma.

BACKGROUND: Patients with liver cirrhosis are recommended ultrasonography screening for early detection of hepatocellular carcinoma to increase the chances of curative treatment. However, ultrasonography alone lacks in sensitivity. Adding plasma biomarkers may increase the detection rate. We performed a broad exploratory analysis to find new plasma proteins with potential applicability for HCC screening in patients with cirrhosis. METHODS: In a protein discovery cohort of 172 patients with cirrhosis or HCC, we screened for 481 proteins with suspension bead array or proximity extension assay. From these, 24 proteins were selected for further analysis in a protein verification cohort (n = 160), using ELISA, Luminex or an electrochemiluminescence platform. A cut-off model and a stepwise logistic regression model were used to find combinations of proteins with the best discriminatory performance between HCC and cirrhosis. RESULTS: Stepwise logistic regression revealed alpha-fetoprotein (AFP), decarboxy-prothrombin (DCP), thioredoxin reductase 1 (TXNRD1), and fibroblast growth factor 21 (FGF21) as the proteins with the best discriminatory performance between HCC and cirrhosis. Adding TXNRD1 to DCP and AFP increased the AUC from 0.844 to 0.878, and combining AFP, DCP and TXNRD1 with age and sex resulted in an AUC of 0.920. FGF21, however, did not further increase the performance when including age and sex. CONCLUSION: In the present study, TXNRD1 improves the sensitivity and specificity of AFP and DCP as HCC screening tools in patients with cirrhosis. We suggest that TXNRD1 should be validated in prospective settings as a new complementary HCC biomarker together with AFP and DCP.

Once-only colonoscopy or two rounds of faecal immunochemical testing 2 years apart for colorectal cancer screening (SCREESCO): preliminary report of a randomised controlled trial.

BACKGROUND: Screening for colorectal cancer is done with lower gastrointestinal endoscopy or stool-based tests. There is little evidence from randomised trials to show primary colonoscopy reduces mortality in colorectal cancer. We aimed to investigate the effect of screening with once-only colonoscopy or two rounds of faecal immunochemical test screening on colorectal cancer mortality and incidence. METHODS: We did a randomised controlled trial in Sweden (SCREESCO). Residents in 18 of 21 regions who were age 60 years in the year of randomisation were identified from a population register maintained by the Swedish Tax Agency. A statistician with no further involvement in the trial used a randomised block method to assign individuals to once-only colonoscopy, two rounds of faecal immunochemical testing (OC-Sensor; 2 years apart), or a control group (no intervention; standard diagnostic pathways), in a ratio of 1:6 for colonoscopy versus control and 1:2 for faecal immunochemical testing versus control. Masking was not possible due to the nature of the trial. The primary endpoints of the trial are colorectal cancer mortality and colorectal cancer incidence. Here, we report preliminary participation rates, baseline findings, and adverse events from March, 2014, to December, 2020, in the two intervention groups after completion of recruitment and screening, up to the completion of the second faecal immunochemical testing round. Analyses were done in the intention-to-screen population, defined as all individuals who were randomly assigned to the respective study group. This study is registered with ClinicalTrials.gov, NCT02078804. FINDINGS: Between March 1, 2014, and Dec 31, 2020, 278 280 people were included in the study; 31 140 were assigned to the colonoscopy group, 60 300 to the faecal immunochemical test group, and 186 840 to the control group. 10 679 (35·1%) of 30 400 people who received an invitation for colonoscopy participated. 33 383 (55·5%) of 60 137 people who received a postal faecal immunochemical test participated. In the intention-to-screen analysis, colorectal cancer was detected in 49 (0·16%) of 31 140 people in the colonoscopy group versus 121 (0·20%) of 60 300 in the faecal immunochemical test group (relative risk [RR] 0·78, 95% CI 0·56-1·09). Advanced adenomas were detected in 637 (2·05%) people in the colonoscopy group and 968 (1·61%) in the faecal immunochemical test group (RR 1·27, 95% CI 1·15-1·41). Colonoscopy detected more right-sided advanced adenomas than faecal immunochemical testing. There were two perforations and 15 major bleeds in 16 555 colonoscopies. No intervention-related deaths occurred. INTERPRETATION: The diagnostic yield and the low number of adverse events indicate that the design from this trial, both for once-only colonoscopy and faecal immunochemical test screening, could be transferred to a population-based screening service if a benefit in disease-specific mortality is subsequently shown. FUNDING: Swedish regions, County Council, Regional Cancer Center Mellansverige, Swedish Cancer Society, Aleris Research and Development Fund, Eiken Chemical.

Cardiac biomarkers in pediatric CKD-a prospective follow-up study.

BACKGROUND: The N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitive cardiac-specific troponin T (hs-cTnT) are associated with abnormal cardiac structure and function and an increased risk of cardiovascular death in chronic kidney disease (CKD) patients. There is limited knowledge about these cardiac markers in pediatric CKD patients. METHODS: Longitudinal levels of NT-proBNP and hs-cTnT were analyzed in 48 pediatric patients, 22 with CKD (GFR range 8.8-68 mL/min/1.73 m2) and 26 transplanted patients (CKD-T; GFR range 30-99 mL/min/1.73 m2). Follow-up was scheduled after 1 and 3 years. Longitudinal patterns and associations to kidney function, cardiovascular risk markers, and echocardiographic parameters were assessed. RESULTS: High NT-proBNP was present in 27% of CKD and 11% of CKD-T patients. Similarly 32% of CKD and 8% of CKD-T patients had elevated hs-cTnT levels. In longitudinal multivariate analyses, high log NT-proBNP was associated with low GFR (ß = - 0.01, p = 0.01) and elevated left ventricular mass index (LVMI; ß = 0.02, p = 0.05). The strong association to LVMI remained when using GFR-adjusted NT-proBNP in similar analysis. Patients with left ventricular hypertrophy (LVH) also had higher NT-proBNP (235 [146-301] ng/L) than patients without LVH (86 [11-477] ng/L), p = 0.02. High hs-cTnT over-time was also associated with low GFR (ß = - 0.007, p = 0.01) and a low cc-TDI e´/a´, indicating a worse LV diastolic function (ß = - 0.09, p = 0.05). This association did not persist for GFR-adjusted hs-cTnT. CONCLUSIONS: NT-proBNP and hs-cTnT are elevated in pediatric CKD and CKD-T patients. GFR-adjusted NT-proBNP was associated with longitudinal levels of elevated LVMI suggesting this might be a marker for early subclinical myocardial damage. A higher resolution version of the Graphical abstract is available as Supplementary information.

High-sensitivity troponins in dialysis patients: variation and prognostic value.

BACKGROUND: Dialysis patients have a high prevalence of cardiovascular mortality but also elevated cardiac troponins (cTns) even without signs of cardiac ischaemia. The study aims to assess variation and prognostic value of high-sensitivity cTnI and cTnT in prevalent dialysis patients. METHODS: In 198 prevalent haemodialysis (HD) and 78 peritoneal dialysis (PD) patients, 4-monthly serum troponin I and T measurements were obtained. Reference change values (RCVs) were used for variability assessment and competing-risk regression models for survival analyses; maximal follow-up was 50 months. RESULTS: HD and PD patients had similar troponin levels [median (interquartile range) troponin I: 25 ng/L (14-43) versus 21 ng/L (11-37), troponin T: 70 ng/L (44-129) versus 67 ng/L (43-123)]. Of troponin I and T levels, 42% versus 98% were above the decision level of myocardial infarction. RCVs were +68/-41% (troponin I) and +29/-23% (troponin T). Increased variability of troponins related to higher age, male sex, protein-energy wasting and congestive heart failure, but not ischaemic heart disease or dialysis form. Elevated troponin T, but not troponin I, predicted death after adjusting for confounders. CONCLUSIONS: A large proportion of prevalent dialysis patients without current established or ongoing cardiac events have elevated levels of high-sensitivity cTns. Mortality risk was doubled in patients with persistently high troponin T levels. The large intraindividual variation of cTns suggests that serial measurements and reference change levels may be used to improve diagnostic utility. However, evidence-based recommendations require more data from large studies of dialysis patients with cardiac events.

Measurement of the alcohol biomarker phosphatidylethanol (PEth) in dried blood spots and venous blood-importance of inhibition of post-sampling formation from ethanol.

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption by action of the enzyme phospholipase D (PLD). PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and forensic applications. However, in blood specimens containing ethanol, formation of PEth may continue after sampling leading to falsely elevated concentrations. This study evaluated the use of dried blood spot (DBS) and microsampling specimens to avoid post-sampling formation of PEth. Filter paper cards and three commercial devices for volumetric microsampling of finger-pricked blood were assessed, using PEth-negative and PEth-positive whole blood fortified with 2 g/L ethanol. PEth (16:0/18:1) was measured by LC-MS/MS. Post-sampling formation of PEth occurred in wet blood and in the volumetric devices, but not filter paper cards, when stored at room temperature for 48 h. Addition of an inhibitor of PLD, sodium metavanadate (NaVO3), eliminated post-sampling formation during storage and drying. In conclusion, the present study confirmed previous observations that PEth can be formed in blood samples after collection, if the specimen contains ethanol. The results further demonstrated that post-sampling formation of PEth from ethanol also occurred with commercial devices for volumetric dried blood microsampling. In order for a PEth result not to be questioned, it is recommended to use a PLD inhibitor, whether venous blood is collected in a vacutainer tube or finger-pricked blood is obtained using devices for dried blood microsampling.

Fecal immunochemical test in cancer screening - colonoscopy outcome in FIT positives and negatives.

Objectives: Fecal immunochemical test (FIT) is used in colorectal cancer (CRC) screening, but evaluations of multiple sample strategies in colonoscopy screening cohorts are rare. The aim of this study was to assess accuracy of FIT for advanced neoplasia (AN) with two fecal samples in a colonoscopy screening cohort. Materials and methods: The study comprised 1155 participants of the colonoscopy arm in SCREESCO (Screening of Swedish Colons, NCT02078804), a randomized controlled study on CRC screening of 60-year-olds from the Swedish average-risk population. Participants provided two FIT samples prior to colonoscopy. First sample, mean of two, and any of the two samples above cut off level were assessed. Colonoscopy findings (CRC, advanced adenoma (AA), AN (CRC + AA) and adenoma characteristics) were evaluated in uni- and multivariable analysis in relation to FIT positivity (at ≥10 µg hemoglobin (Hb)/g). Results: Of 1155 invited, 806 (416 women, 390 men) participated. CRC, AA and non-AA were found in one (0.1%), 80 (9.9%) and 145 (18%), respectively. Sensitivity and specificity for AN were 20%/93%, 25%/92% and 26%/89% for first, mean of two and any of the two samples respectively at cut off level 10 µg/g, corresponding to 60 (74%)-65 (80%) participants with missed AN. The difference in sensitivity between screening strategies was non-significant. The specificity for first sample was significantly higher than for any of the two samples at cut off 10 µg/g (p = .02) and 20 µg/g (p = .04). FIT positivity in participants with adenoma was associated with pedunculated shape (p = .007) and high-risk dysplasia (p = .009). Conclusions: In an average-risk colonoscopy screening cohort of 60-year-olds, sensitivity for AN was modest and did not increase when using two samples instead of one. FIT predominantly detected adenomas with pedunculated shape and high-risk dysplasia, and participants with flat or broad based adenomas may thus be missed in screening.

Gender-specific cut-offs in colorectal cancer screening with FIT: Increased compliance and equal positivity rate.

OBJECTIVE: Using quantitative Faecal Immunochemical Test (FIT) in colorectal cancer screening enables adjustment of the cut-off for a positive test. As men have higher stool blood levels and higher prevalence of colorectal neoplasia, different cut-off levels can be chosen for men and women. We evaluated participation and positivity rates switching from guaiac-based faecal occult blood test (gFOBT) (Hemoccult®) to FIT (OC-Sensor), using gender-specific cut-offs. METHODS: The colorectal cancer screening programme of Stockholm-Gotland, Sweden, started in 2008 and invited individuals aged 60-69 to biennial testing using gFOBT. From 1 October 2015 the test was switched to FIT, with positivity cut-offs of 40 (200) and 80 (400) µg Hb/g (ng/mL) faeces for women and men, respectively. The first year was evaluated for compliance and positivity, number of reminders and incorrect/inadequate tests, compared with gFOBT in the preceding 12-month period. RESULTS: There were 127,030 and 87,269 individuals invited to screening with gFOBT and FIT, respectively. The change of test increased overall participation by 11.9% (95% confidence interval 11.5%-12.3%) from 56.5% to 68.4% ( p < 0.001). The increase was larger in men (14.3%) than women (9.7%), and in those aged 60-64 (14.2%) than those aged 65-69 (8.7%). The positivity rate was 2.6% in women and 2.5% in men. There was a lower rate of reminders and incorrect/inadequate tests with FIT. CONCLUSIONS: Within a well-organised colorectal cancer screening programme, changing the test from gFOBT to FIT markedly increased participation, especially among men, and in the younger age group. With a lower cut-off in women than men, the positivity rate was similar.

Saliva and plasma levels of cardiac-related biomarkers in post-myocardial infarction patients.

AIM: To relate cardiac biomarkers, such as cystatin C and growth differentiation factor-15 (GDF-15) in saliva to myocardial infarction (MI) and to periodontal status, and to investigate the relation between salivary and plasma cardiac biomarkers. MATERIALS AND METHODS: Two hundred patients with MI admitted to coronary care units and 200 matched controls without MI were included. Dental examination and collection of blood and saliva samples was performed 6-10 weeks after the MI for patients and in close proximity thereafter for controls. Analysing methods: ARCHITECT i4000SR, Immulite 2000 XPi or ELISA. RESULTS: The mean age was 62 ± 8 years and 84% were male. Total probing pocket depth, fibrinogen, white blood cell counts and HbA1c were higher in patients than controls. GDF-15 levels correlated with most of the included clinical variables in both study groups. No correlation was found between plasma and saliva levels of cystatin C or GDF-15. CONCLUSION: Salivary cystatin C and GDF-15 could not differentiate between MI patients and controls.

Usefulness of troponin levels below the diagnostic cut-off level for acute myocardial infarction in predicting prognosis in unselected patients admitted to the coronary care unit.

The present study evaluated the prognosis at different troponin concentrations regardless of the underlying diagnosis in an unselected group of patients admitted to the coronary care unit (CCU) and whether the prognosis is dependent on the cause of the troponin increase. Troponin I was measured with the Stratus CS analyzer (Dade Behring) in 468 consecutively and unselected patients admitted to the CCU at Karolinska University Hospital with possible myocardial ischemia lasting <12 hours before arrival. A troponin I concentration below (0.02 to 0.10 microg/L) the diagnostic cut-off level for myocardial infarction predicted mortality after 40 months. In patients with a troponin I level above the diagnostic cut-off level for acute myocardial infarction, the underlying cause of the troponin increase did not affect the prognosis. In conclusion, we report that a troponin I increase below the new cut-off level for myocardial necrosis was associated with increased mortality in unselected patients with possible myocardial ischemia admitted to the CCU at a university hospital. We also observed that the underlying cause of the troponin increase appears to be less important for outcome.

Serum cardiac troponin T, troponin I, plasma BNP and left ventricular mass index in professional football players.

UNLABELLED: Concentrations of cardiac troponins (cTn) in serum or plasma may be elevated in several disease states other than acute coronary syndromes. In heart failure and end stage renal disease, cardiac troponin T (cTnT) correlates positively with left ventricular mass index (LVMI). Exercise-induced elevation of cardiac troponins in well-trained athletes has been confirmed by several reports but the aetiology and clinical significance is unclear. In the present study, we measured baseline concentrations of cardiac markers and investigated whether or not serum cTnT is associated with left ventricular hypertrophy (LVH) in professional football players. METHODS: Twenty-three male professional football players with a mean age of 23 years (range 18-32) were studied. Echocardiography and blood sampling were carried out approx 24h after a training session. Serum cTnT, other cardiac markers and plasma brain natriuretic peptide (BNP) were compared with LVMI. RESULTS: cTnT was only detectable in one subject. The prevalence of elevated cardiac troponin I (cTnI), creatine kinase MB (CKMB) and creatine kinase was higher than for cTnT. cTnI concentrations were higher in football players than in controls. LVMI did not correlate with any of the cardiac markers. Plasma BNP concentrations were normal in all subjects. CONCLUSION: Serum cTnT concentrations were not elevated in healthy professional football players with LVH. This argues against the hypothesis that LVH per se may cause increased cTnT. The finding of higher cTnI in football players than in non-athletic controls should be confirmed and the aetiology elucidated.

A two-center international evaluation of the Immulite 2000 automated serum gastrin assay.

OBJECTIVES: To assess the performance of the Immulite 2000 serum gastrin assay. DESIGN AND METHODS: Two-center validation and comparison with two manual gastrin assays. RESULTS: Serum, serum separator, EDTA and heparin tube results differed <20%. Samples showed <20% degradation for <6 h at room temperature, 3-day-refrigerated, and underwent 3 freeze-thaw cycles. Imprecision was <8% between 20 and 824 ng/L (10-393 pmol/L). Spike recovery was 88.5-106%, and recovery on dilution was 62-135%. There were no interferences from gastrin 1-14, pentagastrin, and cholecystokinin (CCK) 1-33 and CCK 26-33. The fasting reference range was <100 ng/L (<48 pmol/L). Regression slopes to the manual assays were 0.9 and 0.97, with comparable clinical performance. CONCLUSIONS: The Immulite 2000 assay is precise, accurate, and fast and compares well with established gastrin assays.

The new definition of myocardial infarction--can we use it?

BACKGROUND: A joint committee of the European Society of Cardiology and the American College of Cardiology (ESC/ACC) recently redefined myocardial infarction. HYPOTHESIS: The objective of this study was to examine the outcome of diagnoses from more than 500 patients admitted to a university hospital coronary care unit (CCU), when the ESC/ACC committee cut-off levels were compared with the Swedish diagnostic criteria for acute myocardial infarction (MI), comparable with everyday practice in most countries. METHODS: Creatine kinase-MB, troponin I, and troponin T were measured in 525 patients admitted consecutively to the CCU, Huddinge University Hospital, with possible myocardial ischemia lasting <12 h before arrival. RESULTS: The ESC/ACC definition of MI increased the number of MIs by 3-32% compared with the number achieved when Swedish diagnostic criteria for acute MI were used. A significant number of patients with elevated cardiac enzymes presented with acute heart failure, tachycardia, pulmonary embolism, and sepsis as initial symptom. CONCLUSIONS: In this study of more than 500 patients with possible myocardial ischemia admitted consecutively to the CCU at a university hospital, the ESC/ACC definition of MI increased the number of MIs by 3-32% compared with the number achieved when Swedish diagnostic criteria for acute MI were used. A majority of the patients identified with ESC/ACC cut-off levels presented with myocardial ischemia as the primary symptom, whereas many of the other patients had acute heart failure and tachycardia as initial symptom. It is unclear whether patients in this latter group should be labelled as having MI; there are no clinical studies providing guidance in this situation.

Cardiac troponin T content in heart and skeletal muscle and in blood samples from ApoE/LDL receptor double knockout mice.

BACKGROUND: The isolated perfused mouse heart is a useful experimental model, and cardiac troponin T (cTnT) in coronary effluent may be a sensitive marker of myocardial damage. In recent years, the apolipoprotein E/low-density lipoprotein receptor double knockout (apoE/LDLr KO) mice have become valuable tools in atherosclerosis research. The aim of the study was to validate measurements of cTnT in heart, skeletal muscle, and serum of apoE/LDLr KO mice. METHODS: Wild-type C57BL/6J mice were fed with standard diet, and apoE/LDLr KO mice were fed an atherogenic diet. Blood was sampled from the jugular vein or the thoracic cavity. Heart and femoral skeletal muscle were sampled and homogenized. cTnT was measured with the third-generation cTnT assay (Troponin T STAT) on Elecsys 2010 immunoassay analyser (Roche Diagnostics). RESULTS: Median serum cTnT in samples from the thoracic cavity of C57BL/6J mice was about 20-90 times higher, and from ApoE/LDLr KO mice about 30 times higher than serum cTnT in samples from the external jugular vein. There was no difference in cTnT content (microg cTnT/g heart muscle) in hearts from C57BL/6J and apoE/LDLr KO mice. The median cTnT content in skeletal muscle was less than 0.1% of the cTnT content in heart muscle. CONCLUSION: There is no difference in cTnT content of heart muscle comparing C57BL/6J and ApoE/LDLr KO mice, which have larger hearts. Sampling from the thoracic cavity causes unacceptably high cTnT levels. Serum cTnT in samples from the jugular vein is only slightly elevated. Elevated baseline levels of cTnT in mice are not caused by troponin T from skeletal muscle.

Elevated cardiac troponin T in peritoneal dialysis patients is associated with CRP and predicts all-cause mortality and cardiac death.

BACKGROUND: Cardiac troponin T (cTnT) is a highly sensitive and specific marker of myocardial damage. In sera from patients with end-stage renal disease, cTnT may be elevated without other signs of acute myocardial injury. It has been shown that elevated cTnT in haemodialysis patients is associated with poor prognostic outcome. The aim of the present study was to test the hypothesis that elevated cTnT in a single serum sample from peritoneal dialysis (PD) patients is of prognostic importance. METHODS: Blood samples were taken from 26 randomly selected PD patients without signs of acute myocardial ischaemia. Sera were analysed for: cTnT with the second generation TnT ELISA on ES 300; cardiac troponin I (cTnI) with Opus Plus; and for creatine kinase-MB (CKMB) mass and C-reactive protein (CRP). After 4 years, clinical outcomes were evaluated by chart review. The influence on survival was tested with Kaplan-Meier analysis and Cox's proportional regression analysis. RESULTS: Concentrations of cTnT >/=0.04 micro g/l and CRP >/=10 mg/l were strong predictors of all-cause mortality in univariate analysis. Twelve out of 14 patients with cTnT >/=0.04 micro g/l died compared with three out of 12 with cTnT <0.04 micro g/l. Other factors that influenced survival were age and the presence of ischaemic heart disease (IHD). There was a significant positive correlation between cTnT and CRP, and between cTnT and age. Cardiac troponin T was an independent predictor compared with age but not compared with CRP and IHD. Neither cTnI nor CKMB mass concentrations were related to survival. CONCLUSION: Elevated serum concentrations of cTnT significantly predicted poor outcome and there was a correlation between cTnT and CRP concentrations in samples from PD patients. Cardiac troponin I and CKMB mass had no prognostic value.

Spontaneous ischemic events in the brain and heart adapt the hearts of severely atherosclerotic mice to ischemia.

To investigate if spontaneous ischemic events in mice with severe multi-organ atherosclerosis could adapt to ischemia, apolipoprotein E/LDL receptor knockout mice were fed an atherogenic diet for 7 to 9 months. Signs of spontaneous ischemia occurred. One to two days later, hearts were excised, Langendorff-perfused with induced global ischemia, and compared with mice without signs of disease. In vivo heart or brain infarctions were verified by heart histology and/or increased serum levels of cardiac troponin T and S100B. Hearts of mice with spontaneous ischemic events had improved function and reduced Langendorff-induced infarctions. To investigate the remote preconditioning effect of brain ischemia, bilateral ligation of the internal carotid arteries was performed in C57BL6 mice. Twenty-four hours later, their isolated hearts were protected against induced global ischemia. A possible role of inducible NO synthase (iNOS) was studied in iNOS knock out mice, who were not preconditioned by induced brain ischemia. Cardiac iNOS was unchanged 24 hours after preconditioning, suggesting that NO is a trigger rather than a mediator of protection. These findings suggest that spontaneous ischemic events in the brain and heart adapt the heart to ischemia. This can be mimicked by induced brain ischemia, with iNOS as a key factor of protection.