Volume 58, Issue 2

New methods for molecular diagnosis are now available in oncology thanks to the discovery of circulating tumor DNA molecules in the plasma of cancer patients. By utilizing blood samples, rather than traditional tissue sampling, clinical practice is on the verge of new discoveries from the analysis of cell-free DNA (cfDNA). The method, known as a “liquid biopsy”, consists of analyzing therapeutic targets and drug-resistant conferring gene mutations in circulating tumor cells (CTC) and cell-free circulating tumor DNA (ctDNA). These are subsequently released from primary tumors and metastatic deposits into the peripheral blood. The advantages of the method can be observed in the diagnosis, but also in the choice of treatment for solid tumors (e.g. non-small cell lung carcinomas [NSCLC]). In order to interpret the results, an understanding of the biological characteristics of circulating tumor DNA is required. Currently there is no consensus as to how a liquid biopsy should be conducted. In this review, we will assess the pros of ctDNA as analytes in peripheral blood samples and its impact on clinical applications in solid tumors and hematological malignancies. We will also address practical issues facing clinical implementation, such as pre-analytical factors. Moreover, we will emphasize the open questions that remain when considering the current state of personalized medicine and targeted therapy.

Telomere length (TL) is a dynamic marker that reflects genetic predispositions together with the environmental conditions of an individual. It is closely related to longevity and a number of pathological conditions. Even though the extent of telomere research in children is limited compared to that of adults, there have been a substantial number of studies providing first insights into child telomere biology and determinants. Recent discoveries revealed evidence that TL is, to a great extent, determined already in childhood and that environmental conditions in adulthood have less impact than first believed. Studies have demonstrated that large inter-individual differences in TL are present among newborns and are determined by diverse factors that influence intrauterine development. The first years of child growth are associated with high cellular turnover, which results in fast shortening of telomeres. The rate of telomere loss becomes stable in early adulthood. In this review article we summarise the existing knowledge on telomere dynamics during the first years of childhood, highlighting the conditions that affect newborn TL. We also warn about the knowledge gaps that should be filled to fully understand the regulation of telomeres, in order to implement them as biomarkers for use in diagnostics or treatment.

Serum or plasma? An old question looking for new answers. There is a continual debate on what type of sample a clinical laboratory should use. While serum is still considered the gold standard and remains the required sample for some assays, laboratories must consider turn-around time, which is an important metric for laboratory performance and, more importantly, plays a critical role in patient care. In addition, a body of evidence emphasise the choice of plasma in order to prevent modifications of some analytes due to the coagulation process and related interferences. Advantages and disadvantages of serum and plasma are discussed on the basis of current literature and evidence. In addition, data are provided on the current utilisation of the samples (serum or plasma) in Italy and in other countries. Finally, a rationale for a possible switch from serum to plasma is provided.

As it is common practice for laboratories to store patient samples for a predefined period, allowing clinicians to request additional tests on previously collected samples, knowledge about sample stability is indispensable for the laboratorian. A common approach to estimating the maximum storage time is to use a discrete study design, measuring the analyte of interest at various time-points and then checking for significant differences with the help of a statistical test, such as Student’s t-test, Wilcoxon’s test or an analysis of variance (ANOVA) test. Because only discrete time intervals are considered, stability data can just be approximated. Alternatively, a continuous study design, as described by the Clinical and Laboratory Standards Institute (CLSI) for performing stability experiments for in vitro diagnostic reagents, can also be adopted by the clinical laboratory to evaluate the stability of biological samples. The major advantage of this approach is that it allows laboratories to define individual stability limits for different medical situations and offers more flexibility when choosing time-points for measurements. The intent of this paper is to demonstrate the evaluation of sample stability in the clinical laboratory with a continuous study design implemented with linear or non-linear regression analysis. Appropriate statistical modeling and acceptance criteria are presented, stability functions are described briefly, and checking the overall validity of the results is discussed.

Background Simultaneous measurement of 25(OH)D and 24,25(OH)2D is a new tool for predicting vitamin D deficiency and allows evaluating CYP24A1 lack of function. Interpretation of 24,25(OH)2D should be performed according to 25(OH)D levels and a ratio, called the vitamin D metabolite ratio (VMR) has been proposed for such a purpose. Unfortunately, the VMR can be expressed in different ways and cannot be used if 24,25(OH)2D concentrations are undetectable. Here, we propose evaluating the enzyme activity taking into consideration the probability that a normal population presents undetectable 24,25(OH)2D concentrations according to 25(OH)D levels. We thus retrospectively measured 25(OH)D and 24,25(OH)2D in a population of 1200 young subjects to evaluate the 25(OH)D threshold above which the enzyme was induced. Methods Serum samples from 1200 infants, children, adolescent and young adults were used to simultaneously quantify 25(OH)D and 24,25(OH)2D by LCMS/MS. Results Median (interquartile range [IQR]) levels were 20.6 (14.4–27.2) ng/mL for 25(OH)D. 172 subjects (14.3%) presented 24,25(OH)2D values below the LOQ. When 25(OH)D values were <11 ng/mL, 63.1% of subjects presented undetectable 24,25(OH)2D concentrations. Percentage decreased with increasing 25(OH)D values to become 19.7% for 25(OH)D comprised between 12 and 15 ng/mL, 5.1% for 25(OH)D between 16 and 20 and 0.7% for 25(OH)D >21 ng/mL. Conclusions We suggest using a statistical approach to evaluate CYP24A1 function according to 25(OH)D concentrations. Our results also show that vitamin D deficiency, as defined biochemically, could be around 20 ng/mL in infants, children, adolescent and young adults and that vitamin D deficiency could be evaluated on a more individual basis.

Background In 2014, the Joint Croatian Working Group (JCWG) for laboratory diagnostic of chronic kidney disease (CKD) conducted a survey across medical-biochemistry laboratories which demonstrated a large heterogeneity in this area of laboratory medicine. To ensure the tools for the standardization process, in 2017 the JCWG-CKD published the first Croatian recommendations for laboratory diagnostics of CKD. To assess the implementation process, we have repeated a survey to explore how well laboratories adhere to the recommendations. Methods An invitation to the survey was sent to all Croatian medical-biochemistry laboratories (n = 196). The questionnaire was designed in a form of 19 questions and statements, with possible multiple answers. Results The response rate was 98/196 (50.0%). The predominant method for serum creatinine measurement was the standardized compensated Jaffe method (79.2%). There was substantial decrease in the number of laboratories which measure creatinine with the non-standardized uncompensated Jaffe method, compared with the initial 2014 assessment; 7% vs. 40%, respectively. The number of the laboratories that did not report estimated glomerular filtration rate (eGFR) values decreased almost by half compared to the initial data (37.6% vs. 74.4%). However, compared to the 2014 initial assessment, a similar number of laboratories (54/98 vs. 58/80) did not measure urine albumin or protein. Conclusions The collected data showed a substantial improvement in the standardization of the serum creatinine measurement, as well as in the reporting of eGFR. However, albuminuria or proteinuria assessment is still not implemented nationwide, mainly in primary health care laboratories. This demonstrates the importance of promoting and monitoring implementation of guidelines after publication.

Background Lactate dehydrogenase (LD) activity is routinely monitored for therapeutic risk stratification of malignant diseases, but is also prone to preanalytical influences. Methods We systematically analyzed the impact of defined preanalytical conditions on the hemolysis-susceptible parameters LD, potassium (K) and hemolysis index in vacuum blood collection tubes (serum [SE], heparin plasma [HP]). Blood was collected by venipuncture from healthy volunteers. Tubes were either filled or underfilled to approximately 50%, then processed directly or stored at room temperature for 4 h. Potassium (K), sodium (Na), chloride (Cl), LD, creatine kinase (CK), total cholesterol, and indices for hemolysis, icterus, and lipemia were analyzed. Filling velocity was determined in a subset of tubes. Findings in healthy volunteers were reconfirmed in an in-patient cohort (n = 74,751) that was analyzed for plasma yield and LD data distribution. Results LD activity was higher in HP compared to SE. Underfilling led to higher LD values (SE: +21.6%; HP: +28.3%), K (SE: +4.2%; HP: +5.3%), and hemolysis index (SE: +260.8%; HP: +210.0%), while other analytes remained largely unchanged. Filling velocity of tubes was approximately 3-fold higher in the first half compared to the second half in both HP and SE collection tubes. Importantly, plasma yield also inversely correlated with LD in routine patients. By calculating reference limits, the lowest plasma yield quartile of the patient cohort displayed LD values clearly exceeding current reference recommendations. Conclusions Underfilling of tubes leads to a higher proportion of blood aspirated with high velocity and relevant elevations in LD. This finding should be considered in cases of clinically implausible elevated LD activities.

Background Total blood calcium (TCa) is routinely used to diagnose and manage mineral and bone metabolism disorders. Numerous laboratories adjust TCa by albumin, though literature suggests there are some limits to this approach. Here we report a large retrospective study on agreement rate between ionized calcium (iCa) measurement and TCa or albumin-adjusted calcium measurements. Methods We retrospectively selected 5055 samples with simultaneous measurements of iCa, TCa, albumin and pH. We subgrouped our patients according to their estimated glomerular filtration rate (eGFR), albumin levels and pH. We analyzed each patient’s calcium state with iCa as reference to determine agreement rate with TCa and albumin-adjusted calcium using Payne, Clase, Jain and Ridefelt formulas. Results The Payne formula performed poorly in patients with abnormal albumin, eGFR or pH levels. In patients with low albumin levels or blood pH disorders, Payne-adjusted calcium may overestimate the calcium state in up to 80% of cases. Similarly, TCa has better agreement with iCa in the case of hypoalbuminemia, but performed similarly to the Payne formula in patients with physiological albumin levels. The global agreement rate for Clase, Jain and Ridefelt formulas suggests significant improvement compared to Payne calcium adjustment but no significant improvement compared to TCa. Conclusions Total and albumin-adjusted calcium measurement leads to a misclassification of calcium status. Moreover, accurate calcium state determination depends on blood pH levels, whose measurement requires the same pre-analytical restrictions as iCa measurement. We propose that iCa should instead become the reference method to determine the real calcium state.

Background Blood loss from the gastrointestinal (GI) tract is the most common cause of iron deficiency anaemia (IDA) in adult men and postmenopausal women. Gastroduodenal endoscopy (GDE) and colonoscopy are frequently recommended, despite uncertainty regarding the coexistence of lesions in the upper and lower GI tract. The faecal immunochemical test (FIT) measures the concentration of faecal haemoglobin (f-Hb) originating only from the colon or rectum. We aimed to assess whether the FIT was able to select the best endoscopic procedure for detecting the cause of IDA. Methods A prospective study of 120 men and postmenopausal women referred for a diagnostic study of IDA were evaluated with an FIT, GDE and colonoscopy. The endoscopic finding of a significant upper lesion (SUL) or a significant bowel lesion (SBL) was considered to be the cause of the IDA. Results The diagnoses were 35.0% SUL and 20.0% SBL, including 13.3% GI cancer. In the multivariate analysis, the concentration of blood haemoglobin (b-Hb) <9 g/dL (OR: 2.60; 95% CI 1.13–6.00; p = 0.025) and non-steroidal anti-inflammatory drugs NSAIDs (2.56; 1.13–5.88; p = 0.024) were associated with an SUL. Age (0.93; 0.88–0.99; p = 0.042) and f-Hb ≥ 15 μg Hb/g faeces (38.53; 8.60–172.50; p < 0.001) were associated with an SBL. A “FIT plus gastroscopy” strategy, in which colonoscopy is performed only when f-Hb ≥15 μg Hb/g faeces, would be able to detect 92.4% of lesions and be 100% accurate in the detection of cancer while avoiding 71.6% of colonoscopies. Conclusions The FIT is an accurate method for selecting the best endoscopy study for the evaluation of IDA. An FIT-based strategy is more cost-effective than the current bidirectional endoscopy-based strategy and could improve endoscopic resource allocation.

Background Despite that measurement uncertainty data should facilitate an appropriate interpretation of measured values, there are actually few reported by clinical laboratories. We aimed to estimate the measurement uncertainty of some β-lactam antibiotics (β-LA), and to evaluate the impact of reporting the measurement uncertainty on clinicians’ decisions while guiding antibiotic therapy. Methods Measurement uncertainty of β-LA (aztreonam [ATM], cefepime [FEP], ceftazidime [CAZ], and piperacillin [PIP]) values, obtained by an UHPLC-MS/MS based-method, was estimated using the top-down approach called the single laboratory validation approach (EUROLAB guidelines). Main uncertainty sources considered were related to calibrators’ assigned values, the intermediate precision, and the bias. As part of an institutional program, patients with osteoarticular infections are treated with β-LA in continuous infusion and monitored to assure values at least 4 times over the minimal inhibitory concentration (4×MIC). We retrospectively evaluated the impact of two scenarios of laboratory reports on clinicians’ expected decisions while monitoring the treatment: reports containing only the β-LA values, or including the β-LA coverage intervals (β-LA values and their expanded measurement uncertainties). Results The relative expanded uncertainties for ATM, FEP, CAZ and PIP were lower than 26.7%, 26.4%, 28.8%, and 25.5%, respectively. Reporting the measurement uncertainty, we identified that clinicians may modify their decision especially in cases where 4×MIC values were within the β-LA coverage intervals. Conclusions This study provides a simple method to estimate the measurement uncertainty of β-LA values that can be easily applied in clinical laboratories. Further studies should confirm the potential impact of reporting measurement uncertainty on clinicians’ decision-making while guiding antibiotic therapy.

Background Our laboratory obtained the ISO 15189 accreditation for the plasmatic HIV-1, HBV and HCV viral load (VL) using the m2000 RealTi m e™ system, which was recently changed for the platform Panther ® . Here, we discuss a strategy for performing method validation/verification very quickly. Methods We performed the mandatory (repeatability, internal quality assessment [IQA], measurement uncertainty [MU]) and optional technical verifications for CE/IVD assays using the flexible scope range A. We also performed the mandatory assays for the validation of HIV-1 VL in the cerebrospinal fluid (CSF) using the flexible scope range B. The change was checked by following up on the turnaround time (TAT). Results The coefficient of variation (CV%) for repeatability and IQA complied with the limit of 0.25 log. The MU results ranged from 0.04 to 0.25 log copies or IU/mL. The comparisons of methods showed excellent correlations (R 2  = 0.96 for the three parameters) but a delayed centrifugation on HCV VL showed variations of up to 2 log IU/mL. An excellent linearity for HIV-1 in the CSF was obtained from 1.5 to 5 log copies/mL with R 2  = 0.99. The TAT increased (84%–98%) in routine usage. Conclusions The three Aptima assays are well suited for routine laboratory use and can be integrated within less than 2 weeks in accordance with flexible scope range A. Our data allows us to confidently perform HIV-1 VL in CSF following flexible scope range B. Finally, we provide an organizational guide for flexible scope management in molecular virology within a short time frame.

Background Neurofilament light chain (NfL) is a neuron-specific biomarker with prognostic ability in several types of central nervous system injuries. This study investigates if plasma NfL (pNfL) is elevated early after spontaneous intracerebral hemorrhage (ICH) and whether such elevation reflects disease severity and day-30 outcome. Methods pNfL was quantified by single molecule array analysis in 103 reference subjects (RS) and in samples from 37 patients with ICH obtained on admission to hospital and at 24-h follow-up. The primary outcome was day-30 mortality. Clinical status on admission was evaluated by standardized scoring systems. Results Median pNfL among RS was 9.6 (interquartile range [IQR] 6.2) pg/mL. Upon admission, ICH patients had pNfL of 19.8 (IQR 30.7) pg/mL increasing to 35.9 (IQR 44.5) pg/mL at 24 h (all, p < 0.001). On admission, pNfL was higher among ICH non-survivors than survivors (119.2 [IQR 152.6] pg/mL vs. 15.7 [IQR 19.6] pg/mL, p < 0.01) and this difference was observed also on 24 h follow-up (195.1 [IQR 73.9] pg/mL vs. 31.3 [IQR 27.8] pg/mL, p < 0.01). The area under the receiver operating characteristic curve (ROC AUC) for discrimination of day-30 mortality was significant on admission (AUC = 0.83, 95% confidence interval [CI]: 0.56–1.0) and increased on 24-h follow-up (AUC = 0.93, 95% CI: 0.84–1.0). The odds ratio (OR) for death, by each quartile increase in pNfL was significant both on admission (OR = 4.52, 95% CI: 1.32–15.48) and after 24-h follow-up (OR = 9.52, 95% CI: 1.26–71.74). Conclusions PNfL is associated with day-30 mortality after spontaneous ICH when early after the ictus.

Background Recently, several manufacturers have launched automated urinalysis platforms. This study aimed to compare the diagnostic performance of the UF-5000 (Sysmex Corporation, Kobe, Japan) and the cobas ® u 701 (Roche Diagnostics, Rotkreuz, Switzerland) urine sediment analyzers with manual phase-contrast microscopy as the reference method. Methods A total of 195 urine samples were analyzed on both automated platforms and subjected to manual microscopic examination. Agreement was assessed by Cohen’s kappa (κ) analysis. Sensitivities and specificities were calculated. Results The agreement of the UF-5000 with manual microscopy was almost perfect (κ > 0.8) for red (RBC) and white blood cells (WBC), renal tubular epithel cells, hyaline casts, bacteria (BACT) and yeast (YLC), substantial (κ = 0.61–0.80) for squamous epithel cells (SEC) and pathologic casts, and moderate (κ = 0.41–0.60) for transitional epithel cells. The cobas ® u 701 showed substantial agreement (κ = 0.61–0.80) for WBC, moderate agreement (κ = 0.41–0.60) for hyaline casts, and fair agreement (κ = 0.21–0.40) for RBC, SEC, non-squamous epithel (NEC), pathologic casts, BACT and YLC. The UF-5000 sensitivities ranged between 98.5% for RBC and 83.3% for pathological casts. The cobas ® u 701 showed sensitivities between 83.0% for WBC and 31.6% for YLC. Conclusions The UF-5000 (Sysmex) analyzer showed a better diagnostic agreement with manual phase-contrast microscopy compared to the cobas ® u 701 (Roche) module. The Sysmex platform showed reliable results for urine sediment analysis. However, pathological samples should be verified with manual microscopy.

Background The aim of our work was to develop and validate a hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) methods for the quantification of tobramycin (TMC) and lincomycin (LMC)in plasma, microdialysis fluid and urine. Methods Protein precipitation was used to extract TMC and LMC from plasma, while microdialysis fluid and urine sample were diluted prior to instrumental analysis. Mobile phase A consisted of 2 mM ammonium acetate in 10% acetonitrile with 0.2% formic acid (v/v) and mobile phase B consisted of 2 mM ammonium acetate in 90% acetonitrile with 0.2% formic acid (v/v). Gradient separation (80%–10% of mobile phase B) for TMC was done using a SeQuant zic-HILIC analytical guard column. While separation of LMC was performed using gradient elution (100%–40% of mobile phase B) on a SeQuant zic-HILIC analytical column equipped with a SeQuant zic-HILIC guard column. Vancomycin (VCM) was used as an internal standard. A quadratic calibration was obtained over the concentration range for plasma of 0.1–20 mg/L for TMC and 0.05–20 mg/L for LMC, for microdialysis fluid of 0.1–20 mg/L for both TMC and LMC, and 1–100 mg/L for urine for both TMC and LMC. Results For TMS and LMC, validation testing for matrix effects, precision and accuracy, specificity and stability were all within acceptance criteria of ±15%. Conclusions The methods described here meet validation acceptance criteria and were suitable for application in a pilot pharmacokinetic research study performed in a sheep model.

Background Neurological complications are common in the premature and full-term neonates admitted to the intensive care unit, but the diagnosis of these complications is often difficult to make. S100B protein, measured in cord blood, may represent a valuable tool to better identify patients at risk of brain injury. Methods As a first step, we established S100B cord blood serum reference intervals from 183 preterm and 200 full-term neonates. We then measured cord blood serum S100B to identify neurological complications in 272 neonates hospitalized at the neonatal intensive care unit (NICU). Diagnosis of brain injury relied on imaging examination. Results The 95th percentiles of S100B concentration in cord blood were established as 1.21 μg/L for the 383 neonates, 0.96 μg/L for full-term neonates and 1.36 μg/L for premature neonates. Among the 272 neonates hospitalized at the NICU, 11 presented neurological complications. Using 1.27 μg/L as the optimal sensitivity/specificity threshold, S100B differentiate neonates with and without neurological complications with a sensitivity of 45.5% (95% confidence intervals [CI]: 16.7–76.6) and a specificity of 88.9% (95% CI: 84.4–92.4) (p = 0.006). In combination with arterial pH (<7.25), sensitivity increased to 90.9% (95% CI: 58.7–99.8), while specificity was 51.2% (95% CI: 44.8–57.7). The sensitivity is significantly (p = 0.03) increased in comparison to S100B alone. The specificity is significantly higher with S100B only than with pH + S100B (p < 0.001). Conclusions Cord blood S100B protein, in combination with arterial cord blood pH, has the potential to help clinicians to detect at birth neurological complications in neonates hospitalized in an NCIU.

Background Regulatory bodies recommend the use of an assay based on the assessment of the endogenous thrombin potential (ETP) for the investigation of the activated protein C resistance (APCr) in the development of steroid contraceptives in women. However, the assays described in the literature are home-made and not standardized regarding the method, the reagents, the reference plasma and the quality controls. In the absence of any commercially available method, we aimed at validating the ETP-based APCr assay. Methods The validation was performed according to regulatory standards. The method targets a 90% inhibition of the ETP in healthy donors in the presence of APC compared to the same condition in the absence of APC. As a large-scale production of a pool of plasma from well-selected healthy donors is impossible, algorithms were applied to a commercial reference plasma to correlate with the selected pool. Results Repeatability and intermediate precision passed the acceptance criteria. The assay demonstrated a curvilinear dose response to protein S and APC concentrations (R 2  > 0.99). Analysis of plasma samples from 47 healthy individuals (22 women not taking combined hormonal contraceptives [CHC], and 25 men not Factor V Leiden carriers) confirmed the validity of the test, with a mean inhibition percentage of 90%. Investigations in 15 women taking different contraceptives and in two subjects with Factor V Leiden confirmed the good sensitivity and performance of the assay. Conclusions This validation provides the pharmaceutical industry, the regulatory bodies and physicians with a reproducible, sensitive and validated gold-standard ETP-based APCr assay.

Background Non-small cell lung cancer (NSCLC) patients benefit from targeted therapies both in first- and second-line treatment. Nevertheless, molecular profiling of lung cancer tumors after first disease progression is seldom performed. The analysis of circulating tumor DNA (ctDNA) enables not only non-invasive biomarker testing but also monitoring tumor response to treatment. Digital PCR (dPCR), although a robust approach, only enables the analysis of a limited number of mutations. Next-generation sequencing (NGS), on the other hand, enables the analysis of significantly greater numbers of mutations. Methods A total of 54 circulating free DNA (cfDNA) samples from 52 NSCLC patients and two healthy donors were analyzed by NGS using the Oncomine™ Lung cfDNA Assay kit and dPCR. Results Lin’s concordance correlation coefficient and Pearson’s correlation coefficient between mutant allele frequencies (MAFs) assessed by NGS and dPCR revealed a positive and linear relationship between the two data sets (ρc = 0.986; 95% confidence interval [CI] = 0.975–0.991; r = 0.987; p < 0.0001, respectively), indicating an excellent concordance between both measurements. Similarly, the agreement between NGS and dPCR for the detection of the resistance mutation p.T790M was almost perfect (K = 0.81; 95% CI = 0.62–0.99), with an excellent correlation in terms of MAFs (ρc = 0.991; 95% CI = 0.981–0.992 and Pearson’s r = 0.998; p < 0.0001). Importantly, cfDNA sequencing was successful using as low as 10 ng cfDNA input. Conclusions MAFs assessed by NGS were highly correlated with MAFs assessed by dPCR, demonstrating that NGS is a robust technique for ctDNA quantification using clinical samples, thereby allowing for dynamic genomic surveillance in the era of precision medicine.

Background Previous studies have suggested that exercising may induce cardiac damage. Galectin-3 (Gal-3) and soluble suppression of tumorigenicity 2 (ST2) are very interesting biomarkers for heart failure and myocardial fibrosis. We aimed to compare the kinetics of emerging fibrosis cardiac biomarkers as Gal-3 and ST-2 in endurance runners, and recreational runners before and after a running event represented by a marathon and an ultratrail event. Methods Blood samples were taken from 19 healthy non-elite marathon runners (42 km), 27 ultratour runners (67 km), and 14 recreational runners who represented the control group (10 km) just before the run (T0), just after (T1) and 3 h after (T2), in order to analyze Gal-3, ST2, hsTnT, NT-proBNP, CKMB and hsCRP. We compared the percentage of evolution and the slopes obtained from T0 to T1 (pT0T1) and from T1 to T2 (pT1T2), between the different groups of runners participating in three different races. Results Plasma cardiac biomarker concentrations increased significantly from baseline to immediately post-exercise and most of the time decreased over the subsequent 3-h period. For pT0T1 and pT1T2, the markers Gal-3 and ST2 showed a significant difference between types of run (p < 0.05 and p < 0.0001, respectively). During the recovery time, Gal-3 returned to the baseline values but not ST2 which continued to increase. Conclusions Gal-3 and ST2 are considered as a reflection of cardiac fibrosis and remodeling. The evolution of both was different, particularly after the recovery time. ST2 values exceeding cutoff values at any time.