Drainage fluid LDH and neutrophil to lymphocyte ratio as biomarkers for early detecting anastomotic leakage in patients undergoing colorectal surgery.

OBJECTIVES: In this study, we investigated the role of several circulating and drainage fluid biomarkers for detecting postoperative complications (PCs) and anastomotic leakage (AL) in patients undergoing colorectal surgery. METHODS: All consecutive patients undergoing colorectal surgery between June 2018 and April 2020 were prospectively considered. On postoperative days (POD) 1, 3, and 5, we measured lactate dehydrogenase (LDH) in drainage fluid, C-reactive protein (CRP) in serum and drainage fluid, and neutrophil to lymphocyte ratio (NLR). RESULTS: We enrolled 187 patients. POD1 patients with AL had higher serum CRP levels, while on POD3 and on POD5 higher NLR and serum CRP. LDH and CRP in drainage fluid were also significantly higher at both time points. The area under the curves (AUCs) of serum and drainage fluid CRP were 0.752 (0.629-0.875) and 0.752 (0.565-0.939), respectively. The best cut-off for serum and drainage fluid CRP was 185.23 and 76 mg/dL, respectively. The AUC of NLR on POD3 was 0.762 (0.662-0.882) with a sensitivity and specificity of 84 and 63 %, respectively, at a cut-off of 6,6. Finally, drainage fluid LDH showed the best diagnostic performance for AL, with an AUC, sensitivity, and specificity of 0.921 (0.849-0.993), 82 %, and 90 % at a cut-off of 2,186 U/L. Trends in serum parameters between patients with or without PCs or AL were also evaluated. Interestingly, we found that NLR decreased faster in patients without PCs than in patients with PCs and patients with AL. CONCLUSIONS: Drainage fluid LDH and NLR could be promising biomarkers of PCs and AL.

The role of Killer immunoglobulin-like receptors (KIRs) in the genetic susceptibility to non-celiac wheat sensitivity (NCWS).

OBJECTIVES: Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. METHODS: Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. RESULTS: We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, -2DL3, -2DL5, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, -2DS4, and -2DS5 were independent predictors of NCWS. CONCLUSIONS: Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, -2DS4, and -2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings.

Estimation of the reference values and decision limits for growth hormone in newborns using dried blood spots.

OBJECTIVES: Severe deficiency of growth hormone (GHD) of the newborn is a rare but potentially life-threatening disease. GH measured during the first week of life, using dried blood spots (DBS), may offer several advantages. Aim of the study was to estimate the reference values for GH in newborns by a new analytical method using DBS. METHODS: Using a new developed analytical method, GH was estimated from DBS of 1,036 healthy newborns attending the Neonatology Unit of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico of Milan in the period July-October 2021. Reference values for GH deficiency were estimated by the Harrell-Davis bootstrap method, with 90 %CI calculated by the bias-corrected and accelerated bootstrap method. RESULTS: All GH measurements required 33 analytical sessions (8 months) with a CV% for calibration curve slopes equal to 6.9 %. Intermediate precision evaluated by measurement of low (3 µg/L) and high (10 µg/L) quality controls was, respectively, 14 and 6.5 %. GH reference values, estimated at percentiles 1.0st, 2.5th and 5.0th, and their 90 %CI, were, respectively, 4.5 µg/L (90 %CI 3.8-5.1), 5.9 µg/L (90 %CI 5.4-6.4) and 7.0 µg/L (90 %CI 6.7-7.3). GH levels were not associated with sex, standard deviation scores, birth weight, gestational age, type of delivery or mother's variables (age, smoking habit, gestational diabetes). CONCLUSIONS: Validation data suggest that this method can be used to measured GH in newborns using DBS. The reference values estimated in this study are in accordance with previous published works using ELISA and may help confirming the clinical suspicion of neonatal GHD.

Inducible T-cell co-stimulator (ICOS) and ICOS ligand are novel players in the multiple-myeloma microenvironment.

The inducible T-cell co-stimulator (ICOS) is a T-cell receptor that, once bound to ICOS ligand (ICOSL) expressed on several cell types including the B-cell lineage, plays a decisive role in adaptive immunity by regulating the interplay between B and T cells. In addition to its immunomodulatory functions, we have shown that ICOS/ICOSL signalling can inhibit the activity of osteoclasts, unveiling a novel mechanism of lymphocyte-bone cells interactions. ICOS and ICOSL can also be found as soluble forms, namely sICOS and sICOSL. Here we show that: (i) levels of sICOS and sICOSL are increased in multiple myeloma (MM) compared to monoclonal gammopathy of undetermined significance and smouldering MM; (ii) levels of sICOS and sICOSL variably correlate with several markers of tumour burden; and (iii) sICOS levels tend to be higher in Durie-Salmon stage II/III versus stage I MM and correlate with overall survival as an independent variable. Moreover, surface ICOS and ICOSL are expressed in both myeloma cells and normal plasma cells, where they probably regulate different functional stages. Finally, ICOSL triggering inhibits the migration of myeloma cell lines in vitro and the growth of ICOSL+ MOPC-21 myeloma cells in vivo. These results suggest that ICOS and ICOSL represent novel markers and therapeutic targets for MM.

The multicenter European Biological Variation Study (EuBIVAS): a new glance provided by the Principal Component Analysis (PCA), a machine learning unsupervised algorithms, based on the basic metabolic panel linked measurands.

OBJECTIVES: The European Biological Variation Study (EuBIVAS), which includes 91 healthy volunteers from five European countries, estimated high-quality biological variation (BV) data for several measurands. Previous EuBIVAS papers reported no significant differences among laboratories/population; however, they were focused on specific set of measurands, without a comprehensive general look. The aim of this paper is to evaluate the homogeneity of EuBIVAS data considering multivariate information applying the Principal Component Analysis (PCA), a machine learning unsupervised algorithm. METHODS: The EuBIVAS data for 13 basic metabolic panel linked measurands (glucose, albumin, total protein, electrolytes, urea, total bilirubin, creatinine, phosphatase alkaline, aminotransferases), age, sex, menopause, body mass index (BMI), country, alcohol, smoking habits, and physical activity, have been used to generate three databases developed using the traditional univariate and the multivariate Elliptic Envelope approaches to detect outliers, and different missing-value imputations. Two matrix of data for each database, reporting both mean values, and "within-person BV" (CVP) values for any measurand/subject, were analyzed using PCA. RESULTS: A clear clustering between males and females mean values has been identified, where the menopausal females are closer to the males. Data interpretations for the three databases are similar. No significant differences for both mean and CVPs values, for countries, alcohol, smoking habits, BMI and physical activity, have been found. CONCLUSIONS: The absence of meaningful differences among countries confirms the EuBIVAS sample homogeneity and that the obtained data are widely applicable to deliver APS. Our data suggest that the use of PCA and the multivariate approach may be used to detect outliers, although further studies are required.

Prostate health index (PHI) as a reliable biomarker for prostate cancer: a systematic review and meta-analysis.

OBJECTIVES: Prostate cancer (PCa) represents the second most common solid cancer in men worldwide. In the last decades, the prostate health index (PHI) emerged as a reliable biomarker for detecting PCa and differentiating between non-aggressive and aggressive forms. However, before introducing it in clinical practice, more evidence is required. Thus, we performed a systematic review and meta-analysis for assessing the diagnostic performance of PHI for PCa and for detecting clinically significant PCa (csPCa). METHODS: Relevant publications were identified by a systematic literature search on PubMed and Web of Science from inception to January 11, 2022. RESULTS: Sixty studies, including 14,255 individuals, met the inclusion criteria for our meta-analysis. The pooled sensitivity and specificity of PHI for PCa detection was 0.791 (95%CI 0.739-0.834) and 0.625 (95%CI 0.560-0.686), respectively. The pooled sensitivity and specificity of PHI for csPCa detection was 0.874 (95%CI 0.803-0.923) and 0.569 (95%CI 0.458-0.674), respectively. Additionally, the diagnostic odds ratio was 6.302 and 9.206, respectively, for PCa and csPCa detection, suggesting moderate to good effectiveness of PHI as a diagnostic test. CONCLUSIONS: PHI has a high accuracy for detecting PCa and discriminating between aggressive and non-aggressive PCa. Thus, it could be useful as a biomarker in predicting patients harbouring more aggressive cancer and guiding biopsy decisions.

Monocyte distribution width (MDW) as a screening tool for early detecting sepsis: a systematic review and meta-analysis.

OBJECTIVES: Monocyte distribution has recently emerged as a promising biomarker of sepsis, especially in acute setting, such as Emergency Department and Intensive Care Unit. This study aimed to evaluate the accuracy of monocyte distribution width (MDW) for early detecting patients with sepsis by performing a systemic review and meta-analysis of published studies. METHODS: Relevant publications were identified by a systematic literature search on PubMed and Google Scholar from inception to September 07, 2021. Studies were divided into two groups based on the sepsis criteria applied, namely sepsis-2 or sepsis-3. RESULTS: Ten studies including 9,475 individuals, of whom 1,370 with sepsis (742 according Sepsis-2 and 628 according to Sepsis-3), met the inclusion criteria for our meta-analysis. The pooled sensitivity and specificity were 0.789 and 0.777 for Sepsis-2 criteria, 0.838 and 0.704 for Sepsis-3 criteria. CONCLUSIONS: MDW represents a reliable biomarker for sepsis screening.

Tau protein as a diagnostic and prognostic biomarker in amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: To test the hypothesis that total tau (tTau), tau phosphorylated at threonine 181 (pTau) and pTau/tTau ratio in the cerebrospinal fluid (CSF) are diagnostic and prognostic biomarkers of amyotrophic lateral sclerosis (ALS), we performed a retrospective observational study in a large cohort of ALS patients and controls. METHODS: We enrolled 196 ALS patients and 91 controls, who included patients with ALS-mimicking diseases and those with non-neurodegenerative diseases. All patients underwent lumbar puncture for CSF analysis at the time of the diagnostic evaluation or to first referral. We measured tTau and pTau levels in the CSF by chemiluminescence enzyme immunoassay. RESULTS: Patients with ALS showed significantly higher levels of CSF tTau and a lower pTau/tTau ratio than controls (tTau: 245 vs. 146 pg/ml; p < 0.001; pTau/tTau ratio: 0.12 vs. 0.18; p < 0.001, respectively). No differences in pTau levels were detected. Receiver-operating characteristic curve analysis showed a good diagnostic accuracy of tTau and pTau/tTau ratio (tTau: area under the curve [AUC] 0.685, 95% confidence interval [CI] 0.616-0.754, p = 0.039; pTau/tTau ratio: AUC 0.777, 95% CI 0.707-0.848, p < 0.001). Among ALS patients, increased tTau levels were associated with advanced age of onset, increased revised amyotrophic lateral sclerosis functional rating scale (ALSFRS-R) score (ΔFS) rate of progression, and spinal onset. Multivariate analysis showed that in ALS patients, this biomarker was an independent negative predictor of overall survival. CONCLUSIONS: Our findings suggest that tTau and pTau/tTau ratio can be diagnostic biomarkers of ALS. In addition, CSF tTau level at diagnosis might play a relevant prognostic role in the disease.

A new tool for sepsis screening in the Emergency Department.

OBJECTIVES: In this study, we developed and evaluated the diagnostic accuracy of the Sepsis Index for early sepsis screening in the Emergency Department (ED). METHODS: Sepsis Index is based on the combination of monocyte distribution width (MDW) and mean monocyte volume (MMV). Sepsis Index≥1 was selected to define sepsis. We tested its diagnostic accuracy in an ED population stratified in four groups: controls, Systemic Inflammatory Response Syndrome (SIRS), infection, and sepsis, according to Sepsis-2 criteria. RESULTS: Patients with sepsis displayed higher median Sepsis Index value than patients without sepsis. At the receiver operating characterictis (ROC) curve analysis for the prediction of sepsis, the area under the curve (AUC) of MDW and Sepsis Index were similar: 0.966 (95%CI 0.947-0.984), and 0.964 (95%CI 0.942-0.985), respectively. Sepsis Index showed increased specificity than MDW (94.7 vs. 90.6%), without any decrease in sensitivity (92.0%). Additionally, LR+ increased from 9.8 (MDW) to 17.4 (Sepsis Index), without any substantial change in LR- (respectively 0.09 vs. 0.08). Finally, PPV increased from 0.286 (MDW) to 0.420 (Sepsis Index). CONCLUSIONS: Sepsis Index improves the diagnostic accuracy of MDW alone for sepsis screening.

Monocyte distribution width (MDW) as a screening tool for sepsis in the Emergency Department.

Objectives The diagnosis of sepsis in the Emergency Department (ED) is challenging and a reliable biomarker is needed. The current study aimed to evaluate the diagnostic accuracy of monocyte distribution width (MDW) for the early identification of sepsis in the ED. Methods We performed a large observational study including consecutive adult patients (≥18 years of age) presenting to the ED between September and November 2019, with an order for complete blood count (CBC) evaluation. A total of 2,215 patients were enrolled and classified based on Sepsis-2 criteria as the control group (1,855), infection group (172), Systemic Inflammatory Response Syndrome (SIRS) group (100), and sepsis group (88). Results MDW levels were higher in patients with sepsis than in all other groups (p<0.001). ROC curve analysis showed an optimal diagnostic accuracy of MDW for sepsis prediction at a cut-off point of 23.5, with an AUC of 0.964, sensitivity and specificity of 0.920 and 0.929, respectively. Conclusions Our findings encourage further investigation to validate the use of MDW as a screening tool for the early identification of patients at risk of sepsis in the ED.

Diagnostic accuracy of cerebrospinal fluid biomarkers measured by chemiluminescent enzyme immunoassay for Alzheimer disease diagnosis.

In the last decades, an important role of cerebrospinal fluid (CSF) biomarkers for Alzheimer disease (AD) diagnosis has emerged. The evaluation of the triad consisting of 42 aminoacid-long amyloid-beta peptide (Aß42), total Tau (tTau) and Tau phosphorylated at threonine 181 (pTau) have been recently integrated into the research diagnostic criteria of AD. For a long time, the enzyme-linked immunosorbent assay (ELISA) has represented the most commonly used method for the measurement of CSF biomarkers levels. This study aimed to assess the diagnostic accuracy of CSF biomarkers, namely Aß42, tTau and pTau and their ratio, measured by fully automated CLEIA assay (Lumipulse). We included 96 patients clinically diagnosed as AD (48) and non-AD (48). All CSF biomarkers levels were measured on Lumipulse G1200 fully automated platform (Fujirebio Inc. Europe, Gent, Belgium). Aß42 levels, 42/40 ratio, 42/tTau ratio, 42/PTau ratio were significantly reduced, and tTau and PTau levels were significantly increased in AD patients in comparison with non-AD patients. The receiving operator curve (ROC) analysis showed good diagnostic accuracy of all CSF biomarkers and their ratios for discriminating AD patients from non-AD patients, with 42/40 ratio having the best AUC (0.724, 95%CI 0.619-0.828; p < 0.001). Our findings support the use of CSF biomarkers measured by CLEIA method on a fully automated platform for AD diagnosis.

Reference interval by the indirect approach of serum thyrotropin (TSH) in a Mediterranean adult population and the association with age and gender.

Background The serum concentration of thyrotropin (TSH) represents a first-line test in diagnostic algorithms. The estimation of TSH reference intervals (RIs) is still a matter of debate due to the high prevalence of subclinical disease making difficult the definition of truly healthy subjects. The aim of this study was to estimate TSH RIs in healthy subjects and to evaluate the effect of age and gender on TSH concentration. Methods Forty-four thousand one hundred and fifty-six TSH data were collected between July 2012 and April 2018 at the Department of Laboratory Medicine, University-Hospital, Palermo. Common and sex-specific RIs were estimated by Arzideh's indirect method after exclusion of individuals younger than 15 years, subjects with repeated TSH tests and with abnormal free thyroxine (fT4), free triiodothyronine (fT3) or anti-thyroid-peroxidase antibodies. The combined effect of age and gender on TSH values was evaluated. Results RIs estimated in the selected individuals (n = 22602) were, respectively, 0.18-3.54 mIU/L (general), 0.19-3.23 mIU/L (men) and 0.18-3.94 mIU/L (women). Women showed significantly higher median TSH than men (1.46 vs. 1.39 mIU/L; p < 0.0001). Both in men and in women, median TSH decreased along with age; however, although up to 60 years in both men and women showed similar values, afterwards women showed constantly higher TSH than men. Accordingly, statistical analysis showed a significant interaction between gender and age (p = 0.001), suggesting that the effect of age on TSH is different between genders. Conclusions Our findings suggest that the indirect method, with appropriate cleaning of data, could be useful to define TSH RIs.

Polymorphisms Associated with Increased Cardiovascular Risk in the General Population do not Predict Acute Events in Hemodialysis Patients.

BACKGROUND: Cardiovascular diseases are the leading cause of morbidity and mortality in hemodialysis patients (HP). The aim of the study was to analyze a series of polymorphisms (known to be associated with increased cardiovascular risk in the general population) in HP, in order to better understand the mechanisms of cardiovascular disease and to find new prevention strategies. METHODS: 102 hemodialysis patients were investigated for polymorphisms associated with increased cardiovascular risk in unselected population (FV Leiden R506Q; FV H1299R; FII G20210A; PAI-1 var 4G/5G; GpIIIA T1565C; FXIII var G/T; ß-FIBRINOGENO var G/A; ACE I/D; AGT var M/T; ATR-1A1166C; APOE T112C; APOE T158C; MTHFR C677T; MTHFR A1298C; CBS 844ins68). RESULTS: No difference was observed in the prevalence of the analysed polymorphisms between HP and Caucasian unselected population, with the exception of FV H1299R, PAI-1 (4G/5G), and Factor XIII V34L, which were significantly higher in HP. However none of the genetic factors analysed was associated in HP with the cardiovascular events (non-fatal and fatal) recorded at the time of recruitment or during the eighteen months -follow up. CONCLUSIONS: In HP, the traditional genetic risk factors for cardiovascular disease are not able to predict acute cardiac events, peripheral vascular events, and cerebral vascular events recorded during a follow up period of eighteen months.

A comparison between serum carbohydrate-deficient transferrin and hair ethyl glucuronide in detecting chronic alcohol consumption in routine.

AIMS: In heavy alcohol consumption laboratory tests represent an objective evidence. In this study we compared older and newer biomarkers in blood and in hair. METHODS: Carbohydrate-deficient transferrin (CDT), ethyl glucuronide (EtG), AST, ALT, GGT, MCV were measured in a large sample (n = 562). All people declared no alcohol consumption within the last 3 months. Serum CDT was measured by the candidate HPLC reference method and expressed as relative amount of disialotransferrin (%DST: cutoff 1.7%). EtG was measured in hair by a validated in-house method by LC-MS/MS (cutoff 30 pg/mg). RESULTS: Respectively, 42 (7.5%) and 76 (13.5%) subjects were positive to CDT and EtG. In particular, 30 (5.3%) subjects were positive to both tests, 12 (2.1%) only to CDT, while 46 (8.2%) only to EtG. The agreement (positive and negative pairs) between CDT and EtG was 89.7%. Interestingly, 6 out of 12 (50%) CDT-positive subjects had EtG < 15 pg/mg, whereas 27 out of 46 (59%) EtG-positive subjects had CDT < 1.1%. Forty-one out of 76 (54%) EtG-positive subjects display EtG values within 30-50 pg/mg. CONCLUSION: Large variability exists between CDT and EtG in detecting chronic alcohol consumption. We suggest to use CDT, or a combination of different biomarkers, to identify alcohol abuse in a forensic context. EtG results close to the cutoff (30-50 pg/mg) should be cautiously considered before any sanction is assigned.

Therapeutic drug monitoring of levetiracetam: comparison of a novel immunoassay with an HPLC method.

BACKGROUND: Therapeutic drug monitoring of the anticonvulsant levetiracetam may be indicated in patients with conditions that may alter pharmacokinetic characteristics, for tailoring individual dosage regimens or to investigate patient compliance. In this study, the Bio-Rad high-performance liquid chromatography (HPLC) method (in-use method) and the ARK immunoassay method (new method) for levetiracetam monitoring in serum were compared. METHODS: Levetiracetam concentrations were determined in 63 samples using: (1) "Levetiracetam by HPLC" kit by Bio-Rad (Hercules, CA) on the Agilent 1100 HPLC system, and (2) "ARK Levetiracetam" immunoassay by ARK Diagnostics Inc (Fremont, CA) on the CDx90 platform by ThermoFisher Scientific Inc. RESULTS: Within-laboratory imprecision and bias of the new method evaluated over a 20-day period were 7.4% and 0.5% at 7.5 mcg/mL, 4.5% and 1.9% at 30 mcg/mL, and 3.1% and 2.0% at 75 mcg/mL. Passing-Bablok regression analysis (X:Bio-Rad; Y:Ark) showed a nonsignificant intercept of 0.16 [95% confidence interval (CI), -0.55 to 0.72] and a slope marginally significantly different from unity of 0.95 (95% CI, 0.90-0.99), which suggested minimum proportional systematic error. In agreement, Bland-Altman analysis showed minimum systematic bias of 1.0 mcg/mL (95% CI, 0.32-1.69) with 95% of the HPLC-Ark differences ranging from -4.3 (95% CI, -5.52 to -3.16) to 6.3 (95% CI, 5.16-7.52). Our data showed that the 2 methods were identical both within inherent imprecision and analytical quality specifications (maximum allowable error 15%). CONCLUSIONS: The new Ark method on the CDx platform is acceptable and may be used to measure serum levetiracetam concentrations routinely.

An observational study of Venlafaxine and CYP2D6 in clinical practice.

BACKGROUND: Venlafaxine (V) is a serotonin-norepinephrine selective reuptake inhibitor, mainly metabolized by cytochrome P4502D6 (CYP2D6). CYP2D6 polymorphisms result in a variety of phenotypes: poor (PMs), intermediate (IMs), extensive (EMs), and ultrarapid metabolizers (UMs). PMs usually show poor tolerance to drugs metabolized by CYP2D6, while UMs need greater doses. The aim of this study was to evaluate the impact of CYP2D6 genotype on V dosage, therapeutic response, and side effects in a clinical outpatient setting. METHODS: 47 patients with Major Depressive Disorder, treated with V 75 - 300 mg/day, underwent CYP2D6 genotyping using the INFINITI-CYP2D6 assay. Duration of treatment and clinical outcome (Clinical Global Impression [CGI] effectiveness index) were assessed. RESULTS: CGI assessment was performed after 6 weeks, 6 months, and 1 year of treatment with a V median dose of 150 mg/day. CYP2D6 genotyping resulted in 1 PM, 3 IMs, 42 EMs, and 1 UM. The UM took the greatest V dose (375 mg) without side effects; IMs/PMs took moderate/high doses of V (150 - 300 mg) without adverse effects; EMs displayed high response variability. CONCLUSIONS: PM/IM patients responded to V differently than expected according to genotype. However, the UM patient responded to a dosage higher than the usual therapeutic range and without developing side effects, suggesting an association between CYP2D6 gene duplication and the therapeutic efficacy of venlafaxine. The CYP2D6 genotyping may thus provide clinicians with a potential explanation for those patients requiring greater doses of CYP2D6 substrates in order to obtain the same therapeutic efficacy.

Routine coagulation tests are not useful as a screening tool for the FII G20210A polymorphism.

BACKGROUND: The identification of prothrombin G20210A polymorphism (PT20210) is normally included in the thrombophilia laboratory panel and evaluated by DNA-based molecular analysis. To date, a routine coagulation test that helps to identify PT20210 carriers has not been set, in contrast to the FV Leiden mutation, for which a functional coagulation test, the Activated Protein C Resistance test (APCR), is available as a screening tool. More- over the molecular tests are expensive and are used inappropriately. The aim of the study is to characterize the effects of the prothrombin G20210A mutation on routine clotting assays in order to identify, if any, coagulation tests that can be used as a first-line cost-effective assay for prothrombin G20210A polymorphism. METHODS: Our cohort consisted of 80 PT20210 polymorphism carriers and 82 age and gender matched controls. All subjects were investigated for PT-INR, aPTT, dRVVT, FII (%), and Endogenous Thrombin Potential (EPT) parameters. RESULTS: In heterozygotes and wild-type, PT, aPTT, and dRVVT values were not significantly different. The plasma activity of Factor II (%), AUC TG (%), and C max (%) of EPT were significantly higher in heterozygotes than in controls (p < 0.0001, Mann-Whitney test). In the absence of oral anticoagulant therapy and/or heparin, lupus anticoagulants, and liver disease, the discriminating abilities of the FII, AUC TG, and C max (%) to separate properly the study population into carriers and controls were equal to 0.99 (95% CI 0.98 to 1.00); 0.97 (95% CI 0.94 to 0.99), and 0.84 (95% CI 0.77 to 0.90), respectively. CONCLUSIONS: All routine clotting assays performed in the present work are not useful as a screening tool for the G20210A prothrombin gene allele in a general population. Definitely, to date, the exclusive possible approach to identify the PT20210 mutation is molecular genetic testing, but unfortunately it is used inappropriately, contributing significantly to an uncontrolled waste of resources. It is mandatory to restrict the genetic thrombophilia test ordering to when it is actually recommended by the guidelines and to educate clinicians on the waste and danger of over-testing, particularly genetic tests, taking into account the fact that the PT20210 polymorphism is extremely variable (0.7 to 8% in Europe; 1.3-5% in the USA).