49th National Congress of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC – Laboratory Medicine)

UNCONFORMITIES IN DIRECT ORAL ANTICOAGULANTS REQUESTS

P. Calzoni¹, D. Fineschi¹, E. Franceschini¹, A. Silvietti¹, D. Vannoni^1,2, R. Cappelli³, C. Bellini^1,2, L. Terzuoli^1,2, C. Scapellato¹

¹UOC Patologia Clinica, Azienda Ospedaliera Universitaria Senese

²Dip. di Biotecnologie Mediche, Università di Siena

³Dip. Scienze Mediche, Chirurgiche e Neuroscienze, Università di Siena

Introduction: Classic dicumarolics have been replaced by direct oral anticoagulants (DOACs), a new generation of drugs. The Clinical Pathology Laboratory of Siena’s University Hospital introduced a specific assay for DOACs since 2016. In order to orient clinicians towards an appropriate use of requests, an operative protocol has been drawn up, in collaboration with the Thrombosis Centre of the hospital, providing indication about timing and request procedures.

Objective: To assess the protocol effectiveness one year after, analyzing the number and type of unconformities in DOACs requests.

Methods: Thrombin Time Diluted assay for dabigatran and chromogenic anti-Xa activity assay for rivaroxaban and apixaban on BCS XP (Siemens) instrumentation.

Results: To this day we received 15 requests for rivaroxaban, 13 for apixaban and 18 for dabigatran. The overall unconformities were 12: 2 erroneous requests (the patient was not taking the drug); 1 sample not received; 3 samples collected in wrong container; 4 haemolysed or inappropriate sample-anticoagulant volume ratio; 1 double request for the same patient; 1 failure to comply with the operative protocol. In the latter case, despite of the rivaroxaban interruption for three days, its plasmatic levels were still inexplicably high (30 ng/ml) because of the undeclared administration of heparin. It is well known that heparin causes positive interference in rivaroxaban assay, a detail missed by the clinician, although specified in the protocol.

Conclusions: The few number of DOACs requests collected in one year demonstrates both that the patient in DOACs therapy management is considerably simplified and the improvement in request’s appropriateness. The few pre-analytical unconformities are operator dependent, related to difficult blood sampling or erroneous data entry. Our work focuses on the failure to comply with the operative protocol, even though happened only once, with the aim to spread the protocol to the clinicians concerned through personalized audit.

MOLECULAR ADAPTATION INDUCED BY FOOTBALL TRAINING IN SKELETAL MUSCLE: INFLUENCE ON HUMAN HEALTH AND LONGEVITY

A. Mancini^1,2, D. Vitucci³, M.B. Randers⁴, J.F. Schmidt⁴, M. Hagman⁴, T. Rostgaard⁴, E. Imperlini³, S. Orrù^1,2,3, P. Krustrup^4,5, F. Salvatore², P. Buono^1,2,3

¹Dipartimento di Scienze Motorie e del Benessere, Università “Parthenope”, Naples, Italy

²CEINGE-Biotecnologie avanzate, Naples, Italy

³IRCCS SDN, Naples, Italy

⁴Copenhagen Centre for Team Sport and Health, Department of Nutrition, Exercise and Sports, University of Copenhagen, Copenhagen, Denmark

⁵Health Sciences, College of Life and Environmental Sciences, St. Luke’s Campus, University of Exeter, Exeter, UK

Aim: Football training improves cardiorespiratory (VO₂max) and body composition in players (1). Recently, we demonstrated that lifelong football training improves the expression of molecular markers related to oxidative metabolism and senescence suppression in muscle of veteran players (2). Here we performed a comparative analysis of messenger expression profiles in muscle biopsies from veteran soccer players (VSP) versus age-matched healthy untrained (CG; 65-75y) males.

Methods: Total RNA was obtained from 6 VSP and 6 CG muscle biopsies, provided by Copenhagen group; RNA integrity number (RIN) was determined on all samples. Single strand biotinylated cDNA was generated and used to hybridize the Human Genechip HTA 2.0 Array (Affymetrix). Results were filtered for fold change >1.5; statistical analysis was performed by ANOVA test. Validation of differently expressed messengers was performed by RTqPCR on total RNA from 12 VSP and 12 CG muscle biopsies.

Results: 430 messengers (including small non coding RNAs, miRNAs) differentially expressed between groups were identified (p-value <0.05). Among them, we confirmed an increased expression of RAD23A, HSPB6, HSPB1 (HSP25), TRAP1, Sirt2, RAB1B messengers, belonging to auto-lysosomal and proteasome degradation pathways and involved in protein integrity process and of RPL1, RPL4, RPL36, MRLP37, ribosomal messengers, in muscle from VPG compared to CG subjects.

Conclusions: Our preliminary results indicates that Lifelong football training positively affects the regulation of messengers involved in the integrity and function of nervous and muscular tissues and in cellular growth and differentiation pathways, respectively, in VPG. Our results also suggests that football training would be used as a tool to prevent and delay age-related decline.

1. Krustrup et al. 2010.

2. Mancini A, Vitucci D, Labruna G, et al. Eur J Appl Physiol 2017;117:721-30.

EFFICACY OF AN INTENSIVE PHYSICAL RETRAINING PROGRAM IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)

G. Calcagno

University of Molise, Italy

Chronic obstructive pulmonary disease (COPD) is the most common chronic lung disease, and a major cause of lung-related death and disability. This pathology is characterized by chronic airflow limitation associated with an abnormal inflammatory reaction (1). It is also characterized by very disabling and features extra-pulmonary manifestations. An important tool to manage this pathology is the exercise retraining, that is the cornerstone of pulmonary rehabilitation programs because offers many benefits, all well documented in patients with COPD. Physical training improving exercise capacity, muscle strength, body composition, cognitive capacity and quality life, but still there is no consensus about the optimal training strategy, same problem persists to define the ideal working intensity. Some studies report the efficacy of combined training: aerobic exercise associated with strength training, performed at high intensity, for the additional benefits that occurs (2). Aerobic workout provides benefits for exercise tolerance and cognitive capacity while strength training improving peripheral muscle function, body composition and same cognitive domains (3). The high intensity exercise in COPD, offers greater benefits in less time, compared with low intensity (4).

1. Fishman AP, Elias JA, Fishman JA, et al. Pulmonary diseases and disorders, 4th ed. New York: McGraw-Hill, 2008.

2. Daabisa R, Hassan M, Zidana M. Endurance and strength training in pulmonary rehabilitation for COPD patients. Egyptian Journal of Chest Diseases and Tuberculosis 2017;66:231-6.

3. Aquino G, Iuliano E, di Cagno A, et al. Effects of combined training vs aerobic training on cognitive functions in COPD: a randomized controlled trial. Int J Chron Obstruct Pulmon Dis 2016;11:711-8.

4. Morris NR, Walsh J, Adams L, et al. Exercise training in COPD: What is it about intensity? Respirology 2016;21:1185-92.

EFFECTS OF CONCURRENT AEROBIC AND STRENGHT TRAINING ON BREAST CANCER SURVIVORS

A. Parisi

Department of Movement, Human and Health Science, University of Rome “Foro Italico”

Breast cancer is one of the most commonly diagnosed types of cancer in women. Its pathogenesis involves genetic, hormonal, and environmental factors. Large evidences indicate that physical activity has positive effects on every aspect of breast cancer evolution, including prevention, medical treatment, and aftercare clinical settings. Thus, different types of exercise can influence the prevention and progression of the disease. Literature from the past focused major attention on the relationship between malignant disease and aerobic training; fewer studies were conducted on the effects of resistance training on the physical work capacity of cancer patients or survivors. However, resistance exercise should be an integral component of any exercise training program particularly because of its positive effects on muscle atrophy induced by the treatments and the sedentary habits in breast cancer survivors. The aim of our study was to evaluate the effects of a combined aerobic and strength program on physiological and psychological parameters in female breast cancer survivors.

20 patients (age: 45.6±2.7 yrs) surgically treated for breast cancer that had completed all cancer therapies at least 6 months before and with no contraindications to physical activity, were recruited and randomly assigned to an intervention group (n=10) and a control group (n=10). Intervention group patients attended a 24-week combined aerobic and strength training program. Physiological (i.e. VO2max, bioelectrical impedance test, maximal strength of principal muscular groups) and psychological (i.e. functional assessment of chronic illness therapy e fatigue: FACIT-F) parameters were assessed at baseline and after 24 weeks. After 24 weeks the intervention group showed significant improvement in VO2max (38.8%), strength of upper and lower limbs (ranging from 13 to 60%) and decrease in fat mass percentage (6.3%). The FACIT-F showed significant increase in all of the three scores that can be derived (FACIT-F Trial outcome: 13%; FACT-G total score: 18%; FACIT-F total score: 15%) showing patient’s quality of life (QOL) improvement. No significant changes in all the parameters were found for the control group. These results show the positive effects of a combined aerobic and strength training program on breast cancer survivors and underline the importance of the early inclusion of structured physical activity in the rehabilitation protocol.

Future investigation will explore the possible relationship between this and other different protocols of physical activity and disease, on the methylation level and the expression of specific genes involved in the pathophysiology of breast cancer. We strongly believe that the results obtained through this research project will provide real guidelines for a well-structured exercise protocol designed to improve both the survival and quality of life of people affected by breast cancer. Indeed we believe that physical activity could be an important tool to be used alone or in combination with traditional therapies (i.e. medicine, drugs), to improve the efficacy of strategies for prevention and treatment of different chronic diseases.

A SMALL SIDED GAME SESSION AFFECTS SALIVARY METABOLITE LEVELS IN YOUNG SOCCER PLAYERS

M. Pieri¹, D.O. Cicero², S. Di Marino³, V. Dinallo¹, V. Summa³, A. Desideri⁴, A. Bernardini⁵, F. Perondi⁶, D. Stefano⁵

¹Department of Experimental Medicine and Surgery, University of Tor Vergata, Rome, Italy

²Department of Chemical Science and Technologies, University of Tor Vergata, Rome, Italy

³IRBM Science Park, S.p.A., Pomezia, Italy

⁴Department of Biology and Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), University of Tor Vergata, Rome, Italy

⁵School of Sport Sciences and Exercise, Faculty of Medicine and Surgery, University of Tor Vergata, Rome, Italy

⁶SS Lazio, S.p.A., Rome, Italy

High-strength endurance sports such as soccer are known to generate many metabolic changes in athletes. The majority of studies concerning the impact of physical exercise, investigated a limited number of analytes with the aim of discovering biomarkers able to correlate with the actual performance in competition and predict the progress in the improvement. The use of saliva for monitoring metabolic variations in physical exercise and in different sports gained ground in recent years. Several studies showed that saliva reflects biochemical changes useful for analytical purposes in clinical investigations and in physiological research. The aim of this study was to explore the profile of salivary metabolite changes due to a session of small sided games (SSG) in elite soccer players, searching for a correlation between metabolic changes and athlete performance as GPS measured distances covered in the match. Ten under-20 elite soccer players participated to the study. The SSG were conducted with two goalkeepers and a maximum of two ball touches in a 30×40 m pitch. SSG had an overall duration of 24 min and it consisted of 4 bouts of 6 minutes duration with 2 minutes passive recovery between exercise bouts. Saliva samples were collected before and after the game and physiological parameters evaluated, namely the distances covered by players and blood lactate. Samples were analyzed by Nuclear Magnetic Resonance spectroscopy. Multivariate data analysis showed that SSG session affected salivary metabolite levels in players. We observed no relationship between concentrations of hematic and salivary lactate, nor found any changes in the metabolic profiles that correlate with the blood lactate values. Among the identified metabolites, taurine was instead found to correlate with distances covered by players during the game. Taurine level in saliva is a potentially significant marker which needs further investigation to be correlated with physical effort. Altogether, these results point to a potential use of saliva to follow metabolic changes during an athletic competition, and opens the possibility of using this non-invasive bio-fluid for the study of athlete training state and performance.

APPLICATION OF THERAPEUTIC DRUG MONITORING TO ANTIHYPERTENSIVE THERAPY FOR SCREENING OF TREATMENT ADHERENCE IN PATIENTS WITH RESISTANT HYPERTENSION: DEVELOPMENT AND VALIDATION OF A UHPLC-MS/MS METHOD FOR URINE SAMPLES

V. Avataneo, A. De Nicolò, F. Rabbia, E. Perlo, C. Fulcheri, J. Cusato, F. Favata, E. Berra, P. Mulatero, G. Di Perri, F. Veglio, A. D’Avolio

Dept. of Medical Sciences, University of Turin, Italy

The real prevalence of the apparent treatment-resistant hypertension (TRH) is difficult to be measured since very often TRH depends on poor therapeutic adherence (TA). Therefore, by extending the application of Therapeutic Drug Monitoring (TDM) to antihypertensive drugs could be useful to assess the TA, especially for those patients that are candidates to surgery. We hence developed and validated a UHPLC-MS/MS method for the simultaneous TDM of ten antihypertensive drugs in urine samples, with a reduced invasiveness and a great compliance: this can be suitable for a rapid adherence screening before surgery. Amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan were tested. This method has been validated according to FDA and EMA guidelines. A volume of 100 μl of urine sample, calibration standard and quality control was added with 40 μl of internal standard (IS, 6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline) and 860 μl of a mixture of water:acetonitrile 90:10 (v:v, +0.05% formic acid). After vortexing and centrifuging the samples, the resulting supernatant was analyzed through a UHPLC-MS/MS system (Shimazdu, Kyoto, Japan). This method was tested on real samples from patients with apparent TRH, upon informed consent. Results have been than verified with the TDM of plasma sample from the same patients, through an already validated method. Method performances fitted FDA and EMA guidelines for all analytes. Thirty-six patients have been enrolled: 39% of them resulted fully adherent, 39% were only partially adherent and 22% resulted completely non-adherent. TDM of urine samples resulted congruous with TDM of plasma samples about the complete non-adherence but could misunderstand full and partial adherence (lack of one or only a part of prescribed drugs). Concluding, the very low invasiveness and the fast “dilute-and-shot” extraction procedure make this method suitable for screening the adherence of a large population and when it is impossible to perform blood sampling. Nevertheless, the TDM of plasma samples will probably represent a “gold standard”, especially when an accurate and precise quantification of drug concentrations is needed, even considering the poor urinary excretion of some molecules.

ACCREDITATION OF CLINICAL LABORATORIES IN EUROPE - THE EFLM PERSPECTIVE

A.M. Simundic

Zagreb, Croatia

Professional associations in laboratory medicine (e.g. European Communities Confederation of Clinical Chemistry, EC4) in Europe have had an important role already in the early days of the introduction of quality management systems and accreditation in Europe (1). Furthermore, quality management, accreditation of medical laboratories in Europe are among the core values of EFLM (2). Despite certain variations in the approach to accreditation of medical laboratories in Europe, ISO 15189 standard has been widely accepted and is being increasingly implemented throughout Europe. Still, there is a great heterogeneity in the number of laboratories being accredited in different countries in Europe. In some countries, almost all laboratories are accredited (e.g. Finland, Ireland), whereas in others there is only a minority of laboratories which are accredited. Accreditation is still not mandatory in most of Europe. According to EFLM data, in 2016, there were only 3 countries in which accreditation is mandatory for at least some disciplines of laboratory medicine, but not all (e.g. in Belgium, Ireland and Lithuania), whereas only in France and Hungary accreditation is mandatory for all fields of laboratory medicine (in Lithuania it will be mandatory in 2020). In countries where National Accreditation body does not offer accreditation for medical laboratories, there is commonly some other entity providing a formal recognition of competence and quality (e.g. in Slovenia, Ministry of Health provides a licence to medical laboratories). Within EFLM, since 2007, there is a Quality and Regulations Committee whose general goal is to promote and improve the quality and safety of patient care through establishing the highest standards of laboratory medicine. Their specific objectives are to develop initiatives to improve the quality of clinical chemistry and laboratory medicine in Europe, to harmonise standards of practice in quality management through production of guidance documents defining best practice in areas of clinical chemistry and laboratory medicine and to supporting the establishment of effective accreditation schemes in all European countries. One of their aims is also to represent EFLM, liaise and cooperate on accreditation matters with International Organization for Standardization (ISO) and European Accreditation and national accreditation bodies, European co-operation for Accreditation (EA) and European Committee for Standardization (CEN). This Committee and its working groups have so far delivered several documents, recommendations, original articles with results from European surveys (3, 4). The aim of this lecture is to provide an overview of the current achievements and future plans of EFLM in the accreditation of clinical laboratories in Europe.

1. Jansen RT, Kenny D, Blaton V, et al. Usefulness of EC4 essential criteria for quality systems of medical laboratories as guideline to the ISO 15189 and ISO 17025 documents. European Community Confederation of Clinical Chemistry (EC4) Working Group on Harmonisation of Quality Systems and Accreditation. Clin Chem Lab Med 2000;38:1057-64.

2. Zima T. Accreditation in clinical laboratories. Biochemia Medica 2010;20:215-20

3. Boursier G, Vukasovic I, Ghita I, et al; Working Group Accreditation and ISO/CEN standards (WG-A/ISO) of the EFLM. Accreditation process in European countries - an EFLM survey. Clin Chem Lab Med 2016;54:545-51.

4. Thelen MH, Vanstapel FJ, Kroupis C, et al; Working Group Accreditation ISO/CEN standards (WG-A/ISO) of EFLM. Flexible scope for ISO 15189 accreditation: a guidance prepared by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO). Clin Chem Lab Med 2015;53:1173-80.

ACCREDITATION OF MEDICAL LABORATORIES. DUTIES AND OPPORTUNITIES

L. Sciacovelli

Padova

The accreditation of medical laboratories according to the International Standard ISO 15189 aims at demonstrating to its patients and users the reliability of their performances through the implementation of a quality management system but, above all, qualified and technically competent personnel in performing specific examinations. The compliance with the ISO 15189 requirements, therefore, allows going beyond quality as expressed in ISO 9001, which only certifies the compliance with management system requirements. The accreditation ISO 15189, recognizing the technical and organizational competence of the laboratory staff in carrying out specific and well defined performances included in the scope of accreditation, is the tool that increases the confidence of stakeholders about the ability of the laboratory to satisfy its users with a high level of reliability. It assures laboratory compliance in minimizing errors by planning, preventing, implementing, evaluating, and improving its processes. A key element in the accreditation process is the way of assessment, during accreditation visit, that is based on the concepts of independence and peer-review and is representative of all stakeholders. The standard takes into account all the needs of medical laboratories, namely all steps of the entire testing process, starting from the appropriate test request to the right notification of laboratory reports and the role of further clinical advice provided by laboratory professionals. It focuses the attention on both the items of the intra-analytical phase (e.g. verification and validation of examination procedures, measurement uncertainty, metrological aspects, etc.), and to the pre- and post-analytical phases (peculiar features of the medical laboratories in comparison to testing laboratories). The scientific community promotes accreditation with flexible scope for the complete service, based on description of coherent groups of tests (same medical field, same test typology, same analytical principle, same sample type and same medical area). However, the spread of accreditation ISO 15189 has highlighted the need of harmonization of the list of groups in which tests have to be included. The use of the same list assures the clear understanding of the tests under accreditation for the scientific community from different countries and for patients, and allows distinguishing between laboratories with different quality level of service. Similarly, concerning the check-lists used by assessors during the audits, they should be standardized in order to guarantee a congruent evaluation among different countries. Moreover, in order to comply with ISO 15189 requirements the use of approved guidelines and recommendations often do not take into account the costs and the workload needed for their implementation and, therefore, they may represent a type of procedures affected by a high level of complexity and not easy to be performed. A pragmatic approach is, therefore, needed in order to assure the reliability of results and promote the introduction of accreditation process in the Medical Laboratories, balancing technological possibilities, risk and personnel and time constraints.

RISK MANAGEMENT: A MODEL TO BE IMPLEMENTED IN MEDICAL LABORATORIES FOR ISO 15189 ACCREDITATION

A. Aita¹, L. Sciacovelli², M. Plebani1,²

¹Department of Medicine-DIMED, University of Padova, Italy

²Department of Laboratory Medicine, University-Hospital, Padova

Introduction: Assuring patient safety before an injury occurs is the main goal of medical laboratory. The Risk Management, understood as a systematic process of identifying and treating the present/potential risks for patient safety, becomes then an integral part of Quality Management Systems so that it is also required by ISO 15189.

Aim of the work is to propose the workflow planned by the Laboratory Medicine of Padua to implement a risk management process and meet the ISO 15189:2012 requirement (4.14.6).

Method: Available standards/guidelines and literature have been analysed to select technique and scales to apply. Then, was organized a training course consisting of: an in-class stage on risk management principles and an on-the job training to conduct risk identification, analysis, estimation and control. The risk management process was applied to the most errorsprone phase of the process, according to literature data. The preanalytical process, was retrospectively studied through Failure Reporting and Corrective Action System (FRACAS) by a multidisciplinary team using the self-risk assessment approach. Quality indicators (QI) data from IFCC-MQI model were used to identify the critical stages and ISO 14971:2009 scales to estimate the risk.

Results: The results demonstrated that the use of FRACAS improved the processes studied. Concerning the sample acceptance steps, for example, the overall risk index (IR) was 748. Failure in emptying transportation containers, collecting/storage samples, detecting suitability and priority of samples, showed the highest risk priority number (18<RPN>32). After the implementation of two corrective actions (distribution of detailed operating instructions and introduction of a new label to distinguish stat from routine samples) the IR decreased to 685, demonstrating the effectiveness of the implemented actions and the suitability of the proposed model.

Conclusion: To minimize risks, the risk management process has to begin with the staff awareness of safety issues. The self-risk assessment approach allowed, not only to identify the failure modes but also to make the staff more aware and responsible in risk managing. Moreover, the availability of QI data, has allowed to promptly focusing on the more critical stages of the processes.

GESTIONE CLINICA DEL MODY E DELLE ALTRE FORME DI DIABETE MONOGENICO

F. Barbetti

Roma

Monogenic diabetes is more prevalent than previuosly thought, accounting for over 6% of cases in the pediatric diabetes clinic (Delvecchio M et al., JCEM, 2017) (1). In particular, M(aturity) O(nset) D(iabetes) of the Y(oung) (MODY) alone represents 5.5% of a group of 3781 patients referred to Italian pediatric diabetes clinics during years 2007-2012 and confirmed by molecular genetic analysis (1). Monogenic diabetes awareness combined with availability of genetic testing/diagnosis, translated into profound changes of clinical management, at least in the pediatric setting. Heterozygous, loss-of-function mutations of glucokinase (GCK) are the commonest form of MODY in Italy (1) and are easily identified in children (or even neonates; Prisco F et al., Diabetologia, 2000) (2) with mild, not progressive fasting hyperglycemia in the range of impaired fasting glucose or diabetes (between 100 and 140 mg/dl). Individuls carrying these mutations do not require any form of pharmacological therapy, or diet, and are not prone to any diabetic complication. Instead, HNF1A/MODY (previously known as MODY3) often presents with a severe clinical phenotype, resembling type 1 diabetes. Interestingly, HNF1A/MODY patients can respond to oral hypoglycemic agents of the class of sulfonylureas (SU) with optimal metabolic control ((1) and may be switched from insulin to SU upon genetic diagnosis (1). The same applies to patients with neonatal diabetes (diabetes onset before 6 months of age) due to mutations of genes (KCNJ11, ABCC8) encoding for the subunits of ATP-sensitive potassium channel (KATP; subunits:KIR6.2 and SUR1). Sulfonylureas are adiministered in liquid form along with mother’s milk or formula since genetic diagnosis (even at 1 week of age). The metabolic control is incredibly good and HbA1c close or equal to normal values. A 10-y follow-up of a large international cohort of patients with neonatal diabetes due to KCNJ11 mutations shows that this therapy is well tolerated and efficacious on the long term (submitted to NEJM). SU are also useful in patients with mutations of KCNJ11 or ABCC8 with diabetes onset in childhood/adolescence/adulthood (KCNJ11/MODY, or MODY 13 and ABCC8/MODY or MODY 12) and long-standing insulin therapy (Iafusco D et al, Acta Diabetol, 2012, and Barbetti F unpublished observations). In summary, the discovery of more than 25 genes responsible of various forms of monogenic diabetes either in isolation or syndromic has revolutionized the therapeutic approach(es) and genetic counseling of this subtypes of diabetes.

GLYCATED ALBUMIN: RESULTS FROM THE FIRST ITALIAN CLINICAL STUDY

C. Bellia¹, C. Cosma³, M. Zaninotto³, M. Plebani³, M. Ciaccio^1,2

¹Department of Biopathology and Medical Biotechnologies, Section of Clinical Biochemistry and Clinical Molecular Medicine, University of Palermo, Italy

²Department of Laboratory Medicine, University-Hospital, Palermo, Italy

³Department of Laboratory Medicine, University-Hospital, Padova, Italy

Glycosylated hemoglobin (HbA1c) has been established as the gold standard index for long-term glycemic control and, in year 2009, an International Expert Committee reported values of HbA1c ≥48 mmol/mol as the cut-off point for diagnosing diabetes (1). The United Kingdom Department of Health recommends the use of algorithms for diabetes screening in high-risk individuals that include traditional glucose diagnostic criteria or, alternatively, HbA1c measurements combined or not with glucose measurements. In non-symptomatic patients, a confirmed HbA1c ≥48 mmol/mol is enough to diagnose Type 2 diabetes (2). However, this guideline also recommends that patients with HbA1c levels ≥42 mmol/mol and <48 mmol/mol should undergo an oral glucose tolerance test to establish a diabetes diagnosis.

Serum albumin glycation (GA) has been suggested as an additional or alternative clinical parameter to circumvent some of the limitation of HbA1c. Levels of GA are unaffected by conditions that alter erythrocytes lifespan or by genetic variation in hemoglobolin. In contrast to haemoglobin glycation, which resides inside erythrocytes, albumin glycation occurs in the extracellular volume, particular in plasma, but also to a high degree in other fluids such as CSF, interstitial fluid, and amniotic fluid during pregnancy. The much shorter half-lives of albumin compared to haemoglobin makes it more responsive to changes in glycemic status. Based on the half-lives of albumin, GA levels reflect the mean blood glucose levels in the previous 2 week. This makes GA a much more dynamic marker for glycemic control that can be used to assess the efficacy of diabetes therapy. Moreover, GA shows a stronger correlation with continuous glucose measurement over 1 to 2 days than HbA1c, so it may more accurately reflect glycemic variability and glucose excursions. The majority of data regarding clinical use of GA has been obtained in Asiatic populations, especially in Japan where GA has been proposed for diabetes screening in blood donors. Results arising from the ARIC study demonstrated the association between GA and incident diabetes over two decades of follow-up (3), highlighting its potential role in predicting diabetes. At present, it is not clear which relationships exists between GA, the other markers of glucose homeostasis and insulin resistance in subjects at risk of developing diabetes or subjects with diabetes. Moreover, no specific cut off has been established for diabetes diagnosis, nor reference intervals, in European studies. This multi-centric study for the evaluation of the clinical performances of GA is the first large clinical study conducted in Italy aims to 1) define the upper reference limit of GA in blood donors (4); 2) define the contribute of GA to traditional markers (namely HbA1c, fasting plasma glucose and 2h-plasma glucose after OGTT) for diabetes diagnosis; 3) define the role of GA in short term monitoring of diabetics patients. Secondary aim of the study is to explore the association between GA and the metabolic impairment of T2DM evaluated by overweight or obesity and insulin resistance.

1. International Expert Committee. The International Expert Committee report on the role of the HbA1c assay in the diagnosis of diabetes. Diabetes Care 2009;32:1327-34.

2. NHS Health Check. Vascular Risk Assessment and Management Best Practice Guidance. 2009;9:19-24.

3. Selvin E, Rawlings AM, Grams M, et al. Fructosamine and glycated albumin for risk stratification and prediction of incident diabetes and microvascular complications: a prospective cohort analysis of the Atherosclerosis Risk in Communities (ARIC) study. Lancet Diabetes Endocrinol 2014;2:279-88.

4. Bellia C, Zaninotto M, Cosma C, et al. Definition of the upper reference limit of glycated albumin in blood donors from Italy. Clin Chem Lab Med 2017. doi: 10.1515/cclm-2017-0179.

GLYCATION GAP: A NEW LABORATORY PARAMETER USEFUL FOR THE EVALUATION OF GLYCOMETABOLIC CONTROL

A. Mosca

Milano

The determination of glycated albumin (GA) has been suggested as an additional parameter having an independent added value respect to that of HbA_1c. The determination of glycation gap (gg), calculated from the deviation between the measured HbA_1c respect to that calculated from other indexes of glycemic control, such as fructosamine (FA) or GA, has been investigated in some recent studies, and proven to be reliable in diabetic patients with stable glycemic control, significantly associated with retinopathy, nephropathy and mortality in diabetes, although not associated with chronic kidney disease in individuals without diabetes. However, few studies have addressed its potential impact in the routine evaluation of glycometabolic control. In our experience, we have studied a total of 157 subjects presenting normal whole blood cell count, no hemoglobin variants, normal creatinine levels and serum protein electrophoresis patterns in order to estimate a reference range for the gg. In a second phase, a total of 205 subjects with no restrictions as those of the first phase study, were analyzed. HbA_1c was measured by capillary electrophoresis, glycated albumin by an enzymatic method and their gg were then calculated. The correlation between HbA_1c and GA for the subjects of phase 1 was strong (r=0.8927) and significant correlation between gg and age was remarked (r=0.4486). No significant differences between genders were evidenciated. We found 17.1% of phase 2 subjects with gg falling outside the 95% prediction intervals. Various clinical conditions seemed to affect these subjects, in our experience mostly often related to impaired renal function, with typical negative gg values.

In conclusion, we believe that the glycation gap may be useful to alert clinicians about patients under unstable glycemic control or when various pre-analytical conditions my affect the reliability of the measurement of GA or HbA_1c.

– Cohen RM, Holmes YR, Chenier TC, et al. Discordance between HbA1c and fructosamine: evidence for a glycosylation gap and its relation to diabetic nephropathy. Diabetes Care 2003;26:163-7

– Rodriguez-Segade S, Rodriguez J, Garcia Lopez JM, et al. Estimation of the glycation gap in diabetic patients with stable glycemic control. Diabetes Care 2012; 35:2447-50.

– Nayak AU, Nevill AM, Bassett PM. Association of glycation gap with mortality and vascular complications in diabetes. Diabetes Care 2013;36:3247-53.

– Shipman KE, Jawad M, Sullivan KM, et al. The glycation gap and estimated glomerular filtration rate in individuals without diabetes. Clin Chem 2014;60:1346-8.

– Paleari R, Strollo M, Guerra E, et al. Glycaton gap: an additional tool for glycometabolic monitoring. Clin Chim Acta 2016;463:27-31.

MOLECULAR DIAGNOSIS OF NON-AUTOIMMUNE DIABETES MELLITUS (DM) IN PEDIATRIC AGE: AN ATYPICAL CASE OF THE NOVO DELETION ON CHROMOSOME 12q24.31 IN A PATIENT WITH MATURITY ONSET DIABETES OF THE YOUNG (MODY)

F. Iafusco¹, P. De Sanctis¹, D. Pirozzi¹, B. Lombardo¹, L. Pastore¹, D. Iafusco², N. Tinto¹

¹Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università Federico II, Napoli e CEINGE Biotecnologie Avanzate Scarl, Napoli

²Dipartimento di Pediatria, Università della Campania “Luigi Vanvitelli”, Napoli, Italia

MODY is an autosomal dominant disease that includes clinically heterogeneous forms of not autoimmune diabetes. MODY2 and MODY3, due to GCK and HNF1 α mutations respectively, are the most frequent forms. We describe a 15-years-old patient with not-autoimmune DM diagnosed at 11 years with ketoacidosis. The molecular analysis for MODY revealed a whole deletion of HNF1a gene in agreement with a diagnosis of MODY3. The clinical history of the patient also highlighted neurological and cardiac disorders, such as interatrial defect treated surgically at 2 years, a slight mental retardation, epilepsy and Hashimoto thyroiditis. Therefore to clarify this complex phenotype and to define the extension of the deletion, we performed array Comparative Genomic Hybridization analysis that revealed a deletion of 1.04 Mb on chromosome 12q24.31, that encompasses 23 genes including HNF1 α. This deletion was not found in parents and paternity and maternity were confirmed. Few 12q24.31 deletions have been reported in the literature, all with a greater extension and different phenotypes compared to that observed in our patient. In fact, in our case, the phenotype is overall milder probably because the number of genes involved in the deletion is lower. Differently, comparing diabetic symptoms of our patient respect to other MODY patients with HNF1α mutation, different from a deletion, these were more severe and the diagnosis of diabetes was earlier. Despite this, the patient could replace insulin therapy with oral drugs, as in other cases of MODY3. In our proband, the deletion of ACADS gene could be the cause of epilepsy and of mild mental retardation, as well as of cardiac anomalies, while it could be hypothesized that autoimmune thyroiditis that is difficult to associate with a not autoimmune form of diabetes may be due to the deletion of the ORAI1 gene that could have caused a functional abnormality of T lymphocytes resulting in the autoimmune thyroid process. In conclusion our case shows that syndromic symptoms in notautoimmune diabetes should lead to look for deletions involving neighboring genes to better understand the aetiology and pathogenesis of the different defects, to prevent the misclassification of any clinic feature and also to offer appropriate genetic counselling.

DIAGNOSIS OF NEUROMUSCULAR DISORDERS

R. Massa

Center for Neuromuscular Disorders, University of Rome Tor Vergata

Neuromuscular Disorders comprise all those diseases that affect spinal motor neurons, peripheral nerves, neuromuscular junctions and muscle fibers. These entities may be genetically determined or acquired, may develop at any age and may present with acute, subacute or chronic course. The diagnosis of neuromuscular disorders usually relies on neurological and muscular examination, on electrophysiological studies, and on biochemical, histopathological and molecular genetic workup. Clinical chemistry and laboratory medicine techniques may be essential or provide additional information when diagnosing a neuromuscular disorder. Among biochemical markers, creatine-kinase (CK), and its muscular isoform (CK-MM) are by far the most sensitive serum enzymatic correlates of primary muscle fiber damage, indicating cell necrosis or plasma membrane leakage. Very high levels of CK are usually found in Duchenne muscular dystrophy, rhabdomyolysis and myositis. The detection of increased serum lactate levels at baseline or a failure to rise after a forearm ischemic exercise test can reveal defects of mitochondrial functioning or anaerobic glycolysis within muscle fibers, respectively. Moreover, fine tuning in diagnosis of metabolic myopathies may be performed by measuring the activity of single enzymes with different techniques in serum or muscle tissue obtained by biopsy. The search for circulating autoantibodies is now a consolidated aspect of the diagnostic process of autoimmune disorders affecting peripheral nerves and muscles. Indeed, the detection of anti-acetylcholine receptor or anti-muscle specific kinase, two components of the neuromuscular junction, is essential for the diagnosis and classification of autoimmune myasthenia gravis. Moreover, immunoblotting or ELISA techniques may reveal the presence of autoantibodies reacting against glycolipids belonging to the ganglioside family or against sulphatides or the myelin-associated glycoprotein, therefore contributing to classification of autoimmune peripheral neuropathies. Similarly, several autoantibodies defined as myositis-specific or myositis-associated, help differentiating among subtypes of idiopathic inflammatory myopathies, thus facilitating the search for co-morbidities, a better prognostic definition and a correct therapeutic approach. Finally, autoantibodies against the so-called onco-neural antigens may be detected in patients presenting a neurological paraneoplastic syndrome associated with specific malignancies. These antibodies are markers of a molecular mimicry between cell surface antigens shared by neurons subpopulations and some kind of tumours. Although a pathogenic role of these antibodies is still debated, their presence may anticipate the finding of malignancy, therefore prompting an early diagnosis and, possibly, a better outcome.

Progress in the laboratory diagnosis of neuromuscular disorders proceeds at a fast pace: this requires a continuous refresh by the specialists in order to transfer up to date concepts into clinical practice.

LABORATORY DIAGNOSIS OF ACUTE NEUROLOGICAL DAMAGE: GUIDELINES AND NEW PERSPECTIVES

G. Bernardi, E. Corsini

Fondazione IRCCS INN Besta, Milano

Acute neurological damage is mostly due to traumatic brain injury, with millions of people suffering each year worldwide and is recognized as the leading cause of mortality and morbidity in young adults. Despite this, a PubMed search on guidelines for laboratory diagnosis of acute neurological damage identify in the last ten years only one article, published in 2012 by Eastern Association for the Surgery of Trauma (1). Authors suggest that routine use of laboratory biochemical markers for the clinical management of traumatic brain injury is not supported at the present time. Lots of guidelines have been published for the second cause of acute neurological damage: central nervous system infections. A very exhaustive one is the diagnostic pathway proposed by Italian Association of Clinical Microbiologists (AMCLI). Also the European Federation of Neurological Societies (EFNS) published guidelines on diagnosis and management of acute and chronic neurological diseases; they are particularly useful since Clinical Features, Investigations and Therapy are reported. In 2009 EFNS published guidelines on disease-specific CSF investigations, including also diseases with acute neurological damage (2). Italian Association of Neuro Immunology (AINI) published various papers on laboratory diagnosis of neuroimmunological pathologies, reporting preanalytical and analytical procedures, quality control and sample storage. An update will be published in 2017. Revised national (UK) guidelines on Cerebrospinal fluid (CSF) analysis for suspected subarachnoid haemorrhage were published in 2008. The document reaffirmed that for bilirubin detection, due to conversion in vivo of liberated oxyhaemoglobin in a time-dependent manner, spectrophotometry should be used instead of visual inspection. Nine CSF and serum biomarkers were studied in a systematic review published in 2013 on cerebro spinal fluid and serum biomarkers for the clinical differential diagnosis in traumatic brain injury. Authors concluded that at present there are insufficient literature data to support a role for diagnostic biomarkers in distinguishing focal and diffuse injury and in evaluating raised intracranial pressure (3). In the last ten years new perspectives come from studies on neurofilament light (NF-L) protein, first in CSF then also in serum. NF-L are neuron-specific major structural proteins, abundantly expressed in the long myelinated white matter axons. In 2015 Kuhle and coworkers, for the first time, provided evidence that serum NF-L chain is a good biomarker and has prognostic value in Spinal Cord Injury patients (4). In 2016 Shahmin and coworkers demonstrated that measurement of serum NF-L may be useful to assess the severity and outcome of neuronal damage following traumatic brain injury (5). Moreover, NF-L has also very good diagnostic performance as a marker of brain atrophy in Multiple sclerosis and neuronal damage in AIDS patients. We believe that this molecule is a promising biomarker of acute neurological damage in the field of Laboratory diagnosis.

1. Barbosa RR, Jawa R, Watters JM, et al. Evaluation and management of mild traumatic brain injury: an Eastern Association for the Surgery of Trauma practice management guideline. J Trauma Acute Care Surg 2012;73(5 Suppl 4):S307-14.

2. Deisenhammer F, Egg R, Giovannoni G, et al. EFNS guidelines on disease-specific CSF investigations. Eur J Neurol 2009;16:760-70.

3. Yokobori S,Hosein K, Burks S, et al. Biomarkers for the clinical differential diagnosis in traumatic brain injury- A systematic review. CNS Neurosci Ther 2013;19:556-65.

4. Kuhle J, Gaiottino J, Leppert D, et al. Serum neurofilament light chain is a biomarker of human spinal cord injury severity and outcome. J Neurol Neurosurg Psychiatry. 2015;86:273-9.

5. Shahim P, Gren M, Liman V, et al. Serum neurofilament light protein predicts clinical outcome in traumatic brain injury. Sci Rep 2016;6:36791.

CEREBROSPINAL FLUID TAU PROTEIN DISCRIMINATES JAKOB-CREUTZFELDT DISEASE FROM NON-PRION FORMS OF ACUTE AND SUBACUTE RAPIDLY PROGRESSIVE DEMENTIAS

G. Sancesario¹, S. Toniolo², T. Schirinzi², E. Scaricamazza², M. Ghibellini³, M. Perrone³, M. Pieri³, P. Casalino³, A. Martorana², C. Caltagirone¹, S. Bernardini³

¹Dip. Neurologia Clinica e Comportamentale, Fondazione Santa Lucia, Roma

²Dip. Medicina dei Sistemi, Policlinico Universitario Tor Vergata, Roma

³Dip. Medicina Sperimentale, Policlinico Universitario Tor Vergata, Roma

Rapidly progressive dementias (RPDs) are neurological conditions with different etiologies, which typically manifest subacute or acute symptoms over months, weeks or days. The Jakob-Creutzfeldt disease (CJD) is a common form of RPD and is invariably fatal, while some cases are due to other non-prion conditions, many of which are treatable. Moreover, Alzheimer’s disease (AD) is rarely rapid, but unusual presentations can be misdiagnosed as CJD. So, an early diagnosis is essential to identify RPDs that are treatable and potentially reversible. The cerebrospinal fluid (CSF) proteins 14-3-3 and total tau reflect neuronal damage and in several studies have been suggested in the differential diagnosis of CJD. We assessed the potential of Tau as early biomarker to discriminate in a common clinical setting CJD from other non-prion forms of RPDs with acute onset. We evaluate the level of Tau in 26 cases with acute/subacute dementia and 13 non-demented patients, admitted to the Tor Vergata General Hospital during the 2015-2016 and followed prospectively. After one-year of follow up, 11 were diagnosed as CJD, 5 suffered of AD and 10 suffered of other conditions including acute psychotic, dysmetabolic or inflammatory disorders (OD). The presence of 14-3-3 protein in CSF was also investigated. We found that all CJD patients showed level of tau higher than 1300 pg/ml. Oppositely, tau in patients with non-prion conditions were in the pathological range but significantly lower than CJD (AD 517±176; OD 432±321 pg/ml; normal value <350 pg/ml). Level of tau in control patients was in the normal range. Of interest, in one case with clinical suspect of CJD, which was negative for 14-3-3 and had uncommon higher level of tau but lower than 1300 pg/ml (1018 pg/ml), the CSF analysis was repeated after two months to definitely exclude the diagnosis of CJD (tau 341 pg/ml). Our results demonstrate that tau could discriminate CJD from other non-prion forms of acute and subacute dementia. In cases of rapidly presentation of dementia, measurement of tau could be considered as a preliminary step to identify potentially treatable and reversible conditions. The analysis of CSF Tau could be a reliable tool to be included in the diagnostic procedures for the diagnosis of nonprion forms of RPDs.

THE ROLE OF SIBIOC IN OPTIMIZING TEST USE AND DIAGNOSTIC INFORMATION

M. Montagnana

Verona

Laboratory medicine plays a crucial role in the clinical decision making throughout a broad spectrum of human diseases. Although approximately 60-70% of the most important clinical decisions about admission, discharge or therapeutic management entail laboratory tests (1), over- or under-utilization of laboratory diagnostics (i.e., inappropriateness) may impact the quality and the value of laboratory testing. The leading consequences of inappropriate use of laboratory resources include unjustified incremental costs, derangement of laboratory efficiency and patient safety issues related to the possible generation of false positive or false negative results (2).

In a meta-analysis including studies published between 1997–2012, Zhi et al. reported a mean rate of laboratory tests overutilization of 20.6% (95% CI 16.2–24.9%) (3). In Italy the estimated inappropriateness of laboratory tests ordering was also found to be considerably high, in particular in the field of cancer biomarkers (4). Improving appropriateness of laboratory diagnostics is a challenging issue. In the last decade, the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) has published several documents, guidelines and recommendations, and has also organized meetings and seminars for improving and promoting the appropriateness of laboratory tests ordering and interpretation, both for common diseases (i.e. diabetes mellitus, myocardial infarction, cancer) and for less frequent conditions. This important target has been achieved by establishing a large number of valuable collaborations with the other important national and international clinical societies. Among the various examples attesting the important activity of our Society, a close collaboration has recently been established between SIBioC and the Academy of Emergency Medicine and Care (AcEMC) for reaching a tentative consensus about the most informative diagnostic tests in emergency settings (5), along with the publication of ANMCO/ISS/AMD/ANCE/ARCA/FADOI/GICR-IACPR/SICI-GISE/SIBioC/SIC/SICOA/SID/SIF/ SIMEU/SIMG/SIMI/SISA consensus guidelines about the laboratory diagnosis of dyslipidemia. Last but not least, SIBioC has edited the Italian translation of the Website LAB tests Online (6), developed by the American Association for Clinical Chemistry (AACC) and designed to help patients to better understanding the clinical lab tests that are part of routine care as well as diagnosis and treatment of a broad range of conditions.

1. Forsman RW. Why is the laboratory an afterthought for managed care organizations? Clin Chem 1996;42:813-6.

2. Lippi G, Bovo C, Ciaccio M. Inappropriateness in laboratory medicine: an elephant in the room? Ann Transl Med 2017;5:82.

3. Zhi M, Ding EL, Theisen-Toupal J, et al. The landscape of inappropriate laboratory testing: a 15-year meta-analysis. PLoS One 2013;8:e78962.

4. Gion M, Peloso L, Trevisiol C, et al. An epidemiology-based model as a tool to monitor the outbreak of inappropriateness in tumor marker requests: a national scale study. Clin Chem Lab Med 2016;54:473-82.

5. Lippi G, Panteghini M, Bernardini S, et al. Laboratory testing in the emergency department: an Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) and Academy of Emergency Medicine and Care (AcEMC) consensus report. Clin Chem Lab Med 2017 Mar 31. pii: /j/cclm.ahead-of-print/cclm-2017-0077/cclm-2017-0077.xml. doi: 10.1515/cclm-2017-0077 [Epub ahead of print].

6. www.labtestonline.org

A MOBILE APP TO IMPROVE THE APPROPRIATENESS OF LABORATORY TEST REQUEST BY GENERAL PRACTITIONERS

L. Iaboli², F. Borsari², S. Togni³, T. Trenti¹, V. Pecoraro¹

¹Lab. Tossicologia, Dip. Interaziendale ad Attività Integrata Medicina di Laboratorio e Anatomia Patologica, Nuovo Ospedale Civile S. Agostino Estense, Modena

²Corso Formazione Specialistica in Medicina Generale, Regione Emilia Romagna

³Medico di Medicina Generale, ASL Modena

Background: The prevalence of consultations of general practitioners (GP) is huge, and for an unknown number of patients, a consistent number of diagnostic laboratory tests will be requested. A scope of GP is define patients that need or not a specific test, improving the patients outcomes given the available invested resources, and considering as well to reduce the risk of overdiagnosis and to avoid overtreatments. However, GP choices depend on knowledge and time to search for valid (updated, correct and synthetic) information about how a given test can contribute to the diagnosis, prognosis, therapy and prevention of a disease.

Methods: We developed a dedicated software application (APP) for mobile devices to support GPs in their daily practice with valid information, improving the patients referral and satisfaction. The APP contents was developed in collaboration with GP, students of a GP training course and medicine laboratory specialists. We identified the laboratory tests useful for management of the most frequent diseases observed in GPs’ ambulatory, starting from information provided by SIBioC FAD (“Appropriatezza prescrittiva nell’ambulatorio del Medico di Medicina Generale”) and supported by the best available evidence.

Results: In the preliminary phase of the APP development, we included knowledge about hypertension, thyroid and anaemia diseases, completed with data from systematic reviews, guidelines and health technology assessment reports. For each pathology we reported clinical information and laboratory test descriptions to lead GP in appropriate diagnostic test request.

Conclusions: This promising tool could help GP to prescribe suitable laboratory tests in different clinical scenario (diagnosis, therapy, monitoring) and to promote the implementation of best laboratory medicine daily practices, reducing inappropriate tests and accurately identifying patients who need specialist referral.

GENETIC PROFILE AND AGGRESSIVE BEHAVIOR

S. Pellegrini

Department of Experimental and Clinical Medicine, University of Pisa

Recent developments in molecular biology are shedding new light on the neurobiological underpinnings of human social behavior, including moral choices (1) and antisocial behavior (2, 3). Specific genetic variants may increase individual vulnerability to develop impulsive and aggressive behavior by modulating the impact of the environment on behavioral traits. These genetic variants play a role in regulating the cognitive and emotional processes underlying antisocial behavior, mostly in response to markedly negative stimuli and events. More specifically, some allelic variants, reported in literature as being associated with alterations of personality traits, may increase the risk of antisocial behavior in individuals who have been maltreated and abused as children (4). Within a larger collaborative research project aimed at investigating the neurobiological bases of antisocial behavior, here we report preliminary data obtained in a pilot sample by comparing the genetic profiles of 37 criminal males with those of 30 matched controls with no history of aggressive behavior. Six polymorphisms were genotyped: MAOA (Monoamine oxidase A) uVNTR, 5HTTLPR (serotonin-transporter-linked polymorphic region), STin2 VNTR in the SLC6A4 (solute carrier family 6 (neurotransmitter transporter), member 4) gene, COMT (Catechol-O-methyltransferase) Val158Met and DRD4 (dopamine D4 receptor) exon3 VNTR. The combination of three or more “risk” allelic variants had a significantly higher frequency in criminals than in control subjects. Particularly, the concomitant presence of five or six risk variants was observed only in criminals. Thus, we hypothesize that a “risk genetic profile”, instead of single alleles, may predispose to antisocial behavior. Interestingly, according to a recent hypothesis (5), these genetic variants would influence the individual susceptibility to both negative and positive environments, thus being “plasticity genes” rather than “vulnerability genes”. In this light, our findings provide a novel strategy to understand aspects of human behavior that have been a main focus of interest since the very early days of humanity.

1. Pellegrini S, et al. Genetically-driven enhancement of dopaminergic transmission affects moral acceptability in females but not in males: a pilot study. Front Behav Neuroscience, in press.

2. Iofrida C, Palumbo S, Pellegrini S. Molecular genetics and antisocial behavior: where do we stand? Exp Biol Med (Maywood) 2014;239:1514-23.

3. Rigoni D, Pellegrini S, Mariotti V, et al. How neuroscience and behavioral genetics improve psychiatric assessment: report on a violent murder case. Front Behav Neurosci 2010 Oct 13;4:160. doi: 10.3389/fnbeh.2010.00160. eCollection 2010.

4. Caspi A, McClay J, Moffitt TE, et al. Role of genotype in the cycle of violence in maltreated children. Science 2002;297:851-4.

5. Simons RL, Lei MK, Beach SR, et al. Social environmental variation, plasticity genes, and aggression: evidence for the differential susceptibility hypothesis. Am Sociol Rev 2011;76:833-912.

DEVELOPING MEDICAL TESTS FOR IMPROVING PATIENT OUTCOMES

P.M. Bossuyt

Amsterdam, the Netherlands

Like all other interventions in health care, medical tests should be thoroughly evaluated before they are introduced into daily practice. In this era of evidence-based medicine, there should be sound evidence to support the use of laboratory and other tests, to include such tests in practice guidelines, or to provide coverage through reimbursement decisions. For a very long time, such evidence came from evaluations of the analytical performance of medical tests. To be ready for clinical practice, tests should have acceptable precision, trueness, linearity, and limits of detection. Typically, goals for desirable precision and trueness were based on biological variation. Increasingly, however, there is shift towards patient outcomes in the appraisal of health care interventions. Practice guidelines are increasingly based on the effect of clinical actions on patient outcomes, not just on technology or clinicians’ preferences. In more and more countries reimbursement decisions are guided by considerations about effectiveness and cost-effectiveness. Laboratory medicine cannot, must not, and will not escape from this transition towards patient outcomes. In this presentation, we will highlight the evolution from results to consequences in laboratory medicine. We will illustrate how the effectiveness of medical testing – its effect on patient-important outcomes – can be evaluated. We will also discuss how this transition feeds in to evaluations of analytical and clinical performance. To remain effective, laboratory professionals should be aware of these patient-oriented comparative evaluations of outcome, and engage in them whenever possible and needed.

POTENTIAL USE OF BIOMARKERS IN CLINICAL PRACTICE: ALPHA DEFENSIN AND SYNOVIAL FLUID

G. Bartolini, R. Dall’Anese

Prato

Alpha defensin in synovial regards potential biomarker (1,2) for identification of periprosthetic joints infections (PJI). The only commercially available test is an in situ immunocromatographic device. Orthopedics use the device under their direct responsibility, either as part of pre surgery patient assessment or, intra operatively, to make treatment decisions according to the likelihood of infection. In this diagnostic path flow, the availability of a method not prone to interferences, with high sensitivity and specificity, can be of great help to clinicians. LC-MS methods are considered to meet needs in terms of analytical performance and are widely used to measure new biomarkers. The aim of our study was to implement a methods to accurately measure Alpha defensin in synovial fluids. Several issues had to be addressed: the synovial fluid matrix viscosity, the need of a standard Alpha defensin peptide and a valid Internal Standard (IS), the definition of analytical protocol and, finally, the method validation. As preliminary step the uniqueness of peptides derived from trypsin digestion of Alpha defensing was checked by liquid chromatography - time of flight mass spectrometry (LC-QTOF - Agilent Technologies). Data were analyzed by a proteomic software (Spectrum Mill - Agilent Technologies). Synovial fluids samples from primary knee arthroplasty were used as negative matrix; a synthetic matrix (simulant) was produced and measures of spiked samples were run in parallel. The quantitative analysis was performed with two different instruments (liquid chromatography with time of flight or triple quadrupole mass spectrometry, LC-QTOF or LC-MS/MS - Agilent Technologies) and two different methods (negative matrix spike and simulant synovial fluid spike) by spike of different concentrations of synthetic marker peptide (LWAFCC, DBA Italia). The quantitative method was developed with dynamic range from 0.1 to 100 μg/ml. LOD (μg/ml) and LOQ (μg/ml) were respectively 0.14 and 0.47 μg/ml. CQ samples were prepared using both simulant and negative synovial fluid spiked at 5 μg/ml and 50 μg/ml and were run in multiple replicate to determine the intra-day and inter- day accuracy. The mean percent inaccuracy and imprecision at CQs levels were always lower than 10%.

1. Albrethsen J, Bøgebo R, Gammeltoft S, et al. Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study. BMC Cancer 2005;5:8.

2. Gardner MS, Rowland MD, Siu AY, et al. A comprehensive defensin assay for saliva. Anal Chem 2009;81:557-66.

VALIDATION OF AN IMMUNOTURBIDIMETRIC ASSAY FOR MEASURING C REACTIVE PROTEIN IN SYNOVIAL FLUID

G. Balato¹, F. Balboni², A. Baldini³, S. Buoro⁴, G. Lippi⁵, P. Pezzati⁶, M. Quercioli⁶

¹Dipartimento di Sanità Pubblica, Scuola di Medicina e Chirurgia, Università Federico II di Napoli

²Laboratorio Analisi, ³U.O. Ortopedia, Istituto Fiorentino Cura e Assistenza IFCA, Firenze

⁴SMeL Generale di Base - Analisi Chimico Cliniche, Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo

⁵Sezione di Biochimica Clinica, Università degli Studi di Verona, Verona

⁶Centro Regionale di Riferimento SOD Sicurezza e Qualità, AOU Careggi, Firenze

Background: Periprosthetic joint infection (PJI), a rare and severe complication which may occur after total joint arthroplasty, has high morbidity and mortality rates, along with considerable economic implications. Although C-reactive protein (CRP) is a common and inexpensive test for screening PJI, its serum concentration is poorly specific for diagnosing localized infection, since serum CRP can also be increased in several non-infectious inflammatory conditions. A recent study (1) showed that PCR in synovial fluid (SF) may have high sensitivity (0.92) and specificity (0.90) for diagnosing PJI. These promising data are however biased by the fact that the method has not been validated in SF. According to the ISO 15189 standard, method performance should be always validated before changing either the sample matrix or the test purpose. Therefore, we assessed the analytical performances of the Beckman Coulter CRP immunoturbidimetric assay OSR6147 using AU480 Olympus in SF.

Methods: SF samples were collected in serum tubes (Vacuette Grainer Bio-One GmbH Austria) and sent to the local laboratory within 6 hours from collection, at room temperature. The samples were separated and stored at -30°C until further analysis. Hyaluronidase was added the SF immediately before analysis (final concentration, 0.5 mg/mL) and samples were then centrifuged at 3000 rpm for 15 min. Imprecision, linearity, limit of detection (LoD), limit of blank (LoB), limit of quantitation (LoQ) and carry-over were assessed according to CLSI protocols.

Results: The LoD, LoB, LoQ were 1.95, 0.3 and 2.92 mg/l, respectively. Imprecision was comprised between 1.36-3.48 %, linearity was excellent (r=0.99) and carry-over was negligible, dynamic range was between 5 and 250 mg/L in a six point calibration.

Conclusions: We suggest that this immunturbidimetric assay may be suitable for measuring CRP in SF. However, additional research is needed to confirm the clinical significance of measuring CRP in SF to rule out PJI.

– Wang C, Wang Q, Li R, et al. Synovial fluid C-reactive protein as a diagnostic marker for periprosthetic joint infection: a systematic review and meta-analysis. Chin Med J 2016;129:1987-93.

BLOOD GASES ANALYSIS IN CRITICAL CARE SETTINGS: NEW SOLUTIONS FOR OLD ISSUES

M.M. Mion

Department of Laboratory Medicine, University-Hospital of Padova, Italy

Blood gases, pH, glucose, hemoglobin and electrolytes beside to coagulation parameters represent the most important analyses in emergency and intensive care medicine. In particular, arterial and venous blood gases tests provide fundamental insight into a patient’s ability to oxygenate, ventilate and into assess the acid-base status. Fluids, electrolytes and acid-base disorders represent a very common condition in patients presenting to the emergency department as well as in patients admitted to the intensive care units. The risk of death is high because of failure to rapidly identify a life-threatening situation and initiate the appropriate treatment. As a matter of fact, the first step in the acid-base disorders is to evaluate the type and cause of the disorder as well as the degree of compensation, so that the correct treatment could be administered. The need for a rapid blood gas analysis has strongly increased in the past decades. A fast turnaround time (TAT) for laboratory tests has been shown to improve clinical outcomes and decrease time for decision making as well as the medical costs. Point of care testing (POCT) for blood gases and electrolytes reduces the TAT and potentially the pre-analytical errors, shortening the time between blood collection and measurements, in comparison to the testing performed in the central laboratory. However, as for other in vitro diagnostic tests, there are several possible errors that can commonly occur with blood gases analysis, especially in the pre-analytical phase. In fact, identify potentially unsuitable specimens for blood gas analysis is a fundamental activity of the healthcare professionals for preventing inaccurate results from being released. Noteworthy, the crucial challenge in moving critical care testing from the central laboratory to the POCT network is the mandatory assurance of high quality standards for the analytical results across all the POCT sites. Based on a patient centered approach, there is the need to evaluate all steps of the total testing process, irrespective of whether they are related to procedures and personnel outside the laboratory wall. We will present the data obtained in the evaluation of the performaces of the new GEM PREMIER 5000 instrumentation by using an health technology assessment study design.

STANDARDIZATION IN LABORATORY MEDICINE: ADVANTAGES AND CRITICISMS

A. Padoan

Department of Laboratory Medicine, University-Hospital of Padova, Padova, Italy

The most important goal of medical laboratories is to provide reliable and timely information to support the clinical decision-making, allowing the choice of the best available options for patients’ care. Laboratory results are used not only for diagnosis, but also for monitoring patients, for administration of treatments and for risk assessment, thus underlining that the fit-for-purpose of a specific tests result depends also on the clinical needs. Requirements in standards, such as the ISO 13485, ISO 17025 and ISO 15189 have helped in clarifying these concepts, further encouraging the development of methods that follow the intended purposes, validating the appropriated methods performances, and - on the other hand - that laboratories verify the claimed specifications. ISO 15189 not only states that laboratory “shall select examination procedures which have been validated for their intended use”, but also that method performances should be independently verified (if not modified with respect to the manufacturers inserts), or alternatively validated (e.g. for in-house methods). Both of these two processes require that laboratories develop and document objective evidences for verifying that methods performances are appropriated to the intended use. Notably, the sets of documents of the Clinical Laboratory Standards Institute (CLSI) are produced with the aim of providing clear and through guidance for evaluating and characterizing methods performances. The CLSI documents are derived by the consensus of experts from manufacturers, hospitals and reference laboratories and universities. For example, CLSI documents are available for evaluating methods precision (EP15) and for defining limits of detection and limits of quantitation (EP17) (1, 2). Overall, the advantages offered by the CLSI guidelines are broad and regards: a) the standardization of terminology and definitions, b) the proposal of technically and statistically sound, low-end protocols, c) the compliance with international standards and accreditation organizations. Regardless these directives, it should be taken into consideration that the quality of results should be guaranteed independently of the methods and of the instrumentations used, as inadequate laboratory performances may potentially cause patient harms. In order to assure the quality of results, medical laboratories use internal quality controls and external quality assessment schemes. These tools allowed laboratories to evaluate and monitor imprecision (random error) and trueness (systematic error) for quantitative methods, and diagnostic accuracy (in terms of sensitivity and specificity) for qualitative methods (3). Further, measurement uncertainty is also available for all the ISO 15189 accredited laboratories (4). Therefore, the verification by clinical laboratories of imprecision/trueness or sensitivity/specificity appear feasible, even if the choice of appropriate protocols, such as those produced by CLSI, appear advisable to obtain reliable and comparable results. However, overall these processes present several difficulties for labs, as they are time-consuming and require expert technical and statistical skills (3).

1. Clinical and Laboratory Standards Institute (CLSI). User verification of precision and estimation of bias; approved guideline, 3rd ed. CLSI EP15, Wayne, PA, USA, 2014.

2. Clinical and Laboratory Standards Institute (CLSI). Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline, 2nd ed. CLSI EP17, Wayne, PA, USA, 2012.

3. Padoan A, Antonelli G, Aita A, et al. An approach for estimating measurement uncertainty in medical laboratories using data from long-term quality control and external quality assessment schemes. Clin Chem Lab Med 2017. doi: 10.1515/cclm-2016-0896. [Epub ahead of print].

4. Antonelli G, Padoan A, Aita A, et al. Verification of examination procedures in clinical laboratory for imprecision, trueness and diagnostic accuracy according to ISO 15189:2012: a pragmatic approach. Clin Chem Lab Med 2017. doi:10.1515/cclm-2016-0894. [Epub ahead of print].

EVALUATING THE INNOVATION BY MEANS OF SYSTEM FAMILIARIZATION STUDIES: OUR EXPERIENCE

J.-L. Laplanche

Département de Biochimie et Biologie Moléculaire, Hôpital Lariboisière, Assistance Publique Hôpitaux de Paris (APHP), Paris, France

We performed a familiarization study of a manufacturing prototype of the Alinity ci ^® clinical chemistry (c) and immunoassay (i) instrument systems (Abbott, IL, USA). The Alinity ci-series is a fully automated analyzer allowing random and continuous access as well as priority and automated retest for both clinical chemistry and immunoassays tests. Calibration and quality controls including automated quality controls (QC) processing and QC analysis were assessed. The performance of selected assays (seven clinical chemistry parameters: ICT, calcium, glucose, creatinine and urea, and two immunoassays: TSH and hsTn-I) were evaluated through precision on QC, linearity using provided standards, and limits of blank (LoB), detection (LoD) and quantitation (LoQ), based on CLSI guidelines. Method comparison was performed using the ARCHITECT instrument system C8000 and i2000 system. Throughput and internal controls stability on board were also evaluated. Performance analyses were completed using quality control materials from the manufacturer and biological samples from our institution. Statistical analyses were performed using PC SAS 9.3. The CVs for within-run precision for clinical chemistry tests ranged from 0.5 to 2% and were of 1.77% for TSH and 3.35 % for hsTn-I. The CVs for day-to-day precision for clinical chemistry tests ranged from <1 to 2% and were of 1.2% for TSH and 1.94 % for hsTn-I. Linearity testing verified the assay linearity claims for all parameters (r=0.999). Comparison studies showed good correlations with ARCHITECT instruments (r ranging from 0.974 to 1). All estimates of LoB, LoD and LoQ met the manufacturer’s claim. Throughput study revealed 1204 tests/hour and 167 tests/hour capability for clinical chemistry assays and immunoassays, respectively. On board stability of chemistry QC was in the range announced by the manufacturer. Taken together, the familiarization study performed on a selected set of parameters tested on a manufacturing prototype of the Alinity ci-series analyzer revealed satisfactory analytical performances, allied to simplified maintenance procedures, software easy-to-use, shortened hands-on time and overall system reliability.

CARDIAC TROPONINS AND NATRIURETIC PEPTIDES: LAST GENERATION METHODS AND CLINICAL APPLICATIONS

A. Clerico, S. Masotti, V. Musetti, C. Prontera, C. Passino

Fondazione CNR Regione Toscana G. Monasterio and Scuola Superiore Sant’Anna, Pisa

Cardiovascular diseases are the main cause of morbidity and mortality in industrialized countries; therefore, the evaluation of cardiovascular risk factors, as well as an early diagnosis of cardiac diseases, must be considered the first objective of public health in these countries. For this reason, there is an increasing interest in the development of new cardiovascular biomarkers and, consequently, a great number of laboratory tests have recently been proposed, probably more than 100 (1). However, only few of them are recommended by international guidelines, because there are no evidence-based data on their clinical usefulness in the follow-up of patients with cardiovascular disease. Among the huge number of suggested cardiovascular biomarkers, at present time, only cardiac troponin I (cTnI) and T (cTnT) and natriuretic peptides (especially BNP and NT-proBNP) have been demonstrated to be cardiac-specific biomarkers. The cardiac-specificity associated to a high analytical sensitivity are two essential pre-requisites for the diagnosis of heart failure in the first subclinical phases of disease (2), as well as for assessment of very small amounts of injured myocardial tissue (3). In the last 10 years, the analytical sensitivity of both cTn and BNP/NT-proBNP methods has been progressively improved, so that the most recent immunoassay methods are able to detect plasma biomarker concentrations of about 2-5 ng/L (4). For example, it is was calculated that the cTn concentration equal to the 99^th percentile of the reference population actually corresponds to a necrosis of about 40 mg of myocardium, which is a tissue amount too small to detect by cardiac imaging (5). The assessment of amounts of myocardial damage too small to cause specific signs and symptoms of cardiac disease is clinically relevant because only a clinical intervention during the early phases of myocardial remodeling is able to arrest or even to reverse the progression forward to symptomatic heart failure. Several studies demonstrated that progressively increased levels of cTn and natriuretic peptides even in the concentration range below the cut off level values significantly increase cardiovascular risk. Accordingly, the measurement of these biomarkers should be mandatory in al patients before the administration of cardio-toxic drugs, especially the most part of all anti-tumor agents, in order to early demonstrate the progression to heart failure, some months before the demonstration of reduction in left ventricular ejection fraction <50%. It is really important to stress the concept that an increase of cardiac biomarkers is anyway an index of cardiac functional stress or myocardial damage, even in the case of extra-cardiac diseases (including chronic inflammatory disease, end stage renal disease, or treatment with powerful cardio-toxic drugs). Therefore, the detection of increased biomarkers should stimulate a perspicacious clinician to search for a cause for this elevation in order to make an appropriate diagnosis and, when necessary, to initiate a specific treatment.

1. Vittorini S, Clerico A. Cardiovascular biomarkers: increasing impact of laboratory medicine in cardiology practice. Clin Chem Lab Med 2008;46:748-63.

2. Emdin M, Passino C, Prontera C, et al. Comparison of brain natriuretic peptide (BNP) and amino-terminal proBNP for early diagnosis of heart failure. Clin Chem 2007;53:1289-97.

3. Giannoni A, Giovannini S, Clerico A. Measurement of circulating concentrations of cardiac troponin I and T in healthy subjects: a tool for monitoring myocardial tissue renewal? Clin Chem Lab Med 2009;47:1167–77.

4. Clerico A, Passino C, Franzini M, et al. Cardiac biomarker testing in the clinical laboratory: where do we stand? General overview of the methodology with special emphasis on natriuretic peptides. Clin Chim Acta 2015;443:17-24.

5. Marjot J, Kaier TE, Martin ED, et al. Quantifying the release of biomarkers of myocardial necrosis from cardiac myocytes and intact myocardium. Clin Chem 2017;63:990-6.

PHARMACOGENETICS IN PSYCHIATRY

M. Gennarelli

Sez. Biologia e Genetica, Dip Medicina Molecolare e Traslazionale, Università degli Studi di Brescia; Unità di Genetica, IRCCS-Centro S. Giovanni di Dio, Fatebenefratelli, Brescia

In recent years, the study of genetic variability associated with the response and/or side effects of psychotropic drugs has focused on a limited number of candidate genes functional polymorphisms. More recently, the genomic approach through multicentric GWAS studies has allowed to identify new gene variants associated with the therapeutic efficacy of antidepressants, antipsychotics and mood stabilizers. These studies also allowed to identify gene variants associated with endophenotypic and/or specific side effects. These results, if confirmed in stratified populations also derived from the huge sample collected by various international consortia, could produce genomic biomarkers with levels of sensitivity and specificity useful in clinical practice. Furthermore, due to the reduction in the cost of complete genomic sequencing, the study of genetic variability will be completed by the characterization of rare variants with high functional impact. These data will enable, in the near future, the construction of a pharmacogenomic database useful in the development of precision medicine even in psychiatry.

CARDIOVASCULAR PHARMACOGENOMICS

A. Filippelli^1,2, V. Manzo^1,2, C. Sellitto^1,2, T. Iannaccone^1,2, V. Izzo^1,2, F. Dal Piaz^1,2, M. Costantino^1,2, V. Conti^1,2

¹Department of Medicine, Surgery and Dentistry, University of Salerno, Baronissi (Salerno), Italy

²Clinical Pharmacology Unit, University Hospital “San Giovanni di Dio e Ruggi d’Aragona”, Salerno, Italy

Patients’ response to pharmacological therapies depends on a complex interplay among clinical, environmental and genetic factors (1). In this framework, the analysis of the genetic factors is the focus of what is usually referred to as pharmacogenomics (or pharmacogenetics), which deals with the effects of genetic variations (polymorphisms) on drug response. Indeed, polymorphisms such as single nucleotide polymorphisms (SNPs) can influence drugs’ pharmacodynamics and pharmacokinetics, resulting in a range of phenotypes that, in turn, can lead to either lack of effectiveness or toxicity of a bioactive molecule (2). Main aim of pharmacogenomics is to contribute to the so-called personalized medicine, where “the right dose of the right drug” is given to “the right person at the right time”.

The contribution of pharmacogenomic tests is becoming increasingly important in several medical fields including –among others- cardiology. Cardiovascular disease is undoubtedly the leading cause of mortality and hospitalization in developed countries, mainly in elderly patients, and cardiovascular medications are the most prescribed drugs worldwide (3). Since it has been proven that several pharmacogenetic biomarkers influence both the pharmacodynamics and the pharmacokinetics of cardiovascular drugs, pharmacogenomics has recently arisen great interest in this field. Several pharmacogenetic tests are now recommended, including those predicting the response to anticoagulant, (e.g. warfarin) antiplatelet (e.g. clopidogrel) and antihypercholesterolemic drugs (e.g. statins). As an example, many studies have underlined the importance of the pharmacogenetic dosing approach to optimize the starting dosage of warfarin, thereby reducing the risk of both thromboembolic and haemorrhagic side effects. Noteworthy, a specific pharmacogenetic algorithm to evaluate the correct warfarin dosage is currently available, which stratifies patients taking into account genetic variants, demographic and clinical factors (4). Among others, the efficacy of the pharmacogenetic testing has been widely demonstrated in the case of patients affected by peripheral arterial disease or acute coronary syndrome treated with the pro-drug clopidogrel, which is activated by the enzyme CYP2C19 (5). Despite its good efficacy and safety, there is a remarkable variability in the therapeutic response to such antiplatelet agent, partially due to three polymorphisms indicated as CYP2C19-*2; -*3 and -*17. In particular, CYP2C19-*2 and -*3, whose presence identify individuals as intermediate and poor metabolizers, reduce the conversion of clopidogrel to its active form, thereby decreasing the drug’s antiplatelet activity. In this case, the pharmacogenetics analyses that we routinely perform at the University Hospital “San Giovanni di Dio e Ruggi d’Aragona” in Salerno (Italy) allow identifying patients who do not respond appropriately to clopidogrel at standard dosage (5). In conclusion, there is a strong need to promote high power studies to implement the systematic application of the pharmacogenetic analysis in the clinical decision-making process for the treatment of cardiovascular disease.

1. Vesell ES. Genetic and environmental factors causing variation in drug response. Mutat Res 1991;247:241-57.

2. Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999;286:487–91.

3. Mozaffarian D, Benjamin EJ, Go AS, et al. Heart disease and stroke statistics--2015 update: a report from the American Heart Association. Circulation 2015;131:e29-322.

4. Furuya H, Fernandez-Salguero P, Gregory W, et al. Genetic polymorphism of CYP2C9 and its effect on warfarin maintenance dose requirement in patients undergoing anticoagulation therapy. Pharmacogenetics 1995;5:389-92.

5. Hulot JS, Bura A, Villard E, et al. Cytochrome P450 2C19 loss-of-function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects. Blood 2006;108:2244-7.

MUTATIONAL LANDSCAPE PROFILE IN BREAST CANCER BY LIQUID BIOPSY

G.L. Scaglione¹, M. De Bonis², E. De Paolis², D. Gallo³, G. Scambia³, E. Capoluongo⁴

¹Fondazione di Ricerca e Cura “Giovanni Paolo II”

²Polo Scienze delle Immagini, di Laboratorio ed Infettivologiche, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario Agostino Gemelli

³Dipartimento Ostetricia e Ginecologia, “Fondazione Policlinico Gemelli”, Università Cattolica del Sacro Cuore

⁴Dipartimento Diagnostica di Laboratorio e Biologia Molecolare Clinica, Istituto Dermopatico dell’Immacolata - IRCCS

Background: Cancer molecular profile evolves over time in response to a wide variety of endogenous and exogenous selective pressures. The identification of tumor specific molecular landscapes is a crucial aspect of the success of targeted therapy. Tumor dynamic plasticity could not be captured in its complexity by the single molecular source offered by a one-time tissue biopsy. Straightforwardly, diagnostics approaches for cancers clearly benefit from more snapshots at the same time. This cutting-edge clinical applications led to the development of several monitoring tools to study circulating tumor-derived components, including circulating cell-free DNA (cfDNA) and Circulating Tumor Cells (CTCs), widely known as “liquid biopsy”. In this study, we performed liquid biopsy strategy to define the mutational landscape of five breast cancer metastatic untreated women focusing on low frequency pathogenic alleles (³1%) and also on tumor clonality status. Methods.Single EDTA blood tube was collected for each subject (n=5) and prepared for targeted NGS analysis following LiquidBiopsy Platform workflow by ThermoFisher. CTCs, cfDNA, and gDNA (WBC) were processed in a single run (2chip/run) on Ion S5 System. In addition to the variant analysis using the Ion AmpliSeq Cancer Hotspot Panel v2 and Ion Reporter v5.2 Software, an in house UNIX script was designed to gather additional variant annotations from reference diagnostics databases (ClinVar, dSNP, COSMIC) and, to retrieve variant statistics for data reporting.

Result: Custom Ion Reported filter chains coupled with ad hoc UNIX script allowed: a) to filter out ~95% of variants from raw dataset. b) to collect/compare pathogenic variants (~20/sample) in each blood component. c) to highlight unbalanced allelic distributions (up to ~20fold) reflecting potential cancer clonality.

Conclusion: Liquid biopsy approaches could potentially lead to define genomic profile of patients with cancer, to quantify minimal residual disease and to monitor treatment responses. In the era of high-throughput NGS tools, the possibility to better understand the nature of tumor clonality has emerged matching the need of a new diagnostic strategy chasing tumor profile’s remodeling.

– Strauss WM, Carter C, Simmons J, et al. Analysis of tumor template from multiple compartments in a blood sample provides complementary access to peripheral tumor biomarkers. Oncotarget 2016;7:26724-38.

HEMOCYTOMETRIC VIEW OF MIELODISPLASY-INSTRUMENTAL AND MICROSCOPIC MORPHOLOGY

D. Avino

Salerno

Mielodysplastic syndromes (MDS) are a heterogeneous group of clonal hematogical diseases of hematopoietic stem cell characterized by peripheral blood cytopenia(s), ineffective hematopoiesis and increased risk of development of acute myeloid leukemia (1). In the early phase of the disease, the diagnosis of MDS, often occasional, is based essentially on haemochromocytometric examination that evidences cytopenia(s) and on the morphological assessment of dysplasia with standardized methods (2). Morphological alterations of the dysplastic cells, regardless of the technology employed, give different instrumental signals than those generated by normal cells. Morphological abnormalities of red blood cells are: anisocytosis, poikilocytosis, anisochromasia or polychromasia, basophilic stippling, Howell-Jolly bodies, Pappenheimer bodies. Erythroid dimorphism may be present with a normal population and a dysplastic microcytic or macrocytic population. Macrocytosis, often associated with erythroid dysplasia, is well expressed by hematology analyzers by volume measurements (MCV) and the degree of anisocytosis is quantized by RDW (Red Cell Distribution Width). Some analyzers are also able to provide an index of red blood cells anisochromasia (HDW) (3). Morphological alterations of neutrophils may involve the nucleus (abnormal nuclear segmentation, abnormal clumping of the chromatin, presence of nuclear projections) and/or the cytoplasm (reduction of the content of granules) (4). Depending on the specific analytical techniques in use (optical, fluorescence, cytochemical, electrical conductivity) many of these alterations generate numerical and graphical informations that are indicative of these anomalies. Platelet dysplasia morphologically appears with variations in size and density of the granules. MPV parameter measures the size of the platelets while the degree of platelet size variability is expressed by PDW (Platelet Distribution Width) parameter. Some specific-instrument parameters may provide an estimate of the mean platelet component (MPC) that appears to be reduced in myelodysplasia because of platelet granule deficiency or a measure of the fractional platelet immature (IPF) that is increased in MDS and can be a marker for karyotypic abnormalities with a poor prognosis, including chromosome 7 abnormalities. The presence of blasts in the peripheral blood is a decisive factor for WHO classification and prognosis. Definition of a precise and accurate morphological count of blasts in peripheral blood is crucial for values of 1%, <2%, and between 2% and 5% (5). The presence of blasts is well underlined with different accuracy in relation to morphological and structural anomalies and to the thecnology employed but remains a data to be defined exclusively with microscopic observation. The latest generation hematology analyzers are for rapidity, cost and multiplicity of information provided, a powerful screening tool of the population to be subjected to diagnostic deepening. The most important limit of abnormalities found in automation is the technology-dependence of the alarm signals, with difficulty to translate instrumental finds in diagnostic informations understandable to the clinician.

1. Swerdlow SH, Campo E, Harris NL, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC, 2008.

2. Malcovati L, Hellström-Lindberg E, Bowen D, et al. Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European LeukemiaNet. Blood 2013;122:2943-64.

3. Rocco V, Maconi M, Gioia M, et al. Possibility of myelodysplastic syndromes screening using a complete blood automated cell count. Leukemia Research 2011;35:1623-7.

4. Goasguen JE, Bennett JM, Bain BJ, et al. The International Working Group on Morphology of MDS (IWGM-MDS). Proposal for refining the definition of dysgranulopoiesis in acute myeloid leukemia and myelodysplastic syndromes. Leukemia Research 2014;38:447-53.

5. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009;114:937-51.

SATELLITISM OF PLATELETS TO NEUTROPHILS, MONOCYTES, LYMPHOCYTES AND LARGE GRANULAR LYMPHOCYTES ASSOCIATED WITH PLATELET PHAGOCYTOSIS: AN UNUSUAL MULTI-CELLULAR INVOLVEMENT IN A RARE PHENOMENON

A. La Gioia¹, F. Fiorini²

¹Docemus Onlus

²UOC Medicina di Laboratorio Azienda USL Toscana Nord Ovest

Platelets satellitism (PS) is a rare phenomenon (1 of 12,000 blood count. Bizzaro, 1995) consisting in an adhesion of platelets PLTs) to surface of other cells. This fact occurs in vitro and in EDTA anticoagulated samples only. Therefore, a variable number of circulating PLTs are subtracted to automated blood cell count, leading to a pseudo-thrombocytopenia. The recognition of PS in light microscopy is easy because the images of platelets surrounding the contour of leukocytes are unmistakable and evocative. After the first descriptions of PLTs rosetting around neutrophils (NE) (Reisman, 1974; Kjeldsberg, 1974; Mende, 1975), the involvement of other cellular types it was described: basophils (BA) (Liso, 1981); eosinophils (EO) (Fábryová, 1991); monocytes (MO) (Djaldetti, 1978); lymphocytes (LY) (Tun, 2016); LY from lymphomas; (Cesca, 2001); large granular cells (LGC) (Espagnol, 2000). In addition, it was reported PS satellitism around different cell types in the same sample: NE and MO (Greip, 1976); NE, EO and MO (Lazo-Langner, 2002). Rarely, PLT phagocytosis by NE was observed in association whit satellitism (Yoo, 1982; Preethi, 2012) or isolated (Criswell, 2001). In both cases, the phenomenon appeared to be EDTA-dependent by inducing the hypothesis of a common pathophysiology. We report a case of PS and phagocytosis in a 42-year-old- asymptomatic woman presented to our Laboratory for mild thrombocytopenia and neutropenia (50×10⁹/L and 0.90×10⁹/L respectively), absolute monocytosis (1.21×10⁹/L) and relative lymphocytosis (57.3%; 2.58×10⁹/L). PLT count was confirmed but microscopic blood smear examination revealed PLT rosetting NE (100% of these), MO (27.1%), LY (2.7%) and LGC (5.6%). In some case in the NE and in the MO engulfed PLTs were present (20% and 5.9% respectively). Some aspects of “pre-phagocytosis” (platelet that were housed in low lying areas of the nuclear contour) as wells as “post-phagocytic” vacuoles were even observed. BA and EO were not involved. PLT count in sodium citrate was equal to 147×10⁹/L and a finger-pick blood smear demonstrated the demise of PS as well as phagocytosis. Based on this observation we posed the diagnosis of “pseudo thrombocytopenia due to platelet satellitism EDTA dependent”.

EVALUATION OF RESPONSE TO TREATMENT IN A PATIENT AFFECTED BY AL AMYLOIDOSIS WITH LOW FREE LIGHT BURDEN

P. Milani¹, M. Basset¹, F. Russo¹, M. Nuvolone¹, F. Lavatelli¹, T. Bosoni², L. Pirolini², F. Li Bergolis², R. Albertini², G. Palladini¹, G. Merlini¹

¹Amyloidosis Research and Treatment Center, Dep. of Molecular Medicine, University of Pavia, IRCCS Policlinico San Matteo, Pavia, Italy

²Clinical Chemistry Laboratory, IRCCS Policlinico San Matteo, Pavia, Italy

In patients affected by light chain (AL) amyloidosis, the reduction of the concentration of the amyloidogenic circulating light chains translates in improvement of organ dysfunction and is associated with an increase in overall survival. This allowed the definition and validation of the criteria for hematologic response to therapy that are based on FLC quantification (Palladini et al. JCO 2012). A 69 years old man with nephrotic syndrome (proteinuria 5 g/24h), was evaluated in another center in December 2015. A renal biopsy revealed amyloid deposits. In January 2016 at our center, no monoclonal components were detected at serum and urine immunofixation, proteinuria was 6 g/24h with creatinine 0.99 mg/dL (u.r.l 1.17 mg/dL). The λ-FLC concentration was 54 mg/L (κ/λ ratio 0.25, dFLC 41 mg/L) and a bone marrow aspiration revealed 11% of monoclonal plasma cells. Echocardiography did not reveal signs of cardiac amyloidosis. The abdominal fat aspirate for amyloid typing by immune-electron microscopy showed λ-LC deposits. The diagnosis of AL (λ) amyloidosis with renal involvement was made and the patient was started on treatment with cyclophosphamide, bortezomib and dexamethasone. After 2 courses, at our center, the λ-FLC concentration was 59 mg/L (κ/λ ratio 0.24, dFLC 45 mg/L) and an increase of proteinuria (9 g/24h) and creatinine (1.25 mg/dL) was documented. Treatment strategy was changed because of the lack of hematologic response and the progression of renal damage. The patient was stated on therapy with melphalan and dexamethasone. After 3 courses a reduction of the λ-FLC concentration was noticed (κ/λ ratio 1.36, dFLC 0 mg/L). A significant reduction of proteinuria was revealed (5.5 g/24h) with improved serum creatinine (1.0 mg/dL). After 3 other cycles, no monoclonal proteins were detected at serum and urine immunofixation, dFLC was 2 mg/L (κ/λ ratio 1.42) and a further decrease in proteinuria was documented (3.2 g/24h). In this case, as reported in two recent manuscripts (Milani et al. and Dittrich et al. Blood 2017) a reduction of dFLC <10 mg/L after therapy was associated with an increase of organ dysfunction. These data confirm the central role of the FLC assessment in the monitoring also of patients with a low-dFLC burden affected by AL amyloidosis.

INCIDENTAL DETECTION OF A NEW HAEMOGLOBIN BETA VARIANT DURING HbA_1c MEASUREMENT

I. Crespi¹, M.P. Campisi¹, R. Serino¹, E. Saliva¹, M. Marchiando¹, G. Barberio², G. Ivaldi³, M. Mogni³, G. Bellomo¹

¹Laboratorio di Biochimica Clinica, AOU Maggiore della Carità, Novara

²Dipartimento di Patologia Clinica-Medicina di Laboratorio

³Laboratorio di Genetica Umana, Ospedali Galliera, Genova

Introduction: A number of hemoglobin b chain variants has been described. Their presence can be suspected on the basis of typical changes in hemochromocytometric analysis or incidentally detected during chromatographic analysis of glycated hemoglobin (HbA_1c). The availability of capillary zone electrophoresis (CZE) for HbA_1c measurement could improve their detection.

Methods: A non anemic 58-year-old female of sardinian origin was investigated for quantification of HbA_1c. The blood was collected in EDTA-containing tube. CZE was performed with Sebia Tera 3 Capillarys system (Sebia Italia Srl), using programs and reagent for HbA_1c. Standard high performance liquid chromatography (HPLC) was performed with VARIANT II™ Analyzer (Biorad).

Results: The quantification of HbA_1c was invalidated by the presence of a double peak in the HbA₀ zone. HPLC analysis did not allow to separate the variant from Hb A₀. Despite this Hb variant was clinically silent, DNA analysis and b-globin gene sequencing was performed and showed a heterozygous variation of nucleotide sequence HBB:c.376C>A; beta 125 (H3) Pro>Thr. The new variant was denominated Hb Novara. The same variant was found in the daughter of the proband during HbA_1c analysis, together with a marked decrease of HbA₂ (1.4 %). It was associated with a deletion in the a-chain gene. The daughter showed a moderate anemia (Hb 9.0 g/dL, MCV 61.3 fL, MCH 17 pg). The hemoglobin stability tests were normal in both patients. Oxygen affinity was different (P₅₀: 22.68 and 29.38 mmHg respectively for the mother and the daughter).

Conclusion: The discovery of the Hb Novara has been possible by the use of a procedure not originally developed for variant detection. It clearly suggests that sometimes a combination of different technologies (such as HPLC and CZE) can be extremely useful in detection of an increasing number of hemoglobin variants (even those of apparent little clinical interest), in order to better understand the pathophysiology of hemoglobin. Although the carrier of Hb Novara seems to be essentially asymptomatic, is not possible to exclude that this mild defect could produce more relevant hematological phenotypes when associated with other a or b chain defects.

COMBINED PLATELET FUNCTION DISORDER AND FACTOR XI DEFICIENCY

B. Montaruli¹, P. Sivera², G.E. Parvis², I. Canta¹, O. Ruggia¹, N. Renzi¹, M. Migliardi²

¹SC Laboratorio Analisi, ²SCDU Ematologia, AO Ordine Mauriziano, Torino

We present a case concerning a 17 year old boy with positive bleeding history and neck lymphadenopathy scheduled for fine needle aspiration biopsy (FNAB). The patient referred for evaluation of haemostasis status in preparation for biopsy. His medical history included bleeding after appendectomy when he was 6 year old. The family history was significant for a father with bleeding after dental extraction. Laboratory coagulation and platelets profile performed when he was a child showed slightly prolonged PT and APTT ratio (PT ratio=1.25 and APTT ratio=1.22) with normal values of all coagulation factors, normal values of Platelet function analyzer (PFA-100) and abnormal platelet aggregation to ADP. Coagulation laboratory investigation performed in our laboratory showed, normal PT and slightly prolonged APTT ratio (PT ratio=1.08 and APTT ratio=1.22) and reduced factor XI levels (49%, normal values >55%). Platelets investigation showed, normal platelets count (248.000) and morphology, normal PFA-100 closure times to Collagen/ADP and Collagen/Epinephrine and decreased secondary platelets aggregation to ADP and epinephrine on Light Trasmittance Aggregometry. Laboratory findings were consistent with an inherited platelets function defect (storage pool disease or dense granule defect) and hereditary factor XI mild deficiency. Management of this patient included careful monitoring for needle aspiration biopsy bleeding complications, prophylactic tranexamic acid, as well as having platelets readly available during FNAB. In conclusion detailed medical and family history is a key to determining the cause of bleeding, specialized tests including old-fashioned platelet function assays performed as recommended by laboratory guidelines (recommended agonists concentrations) and experienced individuals interpreting tracing and results can provide diagnosis in these patients.

ISCHEMIC STROKE SECONDARY TO SEVERE REACTIVE THROMBOCYTOSIS IN A PATIENT WITH CELIAC DISEASE AND LONG-TERM IRON DEFICIENCY ANEMIA

F. De Liso¹, M. Ronchi¹, C. Matinato¹, M. Fraquelli², M. Ammirabile¹, R. Maiavacca¹, F. Ceriotti¹

¹Lab. Clinical Chemistry and Microbiology, ²Gastroenterology and Endoscopy Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano

We report a case of a 51-year-old woman who was admitted to emergency room due to an ischemic stroke. She underwent to several laboratory and neuroradiological examinations. Computed tomographic angiography showed a thrombus in the right internal carotid artery. Laboratory tests showed an extreme thrombocytosis (PLT: 1963*10⁹/L, RI 130-400*10⁹/L) and a severe microcytic anemia (Hb: 4 g/dL, RI 12-16 g/dL; MCV: 61 fL, RI 78-99 fL). Serum iron (8 μg/dL; RI 37-145 μg/dL) and ferritin levels (1 μg/L, RI 15-150 μg/L) were very low; lactate dehydrogenase was slightly elevated (276 U/L, RI 135-214 U/L); coagulation profile was found in the normal range and antiphospholipid antibodies were absent. The marked thrombocytosis has led to evaluate the presence of myeloproliferative disorders, particularly essential thrombocytemia. Research of JAK-2 gene mutation resulted negative. Bone marrow exam (biopsy and aspiration) and cytofluorimetric analysis allowed to rule out hematological malignancies. Anamnestic data revealed that anemia was present since adolescence and was refractory to oral iron treatment. All these evidences led to hypothesize the celiac disease (CD) as possible cause of anemia due to iron malabsorption. No gastrointestinal symptoms have been referred. CD is an autoimmune disorder triggered by gluten ingestion and targeting small intestine. Among extra-intestinal manifestations of CD iron-deficiency anemia is not so rare and sometimes associated to reactive thrombocytosis. According to guideline, total serum IgA levels were measured, ruling out a deficit of this immunoglobulin isotype; IgA anti-tissue transglutaminase (tTG) and IgA endomysial antibody (EMA) were performed and both resulted strong positive (tTG: 275 U/mL, cut-off <7 U/mL; EMA: 1/80). Endoscopy investigation to confirm the serological diagnosis of CD is in progress. The patient was treated with packed erythrocyte transfusion, intravenous iron and antiplatelet drug with a progressive improvement of neurological signs along with an increase of hemoglobin levels (Hb: 8.0 g/dL) and a reduction of platelet count (PLT: 627*10⁹/L). These results suggest that the heavy thrombocytosis is likely secondary to iron deficiency anemia due to an underlying CD and could represent a risk for thrombotic complications.

A CLINICAL AND SEROLOGICAL STUDY OF IgA DERMATOSIS WITHOUT IMMUNOGLOBULIN DEPOSITION OTHER THAN IgA IN DIRECT IMMUNOFLUORESCENCE: A CASE REPORT

L. Abbracciavento¹, F. Migliucci¹, G. Zanframundo², L. Lospalluti², M. Tampoia¹, R. Fumarulo¹

¹Clinical Pathology Laboratory, University of Bari

²Unit of Dermatology, University of Bari

Linear Immunoglobulin A bullous dermatosis (LABD) is a subepidermal and autoimmune blistering disorder. It is characterized by linear deposits of immunoglobulin (Ig) A along the basement membrane zone (BMZ) that are seen by direct immunofluorescence (IF). We would like to report our experience with a 54-year-old Caucasian woman who presented with a 2 years history of a persistent and steroid-resistant dermatosis. Clinical examination revealed multiple coalescent large pustules, containing clear to slightly cloudy fluid, some of which displayed hypopyon formation. Mucous membranes were uninvolved. Electrophoresis of serum immunoglobulins did not reveal a monoclonal gammopathy. Bacterial cultures were negative. Histopathologic examination of skin biopsy showed a subcorneal cleft with acantholysis. Direct immunofluorescence of the perilesional skin biopsy showed exclusively IgA deposition on keratinocytes cell surfaces. Staining was evident in particular in the superficial layers of the epidermis. Indirect immunofluorescence microscopy performed on monkey esophagus did not detect circulating IgA or IgG anti-cell surface autoantibodies. Enzyme-linked immunosorbent assay (ELISA) for IgG autoantibodies against desmogleins (DsG1, DsG3), performed using commercially available kits (MBL, Nagoya, Japan) showed no reactivity. Commercial IgA ELISAs are not available and so to detect IgA reactivity against desmogleins, ELISA kits were modified applying peroxidase conjugated anti-human IgA. At cut-off value of optical density (OD) more than 0.150, as described previously by Hashimoto et al., IgA reactivity (OD) to DsG1 was 0.206 and to DsG3 was 0.165. Moreover, commercial kits for autoantibodies against desmocollins (Dscs) were not available. In conclusion, basing on clinical appearance and immunopathology results, was made diagnosis of subcorneal pustular dermatosis-type intercellular IgA dermatosis (SPD-type IAD). Lacking circulating IgA specific autoantibodies results, we are not able to confirm this subtype accordingly to Hashimoto’s classification. Recently, a novel IgA ELISAs was developed to detect circulating IgA antibodies anti-Dsc. We hope these IgA ELISAs will introduce in routine practice to facilitate the diagnosis and understanding of pathophysiology in IAD in the future.

STIFF-PERSON SYNDROME CASE REPORT: FROM LABORATORY DIAGNOSIS TO TREATMENT

G. Pedà^1,2, J. Uggeri¹, E. Chierici², G. Giambuzzi¹, M. Amadei¹, L. Ippolito¹

¹U.O. Patologia Clinica, ²U.O. Neurologia, Ospedale di Vaio, Fidenza

Background: Stiff-person syndrome, also called “rigid man syndrome” or “stiff man-syndrome” (SMS), is a rare neurological disease characterized by fluttering chest and limb stiffness, painful muscle spasms, phobia related to certain tasks, abnormal tendency and anchilous deformities. The onset occurs around the age of 45 and the symptoms develop over the course of months or years. The pathophysiology of the disease is autoimmune, this disease seems to be associated with the presence of antibodies to the glutamic acid (GAD) decarboxylase, usually directed against peripheral nervous system but sometimes also against central nervous system parts.

Case report: Woman, age 65, hospitalised in January 2016 at U.O. of Neurology due to a two-to three-month, short-to-left motor lumbar spine, characterized by floating stiffness that appears and accentuates in orthostatism with a tendency to fall, does not exhibit axial rigidity. The patient has always had a mildly disturbed pathological history. Differential diagnosis was performed after hospitalization, after several instrumental assessments, it was decided to perform lumbar puncture with consequent analysis of the liquor and isoelectrophocalization showed absence of bands Intrathecal synthesis, but presence of numerous bands on both liquor and serum, oligoclonal mirror profile, type 4. It was decided to require dosage of anti-GAD antibodies on liquor and serum with “off-normal”, seeking anti Amphiphysin negative antibodies. The patient was discharged with Stiff-person syndrome diagnosis and Diazepam therapy at home and Immunoglobulin e.v. with periodic monitoring.

Conclusion: The patient is in charge of the U.O. Day Service. Neurology of Vaio Hospital, Fidenza and continues the periodic cycles of Immunoglobuline e.v. and now reports clinical stability. It is therefore considered essential to carry out early diagnosis in this type of rare and ingesting pathology so as to set a correct therapy and to move as soon as possible. It is also important that there is a great collaboration between the clinic and the laboratory to reduce the waiting time and orient the clinician in the right diagnosis and in the need for the correct examinations to be performed, always considering the patient at the center.

CALPROTECTIN AND OTHER FECAL MARKERS OF INTESTINAL INFLAMMATION

D. Basso

Department of Medicine - DIMED, University of Padova

Fecal calprotectin (fCal) has challenged in the last decades a more effective diagnostic work-up for patients with inflammatory bowel diseases (IBDs), which comprise Crohn’s disease (CD) and ulcerative colitis (UC). IBDs share intestinal inflammation, which affects the mucosa of the large intestine in UC, while in CD it may occur at any part of the gastrointestinal tract, being not limited to the mucosa, but involving the whole intestinal wall and potentially causing stenosis, perforation and fistulae. IBDs are chronic diseases characterized by alternations of flares and remissions, which onset typically occurs at a young age. To answer the question “how laboratory medicine should help clinicians facing with IBDs?”, three main clinical settings should be considered: 1. Rule-in and rule-out IBDs, i.e. distinguish IBDs from irritable bowel syndrome and make a diagnosis; 2. Define type of IBDs (UC or CD); 3. Monitoring IBDs to early detect flares and predict the response to therapy. A number of blood and fecal biomarkers for IBDs are available. While blood biomarkers allow addressing the second, fecal markers allow addressing the first and the third items. Blood inflammatory biomarkers, as C-reactive protein, although useful are not specific for IBDs, while markers to define the IBDs type, as UC-associated pANCA and CD-associated ASCA, have a diagnostic accuracy which does not exceed 60-70%. The two most extensively studied fecal biomarkers are fCal, a S100A8/S100A9 calcium binding heterodimer, and lactoferrin, an iron binding protein. Both proteins have antimicrobial properties, are released by inflammatory cells - mainly polymorphonuclear cells - infiltrating the gastrointestinal mucosa, and are resistant to proteolysis, which renders their measurement in stool a reliable tracer of intestinal inflammation. However fCal was reported to be more sensitive and specific than lactoferrin. Despite the market offers qualitative fCal assays, to be clinically useful a quantitative result (μg/g) should be provided. Different thresholds allow to address different clinical questions in different settings: values below 50 μg/g in adults and 100 μg/g in children rule-out intestinal inflammation with a high negative predictive value (81% in primary and 98% in secondary care), while values above 150 μg/g in adults and 300 μg/g in children may predict IBDs with high sensitivity (>90%). In patients with an established IBDs diagnosis values above 250 μg/g in adults and 500 μg/g in children predict disease relapse with high sensitivity and specificity (>80%) and may help in predicting the response to anti-TNF treatment. From a clinical laboratory perspective both the pre-analytical (stool storage, weighting and sampling with dedicated devices) and the analytical phase (ELISA, chemiluminescence or turbidimetric assays) require to be strictly monitored through internal and external quality control programs. Since a high inter-method variability has been observed, hence harmonization projects represent a chance in the near future. In conclusion fCal, although not perfect, is actually our best choice for diagnosing and monitoring IBDs.

LABORATORY EVALUATION OF PANCREATITIS

M. Panteghini

Cattedra di Biochimica Clinica e Biologia Molecolare Clinica, Dipartimento di Scienze Biomediche e Cliniche ‘Luigi Sacco’, Università degli Studi, Milan, Italy

Today, laboratory tests not only play a pivotal role in the diagnosis of acute pancreatitis, but also may help to assess the disease severity and its etiology. In the diagnosis of acute pancreatitis, lipase measurement in serum is superior to (P-type) amylase in terms of cost-effectiveness and diagnostic performance. Its clinical sensitivity is 80 to 100% depending on the selected diagnostic cut-off, and the clinical specificity is 80 to 100% depending on the mix of the patient population studied. After an attack of acute pancreatitis, serum lipase activity increases within 4 to 8 h, peaks at about 24 h, and decreases within 7 to 14 days. However, the increase is not necessarily proportional to the severity of the attack. Points to remember for pancreatic enzymes in acute pancreatitis are: a) their diagnostic performance is greatly improved by restricting their use to a population with suspected disease; b) it is recommended that lipase replaces (P-type) amylase as initial test for acute pancreatitis; measuring both serum amylase and lipase is not warranted; c) the measurement of total amylase should be considered obsolete.

1. Panteghini M, Bais R. Serum enzymes. In: N Rifai, AR Horvath, CT Wittwer, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier Saunders: St. Louis, 2018:404-34.

2. Phillip V, Steiner JM, Algül H. Early phase of acute pancreatitits: assessment and management. World J Gastrointest Pathophysiol 2014;5:158-68.

3. Tenner S, Baillie J, DeWitt J, et al. American College of Gastroenterology guideline: management of acute pancreatitis. Am J Gastroenterol 2013;108:1400-15.

4. Panteghini M. Laboratory evaluation of the pancreas. In: Clarke W, ed. Contemporary pratice in clinical chemistry. 2nd ed. Washington DC: AACC Press, 2011:333-41.

FECAL TESTS FOR COLORECTAL CANCER SCREENING: FROM FECAL OCCULT BLOOD TEST TO DNA ANALYSIS

A. Di Leo

Section of Gastroenterology, Department of Emergency and Organ Transplantation, University of Bari, Italy

Colorectal cancer (CRC) is one of the most common malignancies. Due to its biologic behavior, a screening program is easily applicable to CRC. The most used strategies are based on laboratory investigations on stool samples. Blood in the stools is the first and most used strategy. Fecal occult blood test (FOBT) and fecal immunochemical test (FIT) are the main methods. Both are economic, easy to perform with high specificity, and low sensitivity. However, such tests have a low sensitivity and the result may be affected by alimentary and drug interference (1). Based on CRC multi-step process with genetic and epigenetic alterations in large bowel cell DNA, single mutations or panels of alterations have been proposed as alternative stool test. These tests have the advantage of a marked improvement of the sensitivity when compared to fecal blood. However, high costs, poor availability, and correct choice of marker panel represent the major limits. A specific sDNA panel including aberrantly methylated BMP3 and NDRG4 promoter regions, mutant k-ras and β-actin (a reference gene for human DNA quantity), and an immunochemical assay for human hemoglobin has been recently approved by Food and Drug Administration (2). Novel promising biomarkers for CRC screening are represented by microRNAs (miRNAs), a group of 18-25 nucleotide non-coding RNA molecules that regulate gene expression. Reports on these fecal biomarkers are case-control studies, and each of them evaluates single miRNAs or multi-target panels (3). On the other hand, some fecal proteins have been studied as possible CRC screening markers, even though they demonstrated poor results (4). Finally, alterations of estrogen receptor-beta (i.e., dramatic reduction in the early stage of CRC) have been demonstrated in tissue samples (5). In conclusion, specific investigations are warranted in order to add further noninvasive markers to the panel of CRC screening tools.

1. Levin B, Lieberman DA, McFarland B, et al. Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology. Gastroenterology 2008;134:1570–95.

2. Imperiale TF, Ransohoff DF, Itzkowitz SH, et al. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med 2014;370:1287–97.

3. Ahmed FE, Ahmed NC, Vos PW, et al. Diagnostic microRNA markers to screen for sporadic human colon cancer in stool: I. Proof of principle. Cancer Genomics Proteomics 2013;10:93-113.

4. Huang JX, Zhou Y, Wang CH, et al. Tumor M2-pyruvate kinase in stool as a biomarker for diagnosis of colorectal cancer: a meta-analysis. J Cancer Res Ther 2014;10(Suppl):C225–8.

5. Iannone A, Losurdo G, Pricci M, et al. Stool investigations for colorectal cancer screening: from occult blood test to DNA Analysis. J Gastrointest Cancer 2016;47:143-51.

THE OROPHARYNGEAL MICROBIOME DIVERSITY IN HEALTHY INDIVIDUALS AND IN CELIAC DISEASE PATIENTS

L. Iaffaldano¹, I. Granata², C. Pagliuca³, G. Casaburi⁴, M.V. Esposito¹, G. Del Vecchio Blanco⁵, C. Ciacci⁶, G. Salerno³, P. Salvatore³, F. Salvatore⁷, M.R. Guarracino², V. D’Argenio⁷, L. Sacchetti¹

¹Ceinge Biotecnologie Avanzate scarl, Naples, Italy

²LabGTP, ICAR, CNR, Naples

³DMMBM, University of Naples Federico II, Naples

⁴Evolve Biosystems, Inc. Davis, CA 95618

⁵Dep. of System Medicine, University of Rome Tor Vergata, Italy

⁶Dept. of Medicine and Surgery, University of Salerno, Italy

⁷DMMBM University Federico II and Ceinge, Naples

Celiac disease (CD) is a chronic immune-mediated enteropathy of the small intestine triggered by gluten in genetically predisposed individuals expressing the HLA-DQ2/DQ8 molecules, and likely in presence of additional factors not yet completely delucidated. Among these latter gut microbial alteration has been hypothesized to contribute to the CD pathogenesis (Cenit MA, et al. J Clin Gastroenterol 2016). In humans, the gastrointestinal tract contains approximately 10¹⁴ bacteria (>1000 different species, >3 million genes). The gut microbiota provides many functions that are crucial for the well-being of the human host, and gut dysbiosis has been suggested to play a pathogenic role in several diseases, including autoimmune and gastrointestinal disorders. In this context, we recently described a different duodenal microbiome composition in control subjects, active- and gluten free diet (GFD)-CD adult patients (D’Argenio V, et al. AJG 2016). In particular, a significant higher presence of Neisseria flavescens (beta-proteobacteria class), able to induce inflammation in both murine and human dendritic cells, was observed in active CD microbiome with respect to those of the other two groups. As gastrointestinal tract could be considered a single ecosystem extending from the oral cavity to the rectum, we investigated the oropharyngeal microbiome (by 16S rRNA sequencing) in 56 subjects (controls, GFD and active CD patients) and the duodenal microbiome in a small subgroup of CD patients. All collected samples were immediately cooled in dry ice and stored at -80°C until analysis. After DNA extraction, the V4-V6 regions of the 16S rRNA gene was amplified following the Illumina 16S Metagenomic Sequencing Library Preparation workflow, using a specific barcode sequence/sample. After quality and quantity assessment, all the libraries were pooled together and sequenced using the Illumina MiSeq System (PE 300x2). Analysis of the microbiome data was carried out by the QIIME v. 1.9.1. bioinformatics tool. Our findings, in confirming our previously duodenal microbiome data, highlight interesting similarities between the duodenal and oropharyngeal microbial alterations in active CD patients. The latter could have potential applications in CD diagnosis (Grant by 007_FC_2014).