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

LABORATORY MEDICINE: “COST” OR “ADD VALUE”?

G. Lippi

Verona

The panorama of laboratory medicine, as part of the growing complexity of health care in the third millennium, is variegated. Laboratory medicine has been an irreplaceable element in the clinical decision-making throughout its almost millennial history. Despite its essential role, the general perception of laboratory medicine, especially by politicians, administrators and some clinicians, is not always favourable because this “science” is often considered on the basis of its costs and not according to the added value generated. The increased complexity and volume of testing, coupled with an increasing aspiration for efficiency and quality by patients, may even compromise the survival of clinical laboratories. Nonetheless, it now clearly demonstrated that the economic impact of laboratory diagnostics on general healthcare expenditure is very modest in the most advanced countries, usually comprised between 1.4% and 2.8%. Nevertheless, the comparison between global cost (personnel, consumables, services, ordinary operating expenses) and revenues, clearly shows that laboratory diagnostics can generate twofold economic resources than those consumed, thus representing one of the areas with higher profitability not only in healthcare, but also in comparison with other human activities, wherein the net profit of the most profitable global industries does not usually exceed 30%. Realistically, laboratory diagnostics can therefore guarantee a net profit almost seven times greater than any other human sector. Alongside a genuine economic perspective, the added value that laboratory diagnostics offers in diagnostic and treatment pathways, especially in the era of personalized medicine, emerges strongly, since precision medicine strongly relies on results of genetic, epigenetic and biochemical tests. The role of laboratory medicine in the field of diagnostic and therapeutic care pathways is unavoidable, especially in some areas of medicine in which the diagnosis or therapeutic management are subsidiary to result of laboratory tests (e.g., anticancer therapies, diagnostics of acute coronary syndrome, and so forth). A major awareness of the added value of laboratory diagnostics is now unavoidable to reaffirm the role of the clinical laboratory in the modern healthcare system.

APPROPRIATENESS IN LABORATORY MEDICINE

M. Ciaccio

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

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

Laboratory Medicine plays a central role today in the entire decision-making process in Medicine, from diagnosis and prognosis definition to therapy monitoring. Laboratory Medicine is not limited to analytical aspects but has the primary objective of improving patient outcomes.

In this context, appropriateness is a very relevant issue. In fact, the inappropriate use of laboratory tests induces the deterioration of quality of care since the lack or excess of information related to the clinical problem can often be misleading or even harmful. Moreover, an inappropriate test involves a significant waste of resources. For these reasons appropriateness has become a strategic topic today, when the containment of costs for health services has became mandatory.

Appropriateness consists in requesting the right test, for the right patient, at the right time, thus avoiding the overuse of laboratory tests with the aim of providing the most effective intervention for a specific individual. It also should be noticed that the appropriateness of a specific test can be evaluated only in the context of the specific clinical question, and for the individual patient. Thus, in most circumstances, appropriateness depends by pre-test probability of having the disease.

Some examples of appropriateness that are often overlooked in the field of Laboratory Medicine are:

1. Free PSA should be proposed only to patients with total PSA ranging from 3 to 10 ng/ml. Laboratories should implement adequate instruments, such as reflex test, to limit inappropriate requests.

2. The use of AST, LDH, CK or myoglobin in patients suspected of acute myocardial infarction is not appropriate. For the diagnosis of acute myocardial infarction the only effective marker is troponin measured with high sensitivity assays.

3. Glycosuria for diabetes monitoring is not appropriate. For this purpose HbA1c should be used and it should be repeated only twice per year in patients with type 2 diabetes with stable glycemia well within target.

4. The basic evaluation of thyroid disease requires the execution of TSH only. The dosage of fT4 or antithyroperoxidase (anti-TPO) antibodies should be performed only when an alteration of TSH levels is detected.

5. Genetic tests for thrombophilia screening, namely Factor V Leiden and Factor II G20210A, should be performed only when a personal and family history of thromboembolic diseases is ascertained and they must not be repeated during the life.

Great efforts are needed to achieve appropriateness in laboratory tests requesting. Intervention strategies include educational programs; development of Evidence Based Medicine; sustain to large randomized clinical trials; promotion of formulation and dissemination of guidelines and consensus documents.

In conclusion, appropriateness is a cornerstone in high quality medical practice. Inappropriate use of laboratory testing induces high medical costs due to unnecessary diagnostic test, patients discomfort, and definitely poor outcomes for patients.

INNOVATION IN TYPE 2 DIABETES PATIENTS CLINICAL PATHWAY: THE ROLE OF GLYCATED ALBUMIN

L. Ferrario¹, E. Foglia¹, F. Schettini¹, A. Avogaro², C. Bellia³, F. Bertuzzi⁴, G. Bonetti⁵, A. Ceriello^6,7, M. Ciaccio^3,8, M. Corsi Romanelli^9,10, E. Dozio^10,11, L. Falqui¹², A. Girelli¹³, A. Nicolucci¹⁴, G. Perseghin^15,16, M. Plebani¹⁷, U. Valentini¹³, M. Zaninotto¹⁷, D. Croce^1,18

¹Centre for Health Economics, Social and Health Care Management, Università Carlo Cattaneo - LIUC, Castellanza, Italy

²Department of Medicine, University-Hospital, Padova

³Clinical Biochemistry and Clinical Molecular Medicine, Dept. Biopathology and Medical Biotechnologies, University of Palermo, Italy

⁴Diabetology Unit, ASST Grande Ospedale Metropolitano Niguarda, Milan, Italy

⁵Central Clinical Chemistry Laboratory, ASST Spedali Civili, Brescia, Italy

⁶Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS) and Centro de Investigación Biomedica en Red de Diabetes y

Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain

⁷Department of Cardiovascular and Metabolic Diseases, IRCCS Multimedica, Sesto San Giovanni, Milan, Italy

⁸Department of Laboratory Medicine, University-Hospital, Palermo, Italy

⁹Service of Laboratory Medicine 1-Clinical Pathology, I.R.C.C.S. Policlinico San Donato, Milan, Italy

¹⁰Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, Italy

¹¹Laboratory of Molecular Pathology, I.R.C.C.S. Policlinico San Donato, San Donato Milanese, Milan, Italy.

¹²Department of Medicine, Diabetes and Endocrinology, IRCCS Multimedica, Sesto San Giovanni, Milan, Italy

¹³Diabetes Care Unit, ASST Spedali Civili, Brescia, Italy

¹⁴Center for Outcomes Research and Clinical Epidemiology, Pescara, Italy

¹⁵Department of Medicine and Surgery, Università degli Studi di Milano Bicocca, Milan, Italy

¹⁶Department of Medicine and Rehabilitation, Unit of Metabolic Medicine, Policlinico di Monza, Monza, Italy

¹⁷Department of Laboratory Medicine, University-Hospital, Padova

¹⁸School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

AIMS:

The study proposed a multi-dimensional assessment, exploring clinical, economic, ethical, social and organisational implications, related to an innovative glycemic marker introduction (glycated albumin – GA), in the Italian clinical practice, as an add-on technology to the traditional glycemic monitoring systems (Hb1Ac and FPG), for insuline-naïve patients affected by tipe 2 diabetes, assuming oral therapy.

METHODS:

The HTA approach considers the Italian NHS perspective. All the 9 dimensions derived from EUnetHTA Core Model, were deployed considering: i) a systemic literature review, ii) administration of qualitative questionnaire filled by 15 healthcare type 2 diabetes experts (evaluation scale, ranging from -3 to +3) and iii) quantitative approaches, useful for the economic evaluation of the clinical pathway and for budget impact analysis.

RESULTS:

Literature review showed that AG implementation could reduce the risk to develop diabetes complications (such as diabetic retinopathy, nephropathy and cardiovascular problems) and improve the number of patients achieving a therapeutic success (97% vs 72%).

Professionals’ perception confirmed the superiority of AG, in terms of safety (0.11 vs 0.65) and effectiveness (0.53 vs 1.80) profiles. At the 12-month time point to the base-case scenario for market penetration, AG introduction would lead to fewer amount of patients experiencing a therapy switch (-89%), with a significant economic saving equal to 1.06% (€3,551,275), for the NHS, thus presenting a better cost-effectiveness trade-off with respect to the comparator (CEV: 225.53 vs 237.74). Whilst the use of traditional systems would be more advantageous from an equity point of view (0.13 vs 0.72), due to GA limited accessibility, GA would improve both patients (1.33 vs 2.17) and caregivers (0.83 vs 1.50) quality of life. Despite, in the short-term, GA required training courses and equipment update, in the long-term it would be the preferable technology, from an organizational point of view (0.01 vs 0.30), freeing up organizational resources.

CONCLUSIONS:

Results demonstrates the strategic relevance related to AG introduction into the Italian clinical practice, its economic sustainability and feasibility, as well as the potentialities in clinical pathway improvement.

INVESTIGATIONS’S MANAGEMENT PROTOCOL IN THE COAGULATIVE FIELD ON THE PREOPERATIVE PATHWAY

L. De Valentin¹, S. Luzi¹, R. Turrini¹, L. Dumitrescu¹, S. Bonaguro¹, L. Zardo¹

¹Lab. Analisi Osp. S. Giacomo, Castelfranco V.to (Treviso) - ULSS2 Marca Trevigiana, Italy

The request of the basic coagulation tests (PT and aPTT) falls within the normal preoperative pathway. The aim of the work is to highlight the effectiveness of the management of patients with altered screening tests through a protocol shared among the Intensive Care Unit, which coordinates the preoperative pathways, Hematology Unit, which performs the consultations, and the Laboratory, which performs the I and II level coagulation tests. The protocol causes a role reversal of the laboratory, from a passive element to the proposer of the diagnostic path. After the first finding of alteration of one or both PT and aPTT tests, patient’s blood is collected in 3 sodium citrate tubes on which the laboratory performs both first level and mixing test. The results, according to the evaluation of the laboratory specialist and a specific algorithm, trigger the most suitable second level tests. At the end of the diagnostic procedure the Haematologist, on the basis of the results, decides whether the haematological examination is needed. In case of negative bleeding history and LAC positive or factor XII defect, the patient undergoes surgery. If an indicator of hemorrhage, such as non-LAC inhibitor, a factorial defect or vWF desease is found the haematologist collects a thorough anamnesis of the patient for a correct evaluation of the bleeding risk. In 2017 the protocol was applied to 37 cases of altered PT/aPTT. In 8% of cases the alteration was not confirmed, in 11% of cases the investigations didn’t find a cause of the PT/aPTT prolongation, but in 81% of cases an explanation of the prolongation was found. In some patients there was not haemorrhagic risk-associated defects/alterations but whose definition was useful for the evaluation of the patient. With the new protocol, two or three hospital admissions are required instead of the previous five, with patient’s stress and resources saving. Furthermore the implementation of the mixing test-based diagnostic algorithm allows the execution of appropriate tests suggested by hemostasis laboratory with the decrease of useless tests.

THERAPEUTIC MONITORING OF TNFA INHIBITOR DRUGS AND AUTOANTIBODIES: SIBIOC RECOMMENDATIONS

V. Pecoraro

Medicina di Laboratorio- Dipartimento Interaziendale ad Attività Integrata “Medicina di Laboratorio e Anatomia Patologica” Ospedale Civile S. Agostino Estense, Modena

ABSTRACT:

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory disease, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Chrons disease and ulcerative colitis. TNFα inhibitors (anti-TNFα) are monoclonal antibodies drugs directed against TNFα. They are adalimumab, infliximab, etarnecept, golimumab and certolizumab (1). Their effect consists in reducing the inflammatory response of autoimmune diseases. We evaluated evidence about the therapeutic efficacy of these drugs on patients affected by chronic inflammatory disease, highlighting their clinical benefit, but also a high risk of injection site reactions and infections (2). Furthermore, these drugs, due to their immunogenicity, can cause the formation of anti-drug antibodies (ADA). ADA could interfere with drugs compromising their effects, and resulting in a loss of clinical response (2). The determination of serum TNFα inhibitors and ADA levels could improve the patient’s management, allows improving the treatment and reducing the risk of adverse effects. Even if the loss of therapeutic response, due to ADA production and the variation of TNFα inhibitors concentration are well documented, the clinical benefit of their serum determination remains unclear due to lack of standardization of laboratory methods (3, 4). The drug and ADA monitoring could be useful to reduce the loss of response and to avoid immunogenicity in patients’ either responders or not responders to TNFα therapy, but the monitoring implementations needed of standardization of determination methods and agreement in the interpretation of results (5).

Members of SIBioC Study Group “Autoimmunità e Immunologia Clinica” drafted a documented showing the potentiality and level of criticality of TNα inhibitors and ADA monitoring. There are any indications about the use of immunogenicity test to guide the therapy, but there is the need of more information before implementing this test in clinical practice.

REFERENCES

1. Kalliolias GD, Ivashkiv LB. TNF biology, pathogenic mechanisms and emerging therapeutic strategies. Nat Rev Rheumatol. 2016; 12:49-62.

2. Pecoraro V, De Santis E, Melegari A, et al. The impact of immunogenicity of TNFα inhibitors in autoimmune inflammatory disease. A systematic review and meta-analysis. Autoimmun Rev 2017;16:564–75

3. Lombardi G, Perego S, Sansoni V, et al. Anti-adalimumab antibodies in psoriasis: lack of clinical utility and laboratory evidence. BMJ Open 2016; 6: e011941.

4. Keiserman M, Codreanu C, Handa R, et al. The effect of antidrug antibodies on the sustainable efficacy of biologic therapies in rheumatoid arthritis: practical consequences. Expert Rev Clin Immunol 2014;10:1049-57.

5. Krieckaert C, Rispens T, Wolbink G. Immunogenicity of biological therapeutics: from assay to patient. Curr Opin Rheumatol 2012;24:306-11.

VITAMIN D PLASMA LEVEL IS ABLE TO AFFECT NIVOLUMAB DRUG EXPOSURE IN A COHORT OF PATIENTS WITH NON SMALL CELL LUNG CANCER

J. Cusato¹, P. Carrega^7,8, C. Tomasello³, C. Genova⁹, S. Ottonello^4,5, I. Cossu⁶, G. Pietra^4,6, M.C. Mingari^4,5,6, F. Grossi⁹, G. Ferlazzo^7,8, G. Di Perri², A. D’Avolio¹

¹ Lab. di Farmacologia Clinica e Farmacogenetica, Univ. Studi di Torino, Dip. Scienze Mediche, Osp. Amedeo di Savoia,

Torino

²Unità di Malattie Infettive, Univ. Studi di Torino, Dip. Scienze Mediche, Osp. Amedeo di Savoia, Torino

³S.C. Farmacie Ospedaliere, Osp. Maria Vittoria, ASL CITTA’ DI TORINO, Torino

⁴Dip. di Medicina Sperimentale (DiMES), Univ. di Genova, Genova

⁵Centro di Eccellenza per Ricerche Biomediche (CEBR), Univ. di Genova, Genova

⁶Unità di Immunologia, Osp. San Martino, Genova

⁷Lab. di Immunologia and Biotecnologie Terapeutiche, Dip. di Patologia Umana, Univ. di Messina, Messina

⁸Cell Factory Center and Division of Clinical Pathology, Univ. Osp. Policlinico G.Martino, Messina

⁹UOS Tumori Polmonari, Ospedale Policlinico San Martino, Genova

Immune-checkpoint inhibition using programmed cell death-1 and its ligand drug inhibitors have improved survival among patients with advanced non small cell lung cancer: nivolumab (NV) is one of the most used and the presence of its antiantibody is considered a negative prognostic factor. Vitamin D (VD) deficiency (<20ng/mL) is frequent in lung cancer patients and studies showed this hormone modulates the expression of genes involved in drug metabolism/elimination and in the immune system regulation. No data are present in literature concerning NV and its relationship with VD. For these reasons, aim of this study was to quantify 25-hydroxyVD, NV and its anti-antibody in patients’ plasma before starting (baseline) and at 15, 45 and 60 days of therapy. Molecules were quantified through enzyme-linked immunosorbent assay. We enrolled 45 patients: median NV concentrations were 12.48ug/mL (interquartile range, IQR:9.54-17.13), 22.31ug/ mL (IQR:18.30-34.88) and 27.05ug/mL (IQR:17.43-39.38) respectively at 15, 45 and 60 days. No anti-NV antibodies were found. VD median concentrations were 12.78ng/mL (IQR:10.09-16.55), 13.62ng/mL (IQR:10.86-16.11), 11.78ng/mL (IQR:10.13-18.87) and 12.92ng/mL (IQR:10.79-17.03) respectively at baseline, 15, 45 and 60 days. Correlations wereobserved between NV concentrations at 15 days and VD levels at baseline (p=0.024, Pearson’s coefficient (PC) 0.451) and at 15 days (p=0.017, PC=0.542); NV exposure at 60 days was correlated with VD at baseline (p=0.001, PC=0.730), at 15 (p<0.001, PC=0.858), 45 (p=0.001, PC=0.779) and 60 days (p<0.001, PC=0.900). Furthermore, in a sub-population we stratified patients according to VD deficiency: baseline VD levels<20ng/mL were associated with lower NV concentrations at 15 (p=0.103, a trend without statistical significance), 45 (p=0.018) and 60 days (p=0.021); 15 days VD<20ng/mL with 15 (p=0.019), 45 (p=0.019) and 60 days (p=0.028) NV lower concentrations; finally, 60 days VD<20ng/mL with 60 days lower NV levels (p=0.030). This is the first study showing VD is able to predict NV concentrations and describing nivolumab levels in real-life context of non small cell lung cancer. Further studies in bigger and different cohorts are needed to clarify these aspects and to relate them to clinical features.

FROM COMPLETE BLOOD COUNT TO MOLECULAR BIOLOGY: YESTERDAY, TODAY AND TOMORROW

S. Buoro

Bergamo

The qualitative and quantitative cells evaluation on the peripheral blood smear by optical microscopy was the first step taken by the scientific community towards the current concept of liquid biopsy.

The cellular component in the peripheral blood is the result of fine regulation of hematopoiesis on the bone marrow that depends of the ability of the stem cell and other cell precursors to replicate, to differentiate and to migrate into the peripheral blood. The evaluation of the cells in the blood allows verifying the alteration of homeostasis of the hematopoiesis due to infections, nutritional alterations, neoplasia or paraneoplastic reactions etc.

The number and type of peripheral blood and bone marrow cells, associated with the patient’s clinical condition, have been the basis of the development of modern hematology. Cell morphological evaluation was a fundamental criterion to FAB classification of hematological diseases (1)

Another important step of laboratory hematology has been the development of flow cytometry with the availability of the first complete blood count profile (CBC). This method of analysis allows the cells count and characterization, obtaining robust results in a short time, at low cost and all day (2)

The development of flow cytometry in the new hematology analyzers has been very rapid in the last decade. Today, the new CBC-profile, integrated with the latest generation erythrocyte, platelet and leukocytes parameters provides increasingly detailed quantitative and qualitative information about the cells (3), allowing the recognition also of poorly represented neoplastic cells or alterations induced by solid neoplasms.

The development of multiparametric flow cytometry, with the classification of cells based on the protein biomarkers expressed on their surface, as a differentiation cluster (CD), allowed the “immunological” classification of cells (4). The last two decades have been also characterized by the rapid development of molecular biology including the evaluation of gene expression with “Next Generation Sequencing” (NGS), and the improvement of methods for detection of free circulating DNA (5). This technology progress have allowed a better definition of etiopathogenesis, diagnosis, prognosis and therapy of hematological and non-hematological neoplasms (5). The high quantity and the increasingly detailed information from cells analysis, up to the data of gene expression, have revolutionized the classification of hematological diseases (Hematology Tumors Classification of 2016 proposed by the World Health Organization). Thanks to a more refined characterization of neoplasms, we are seeing the development of personalized therapies, with biological drugs aimed at correcting the gene defect that causes the disease. The reference model is chronic myeloid leukemia (CML), a clonal neoplasm caused by t(9;22) translocation with the generation of the BCR/ABL fusion gene that generally leads to the increase of neutrophils and immature granulocytes in peripheral blood, with the presence of blasts. In this case, the knowledge about of the genetic lesion led to the development of a targeted biological drug that allowed to chronic the disease.

REFERENCE

1. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C.Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol. 1976;33:451-8.

2. Green R, Wachsmann-Hogiu S. Development, history, and future of automated cell counters. Clin Lab Med. 2015;35:1-10. doi: 10.1016/j.cll.2014.11.003

3. Lecompte TP, Bernimoulin MP.Novel parameters in blood cell counters. Clin Lab Med. 2015;35:209-24. doi: 10.1016/j.cll.2014.11.001.

4. Lukes RJ.Functional classification of malignant lymphoma of Lukes and Collins. Recent Results Cancer Res. 1978;64:19-30

5. Kubaczkova V, Vrabel D, Sedlarikova L, Besse L, Sevcikova S. Cell-free DNA - Minimally invasive marker of hematological malignancies. Eur J Haematol. 2017;99:291-299. doi: 10.1111/ejh.12925

MOLECULAR BIOMARKERS IN ACUTE AND CHRONIC LEUKEMIAS

B. Izzo

Napoli

Acute and chronic myeloid leukemia are hematological disorders characterized by different genetic profiles, for which several predictive and prognostic biomarkers have been identified and characterized in leukemic blood cells. Predictive biomarkers using molecular diagnostics are currently in use in clinical practice of personalized therapy of acute and chronic leukemia. In particular, chronic myeloid leukemia (CML), was the first human malignancy found to be associated with a recurrent chromosomal abnormality(1,2),genetically characterized by the presence of the reciprocal translocation t(9;22)(q34;q11) resulting in a BCR-ABL1 gene fusion, which produces a constitutively active tyrosine kinase that drives leukemic transformation. This specific molecular biomarker paved the way to targeted tyrosine kinase inhibitor (TKI) treatment, which is currently the goal standard of modern therapy for this hematologic malignancy. Multiple BCR-ABL1 TKIs are currently approved for newly diagnosed CML patients (imatinib, dasatinib and nilotinib) and for patients with relapsed or refractory disease (dasatinib, nilotinib, bosutinib, ponatinib)(3). By coupling the optimal use of currently available therapeutic options and appropriate monitoring through reverse transcriptase-quantitative polymerase chain reaction (RT-QPCR), the life expectancy of the majority of patients diagnosed with CML is, today, predicted to be comparable to that of the general healthy population (4). Furthermore, the continuous therapeutic improvement, has led to increasingly ambitious treatment endpoints, such as the treatment free remission (TRF), that identifies the possibility of discontinuing TKIs in patients with CML after achieving a consolidated and stable molecular response. The advent of high-throughput sequencing techniques has allowed new insights also into the molecular basis of acute myeloid leukemia (AML) showing this latter to be a complex, dynamic disease, characterized by multiple somatically acquired driver mutations, coexisting competing clones, and disease evolution over time (5). The 2017 ELN recommendations for AML shed light on the diagnostic workup that should include, in addition to morphological, immunophenotypical and cytogenetic evaluation, the molecular screening for different prognostic molecular biomarkers such as mutations in NPM1, CEBPA, and RUNX1 genes because they define risk stratification categories. In addition, particular attention should be devoted to mutations in FLT3 that may beneficially be affected by tyrosine kinase inhibition and mutations in TP53 and ASXL1 because they consistently have been associated with poor prognosis. Furthermore, minimal (or measurable) residual (MRD) disease assessed by molecular techniques (RT-qPCR, NGS) provides an important molecular biomarker of response quality for patients with AML. In conclusion, the therapeutic approach to haematological cancers is currently being dramatically influenced by the “precision medicine”approach, which allows to associate specific genetic anomalies responsible for the tumor onset with even more specific molecular biomarkers and increasingly advanced diagnostic molecular techniques.

BIBLIOGRAFIA

1. Nowell PC, Hungerford DA. Chromosome studies on normal and leukemic human leukocytes. J Natl Cancer Inst 1960; 25:85–109.

2. Gordon JE, Wong JJ, Rasko JE. MicroRNAs in myeloid malignancies. Br J Haematol 2013; 162:162–76)

3. Baccarani, M., Deininger, European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013.Blood, 122, 872–884.

4. Gambacorti-Passerini, C., Antolini, 2011 Multicenter independent assessment of outcomes in chronic myeloid leukemia patients treated with imatinib.2011 Journal of the National Cancer Institute, 103, 553-561.

5. Dohner H, E. Estey Diagnosis and management of AML in adults: 2017 ELN Recommendations from an International Expert Panel, Blood 2017,129:424-447

EMERGENCY TESTS MANAGEMENT: SEPARATED OR CONSOLIDATED LABORATORY SECTOR?

D. Giavarina

Laboratory Medicine, St. Bortolo Hospital, Vicenza, italy

Clinicians are interested in a laboratory service that encompasses analytical quality, full availability and timeliness; among these characteristics, timeliness of result reporting is considered the most relevant . The main causes of laboratory turnaround time (TAT) delay can be ascribed to order entry, responsible for about 30% of all reports that are delayed. Sample collection causes 27% of delays, analytical phases 28%, report transmission 2%; the remaining 13% was not determined. There are three options to manage and perform “emergency” tests: a) in dedicated (“emergency”) laboratories in the central laboratory, together with other tests, b) in the central laboratory, together with other tests, and c) at point-of-care (POC). The first model has been certainly the most popular until the nineties, but during the past decades, the healthcare systems have rapidly changed and today hospital care is primarily advocated for critical patients and acute treatments, for which laboratory test results are crucial and need to be always reported in predictably short. However, not only hospital care need of short TAT. For both the “acute care” and outpatient offices, laboratory services must ensure a short TAT (usually less than 1 hour) to guarantee timely patient care. In addition, outpatients being cared for by general practitioners require rapid response for test requests since most of these patients are affected by chronic conditions which require continuous monitoring in order to guide effective and prompt adjustments to therapy (i.e., Oral Anticoagulant Therapy, Diabetes, Heart failure). Therefore, in the current healthcare system, most laboratory tests should be considered “STAT”. POCT for immediate results, and consolidate corelab, for a high throughput have been progressively considered as possible alternative solutions. Consequently, laboratories in the hospital setting as well as in territorial service can face this challenge by changing their organization from a compartmentalized laboratory department toward a decision making-based laboratory department. This requires the implementation of a core laboratory, that exploits total laboratory automation (TLA) using technological innovation in analytical platforms, track systems and information technology, including middleware, and a number of satellite specialized laboratory sections cooperating with care teams for specific medical conditions. In this laboratory department model, the short TAT for all first-line tests performed by TLA in the core laboratory represents the key paradigm, where no more stat testing is required because all samples are handled in real-time and (auto) validated results dispatched in a time that fulfils clinical needs. To optimally reach this goal, laboratories should be actively involved in managing all the steps covering the total examination process, speeding up also extra-laboratory phases, and such sample delivery. Furthermore, to warrant effectiveness and not only efficiency, all the processes, e.g. specimen integrity check, should be managed by middleware through a predefined set of rules defined in light of the clinical governance. We are now moving towards a new model of healthcare systems, where every patient has a critical pathology and treatment is “urgent” for everyone. In this scenario, laboratories need to consider every test as a Stat test, so that the separation between routine and stat organizations will be abolished. Automation, particularly TLA, represents a formidable tool to meet the increasingly more demanding critical needs and, even more importantly, improve patient outcomes. POCT could be variously integrated into Stat test activities, usually (but not solely) in the evaluation of vital functions.

1. R.C. Hawkins, Laboratory turnaround time, Clin Biochem Rev 2007;28:179–94.

2. Steindel SJ, Novis DA. Using outlier events to monitor test turnaround time. Arch Pathol Lab Med 1999;123:607-14.

3. Dolci A, Giavarina D, Pasqualetti S, Szőke D, Panteghini M. Total laboratory automation: Do stat tests still matter? Clin Biochem 2017;50:605-11.

WHICH BIOLOGICAL MATRIX TO USE FOR BLOOD TESTING: SERUM OR PLASMA?

A. Padoan

Department of Laboratory Medicine, University-Hospital of Padova, 35128, Padova, Italy

Clinical laboratories can routinely use different biological matrices even if the majority of tests are performed on serum or plasma. Serum represents the liquid part of the blood originating from the clotting process that is activated by the transformation of fibrinogen in fibrin, a meshwork that can trap inside some cellular components (e.g. platelets). Plasma is obtained by the centrifugation of blood treated with anticoagulants and contains all the intact coagulation factors. Different anticoagulants agents are commercially available, such as EDTA salts, sodium citrate and lithium heparin, but their specific utilization largely depend on the test type, as they can interfere with specific analytical reactions. For example, EDTA salts, being a chelating agent, is known to interfere with several biochemical reactions, while lithium heparin, with a non-competitive mechanism, is one of the most inert anticoagulant. Because serum does not contain anticoagulants, it is free of platelets and clotting-derived proteins, but contains clotting metabolites. Both serum and plasma are usually collected in test tubes containing a gel barrier, which allowed to separate the cellular elements by centrifugation. Historically, serum has been the preferred assay material for determining measurands concentrations in blood specimens and it is still widely used. Today, many clinical laboratories are facing a change from serum to plasma, as the plasma constituents are thought to better represent the patient’s pathological situation than serum [1]. However, both matrices present advantages and disadvantages. Firstly, plasma samples can be centrifuged immediately after collection, while serum needs 30 minutes for completing coagulation. This time saving property of plasma is particularly relevant for those situations, e.g. emergency departments, in which physicians need urgent testing. Plasma allowed also to obtain a higher volume yield than serum (15%-20% or more), and prevents the coagulation-induced interferences, such as the decreased glucose, the increased lactate dehydrogenase, etc… On the other hand, sample centrifugation and the usage of gel barrier tubes are not sufficient for removing all blood cells and the presence of plasma residual cells, even if pelleted over the gel, may alter some constituent concentrations. Serum is almost completely lacking in cellular constituents. Further, the widespread usage of serum over plasma, have contributed to the development of many analytical methods validated for serum but not for plasma.

In conclusion, despite the wider usage of serum over plasma, the advantages of plasma are encouraging clinical laboratories moving from serum to plasma. However, manufactures should validate analytical methods also for plasma as this is step is expensive and time consuming for clinical laboratories.

Reference

1. World Health Organization (WHO). Diagnostic Imaging and Laboratory Technology. Use of anticoagulants in diagnostic laboratory investigations. Geneva, 2002

A COLLABORATIVE STUDY PLANNED BY THE WORKING GROUP ON HEMOSTASIS AND THROMBOSIS OF THE ITALIAN SOCIETY OF CLINICAL BIOCHEMISTRY AND CLINICAL MOLECULAR BIOLOGY (SIBIOC ) ON HEMOLYSIS INTERFERENCE ON FIVE ROUTINE HEMOSTASIS TESTS

C.A.E. Novelli¹, M. Vidali², B. Morelli³, B. Brando¹, G. Andreani⁴, M. Arini⁵, P. Calzoni⁶, R. Giacomello⁷, B. Montaruli⁹, E. Muccini⁹, A. Papa¹⁰, P. Pradella¹¹, L. Ruocco¹², F. Siviero¹³, F.G. Viola¹⁴, M. Zanchetta¹⁵, L. Zardo¹⁶, G. Lippi¹⁷

¹Transfusion Center and Haematology Laboratory, Western Milan Area Hospital Consortium, Legnano General Hospital, Legnano, Italy

²Clinical Chemistry Unit, Maggiore della Carità Hospital, Novara, Italy

³Synlab Laboratory, Castenedolo, Italy

⁴Clinical Chemistry Laboratory, Apuan Hospital, Massa, Italy

⁵Clinical and Microbiological Analysis Laboratory, Dell’Angelo Hospital, Mestre, Italy

⁶Clinical Pathology, University Hospital of Siena, Siena, Italy

⁷Department of Medical Area, University of Udine, Department of Laboratory Medicine, ASUI UD, University Hospital, Udine, Italy

⁸Laboratory Analysis, A.O. Ordine Mauriziano, Turin, Italy

⁹Clinical Biochemistry Laboratory, Azienda O.U. S. Giovanni Battista, Turin, Italy

¹⁰Laboratory Medicine, G.Monasterio Foundation, CNR-Regione Toscana, Pisa, Italy

¹¹Transfusion Medicine, University Hospital “Ospedali Riuniti” of Trieste, Trieste, Italy

¹²Clinical Analysis Laboratory, University Hospital of Pisa, Pisa, Italy

¹³Laboratory Medicine, Bassano del Grappa Hospital, Bassano del Grappa, Italy

¹⁴Department of Laboratory Medicine, Tor Vergata University Hospital of Rome, Rome, Italy

¹⁵Laboratory Analysis, Degli Infermi Hospital, Ponderano, Italy

¹⁶Laboratory Analysis, San Giacomo Apostolo Hospital, Castelfranco Veneto, Italy

¹⁷Section of Clinical Biochemistry, University of Verona, Verona, Italy.

INTRODUCTION:

Hemolysis is the leading cause of sample rejection in laboratory hemostasis testing. Many studies demonstrate the effect of hemolysis on coagulation tests using artificially hemolyzed samples. New coagulometers are able to detect optical interferences replacing visual inspection of the sample. The aim of this study was a prospective assessment of spontaneous hemolysis during sampling on hemostasis tests, by comparing results of hemolyzed (H) versus new, nonhemolyzed (NH) specimens, collected within 4 hours afterward, from the same patient. Moreover, visual assessment of hemolysis by two operators was also compared with instrumental hemolysis index.

MATERIALS AND METHODS:

The study was planned by the Working Group on Hemostasis and Thrombosis of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC), and thus involved 15 hospital laboratories allocated in 6 different Italian Regions. A total of 269 pairs of plasma samples, mostly from inpatients, were analyzed using ACL TOP 750-CTS (IL, Bedford, USA), for the following tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), DDimer (DD), fibrinogen (Fib) and antithrombin (AT). The critical difference (CD) and bias between H and NH was calculated for each test. Differences between paired samples were evaluated by non-parametric paired sample test and Bland-Altman analysis. Results: Mean bias was -0.1s for PT (P=0.057), -1.1s for aPTT (P<0.001), 1025ng/mL for DD (P<0.001), -0.04g/L for Fib (P=0.258) and 1.4% for AT (P =0.013). Bias exceeding the CD varied according to the method, with larger differences for aPTT (36.1%) and DD (17.1%). No correlation was found between free hemoglobin values and difference in hemostasis tests between H and NH samples. The agreement of visual hemolysis assessment among different operators, and between visual and instrumental assessment, evaluated by Cohen’s kappa, was 0.85 and 0.62, respectively.

CONCLUSION:

According to our data, PT seems scarcely influenced by spurious hemolysis. Nevertheless, a larger unfavorable impact of spurious hemolysis was observed for other routine coagulation tests, manly for aPTT, DD and lesser extent for AT and Fib, suggesting that these test results should be suppressed in hemolyzed plasma samples. A good correlation of hemolysis assessment was found between the two operators, but only moderate compared to instrumental hemolysis evaluation. The use of instrumental hemolysis index seems today more raccomandable.

CHOISE OF THE RIGHT SAMPLE TYPE FOR PLASMA GLUCOSE DETERMINATION IN ORAL GLUCOSE TOLERANCE TEST: AN IMPORTANT PRE-ANALYTICAL TOOL FOR A CORRECT CLASSIFICATION OF GESTATIONAL DIABETES MELLITUS

G. Bonetti¹, D. Gavarina², D. Urbani², C. Marchetti², M. Marini¹, M. Carta²

¹Laboratorio Centrale Analisi Chimico Cliniche, A.S.S.T. Spedali Civili, Brescia

²Laboratorio Analisi, Ospedale S. Bortolo, Vicenza

BACKGROUND-AIM:

The aim of the present study was to evaluate the effect of the use of acidified mixture of NaF, EDTA and citrate, as suggested in recently published Italian recommendations of WG Diabete of SIBioC-SIPMeL and SID, on OGTT for screening and subsequent diagnosis of gestational diabetes mellitus (GDM).

METHODS:

A total of 82 pregnant women were submitted to OGTT 75 g for screening of GDM in Vicenza [n=51, mean age (range): 33 (18-44) years] and in Brescia [n=31, mean age (range): 32 (22-43) years] were included in the study. All subjects signed written informed consent to participate to it.Glucose determination was performed in currently in use NaF containing tubes and in FC-MIX tubes from reiner BioOne, with a lyophilized ternary mixture, in Vicenza and in GlucoEXACT tubes from Sarstedt, with a liquid one, in Brescia, using an hexochinase method on Dimension Vista systems from Siemens Healtheers.Screening for GDM was considered positive if at least one of the three blood samples exceed clinical cut-off derived from HAPO study.

RESULTS:

Using NaF 6/51 (11.8%) and FC-mix tubes 12/51 (23.5%) women, respectively, were diagnosed having GDM in Vicenza. Using NaF 2/31 (6.5%) and GlucoEXACT tubes 5/31 (16.1%) women, respectively, were diagnosed having GDM in Brescia.Median (IQR) of glucose in NaF tubes was: 76 (70.3-83.8) at T0, 130.0 (109.3-150.5) at T60’ and 106.0 (91.3-125.5) mg/dL at T120’, respectively in FC-MIX tubes was: 81.0 (77.0-89.0) at T0, 131.0 (115.3-154.0) at T60’ and 109.0 (96.5-129.8) mg/dL at T 120’ in Vicenza. Median (IQR) of glucose in NaF tubes was: 74.3 (71.2-77.5) at T0, 122.2.0 (102.4-136.8) at T60’ and 106.7 (98.4-128.5) mg/dL at T120’, respectively; in GlucoEXACT tubes was: 83.0 (78.7-85.4) at T0, 126.9 (111.1-143.8) at T60’ and 116.5 (105.8-137.1) mg/dL at T 120’ in Brescia.There was a statistically significant difference (P<0.05) between NaF and ternary mixture at all time points.

CONCLUSIONS:

The use of the new tubes containing a mixture of NaF, EDTA and citrate is an useful and necessary pre-analytical tool for a right OGTT for the screening GDM. NaF containing tubes should no longer been used for screening of GDM because their use results in underdiagnosis of GDM.

PRESENTATION OF THE CONSENT DOCUMENT ON THE BLOOD AS BIOLOGICAL MATRIX TO EVALUATE THE DRIVING UNDER THE INFLUENCE OF DRUGS AND PSYCHOTROPIC SUBSTANCES

R. Pacifici

Roma

The prohibition of driving under the influence of drugs and psychotropic substances is governed by art. 187 (Legislative Decree No. 285/1992 and subsequent amendments and additions) which establishes both illicit conduct and the sanctions. With the Law of 23 March 2016, n. 41 new types of crimes were introduced in the penal code with reference to articles 589-bis and 590-bis, respectively entitled “vehicular homicide” and “serious or very serious road personal injury”.

Among the multiple problems of interpretation and practical application of the legislation one of the most difficult concerns the lack of uniformity on the methods of investigations to establish the state of psycho-physical alteration of a driver due to the use of drugs and psychotropic substances and the interpretation of the toxicological-analytical result. For these reasons it was necessary to establish a technical table at the Istituto Superiore di Sanità with the experts of the same Institute, the Ministry of Health, the Health Commission and representatives of the major Italian Scientific Societies, competent in the subject. whose task is being to elaborate a technical document on analytical protocols and on the cut-off values that can be used for the analysis of drugs and psychotropic substances in the blood, in order to evaluate the psychophysical alteration of the guide due to the use of these substances. Hence the need of a document which treats four basic points:

1. The choice of the biological matrix: the blood is the only biological matrix to evaluate the driving under the influence of drugs and psychotropic substances.

2. The timing: The time between the road check, the request to carry out the blood sampling and the carrying out of a medical facility of the sample must be as short as possible and traced.

3. The methodology used for the analysis of drugs and psychotropic substances in the elective matrix; once the collection has been carried out, the laboratory, where it is possible, proceeds with screening tests, followed, in the case of positive results, by a confirmation analysis with a toxicological-forensic value in chromatography coupled to mass spectrometry.

4. The choice of the cut-off values for the different drugs and psychotropic substances in the elective matrix correlated with driving disability. The methods for identifying cut-off values in the context of driving under the influence of drugs and psychotropic substances are different and based on different criteria. The Technical Table has therefore established some minimum cut-off values that can be correlated with an inability to drive.

REFERENCE

1. Pacifici R, Gori P, Martucci L, et al. Considerazioni sulle matrici biologiche idonee alla valutazione dell’“attualitàd’uso di sostanze illecite” ai fini degli articoli 186 e 187 del nuovo Codice della Strada. Biochim Clin 2014; 38:27-31

2. Linee Guida per Strutture dotate di Laboratori per gli accertamenti di Sostanze d’Abuso con Finalità Tossicologico-Forensi e Medico-Legali - Gruppo Tossicologi Forensi Italiani (GTFI) Revisione n. 5 del 29 maggio 2017

3. Pichini S. e Pacifici R. Linee guida per la determinazione delle sostanze stupefacenti e psicotrope nelle urine Istituto Superiore di Sanità 2013

4. Pichini S. e Pacifici R. Linee guida per la determinazione delle sostanze stupefacenti e psicotrope nella matrice pilifera Istituto Superiore di Sanità 2013

5. Pichini S. e Pacifici R. Linee guida per la determinazione delle sostanze stupefacenti e psicotrope nella saliva Istituto Superiore di Sanità 2013

THE CONSUMPTION OF AMAZING SUBSTANCES FOR THERAPEUTIC OR RECREATIONAL PURPOSE: THE CRITICALITY RELATED TO DEPENDENCE AND TOLERANCE

C. Leonardi

Roma

The concern to determine a condition of complex dependence or addiction induced by drugs that can be used for analgesic purposes in patients suffering from chronic non-cancer pain, represents an impediment that is not infrequent in everyday clinical practice. In fact, opioid drugs or cannabis, in addition to having an antalgic effect, are able to modulate the brain system of gratification and reward of any individual. These effects can be “evaluated” and experienced by the subject in different ways: in some cases they are not of significant importance and do not determine any alteration of the psychic and behavioral balance of the subject in others, instead, they can represent the basis for the beginning of a misuse of the drug, of the worsening of the phenomenon of tolerance and of the progressive establishment of a state of addiction proper. Therefore, although both opioid drugs and cannabis are able to determine a iatrogenic dependence related to their pharmacodynamic characteristics, this does not in itself represent a pathological condition, but a normal physiological process linked to the progressive establishment of the phenomenon of tolerance that inevitably, these drugs determine, in the same way as other molecules that insist on any type of brain receptor. Therefore, being affected by a condition of “dependence” during the treatment of pain, does not mean becoming “sick” nor, even less, being in the precondition of becoming “addicted” subjects. The same condition of pseudoaddiction, generally determined by low non-therapeutic dosages of the prescribed antalgic treatment, does not represent a risk of addiction and, it becomes, only if incorrectly maintained, as responsible for the misuse of the prescribed drug that the patient starts independently, looking for a “real” antalgic dose, in total absence of a control and a series of specialized tests. All this would also be supported by recent studies of a neurobiological nature in which a reduction of dopamine production and, therefore, of the “reward” effect of opiates in animal models affected by neuropathic pain and treated with an antalgic therapy would have been demonstrated. based on opioids. They must therefore be specific organic, psychic and social factors, related to the additive effects of opioids or cannabis, to make patients undergoing analgesic treatment more vulnerable to the additive effects proper of these drugs.

BIBLIOGRAFIA

1. Niikura K, Narita M, Butelman ER, Kreek MJ, Suzuki T. Neuropathic and chronic pain stimuli downregulate central μ-opioid and dopaminer- gic transmission. Trends Pharmacol Sci 2010; 31: 299-305.

2. Solanki DR, Koyyalagunta D, Shah RV, Silverman SM, Manchikanti L. Monitoring opioid adherence in chronic pain patients: Assessment of risk of substance misuse. Pain Physician 2011; 14:E119-E131

3. Sairam Atluri, Hammam Akbik, Gururau Sudarshan Prevention of Opioid Abuse in Chronic Non- Cancer Pain: An Algorithmic, Evidence Based Approach. Pain Physician 2012; 15:E177-189

4. Katrin Skala et Al.. Can We Predict Addiction to Opioid Analgesics? A Possible Tool to Estimate the Risk of Opioid Addiction in Patients with Pain. Pain Physician 2013; 16:593-601

5. Roger Chou et Al.. Methadone Safety: A Clinical Practice Guideline From the American Pain Society and College on Problems of Drug Dependence, in Collaboration With the Heart Rhythm Society. The Journal of Pain, Vol 15, No 4 (April), 2014: pp 321-337.

HELICOBACTER PYLORI: FROM BENCH TO BEDSIDE

C.F. Zambon, D. Basso

Padova

Helicobacter pylori infection, which affects more than 50% of the human population, is usually acquired during childhood, lasting a lifetime if not treated. The prevalence of infection varies greatly worldwide being higher in the developing, where it is estimated at 70%, than in developed countries, where it affects about 30 to 40% of population. H. pylori is not spontaneously cleared from the infected stomach, and can survive for decades in this ecologic niche, causing gastric damage and disease. All H. pylori infected individuals have mild to severe gastric mucosal inflammation but, in a subset of subjects, it can also cause peptic ulcer, gastric adenocarcinoma or MALT lymphoma. Ninety-five per cent of duodenal and 70% of gastric ulcers are associated with H. pylori while from 75% to 90% of non-cardia gastric cancers and 75% to 100% of gastric diffuse large B cell lymphomas are attributable to the infection. However, these severe diseases are present in only 10 to 20% of infected patients, those affected by malignant disease being a very small minority (1-2%).

Many factors, often interplaying, have been associated so far with these severe H. pylori-associated clinical outcomes: the duration of the infection, host related factors (both genetic and non genetic) and bacterial related factors (virulence factors). Moreover, the human stomach is known to have its own microbiota regardless of H. pylori colonization, being the most relevant phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroides and Fusobacteria as in many other site of human host. H. pylori presence and its infection density are important in modifying microbiota relative composition. Progression to severe H. pylori associated disease is accompanied by a series of histopathological changes (Correa’s pathway) that modify gastric niche reducing progressively H. pylori colonization density, modifying microbiota relative composition and exiting in some cases to dysbiosis particularly in gastric cancer patients. The possible role of microbiota changes in gastric carcinogenesis is therefore an important medical question. Studies on humans, however, have not allowed scientists to verify whether the observed specific changes in microbial communities along Correa’s pathway are a cause or simply an effect of disease evolution. Studies on well established H. pylori mouse models with H. pylori infected INS-GAS mice showed that gastric atrophy, metaplasia and dysplasia were more severe and gastrointestinal intraepithelial neoplasia (GIN) more frequent in animals hosting commensal flora than in germfree animals underpinning a role of microbiota at least in magnifying H. pylori effect. Multiple interactions taking place between H. pylori and other bacterial species might modulate the overall inflammatory response to H. pylori infection and induce inflammatory processes related to cancer development at late stage of carcinogenesis when H. pylori density is dramatically reduced. These interactions might be a potential target for manipulation aiming to prevent disease and improve outcome.

MICROBIOTA AND OBESITY

G. Labruna

Napoli

Obesity represents a leading health problem worldwide that fundamentally arises from a positive energy balance under a complex interaction of genetic and environmental factors regulating weight gain. Although obesity is highly heritable, genetic variants so far identified only explain a small proportion of the obesity risk. Moreover, the genetic and epigenetic contribution to obesity are profoundly influenced by behavioral and environmental aspects, such as a reduction in physical activity and/or the high availability of low-cost and high-energy foods. In the last years, the gut microbiota has been proposed as a relevant factor connecting genes (and their functions) and environment. In humans, the gastrointestinal tract contains approximately 1014 bacteria, belonging to more than 1000 different species that represent a potential genetic space of more than three million bacterial genes. The microbiome (the totality of all the genomic elements of a specific microbiota) retains a high degree of plasticity and its composition changes adaptively with age, diet and the use of medications as well as in relation to the healthy status of the host in a bi-directional way. Accumulating evidences suggest that composition and functions of gut microbiota differ between healthy lean subjects and obese patients. The mechanisms that have been proposed to link the gut microbiota to obesity include energy extraction capacity from food, influence on the integrity of the gut barrier, modulation of the immune system and production of specific metabolites. These latter not only have a local effect on the gut-associated immune system and intestinal barrier, but also function as signals to other tissues and organs including brain, liver and adipose tissue, where they can regulate hunger/satiation stimuli and energy metabolism. To date, the vast majority of the studies conducted in obese patients only report data concerning microbiota composition evaluated on fecal samples and few data are available regarding the functional activity of microbiota especially at level of small intestine, in particular in duodenum, that has a critical physiological and pathophysiological role in metabolic homeostasis. To shed light on the functional correlation between microbiome and obesity, as well as on the regulatory mechanisms involved in this complex interplay, we performed a metatranscriptomic analysis of duodenum samples obtained both from obese patients and normal weight subjects. By next generation sequencing we analyzed the duodenal bacterial composition (analysis of 16S rRNA), as well as the whole bacterial and host transcriptome. Bioinformatic tools evidenced different bacterial composition as well as gene expression levels between obese and control groups. In particular, genes involved in energy production and conversion resulted to be up-expressed in obese patients suggesting that microbial energetic metabolites could be mediators between gut microbiota and obesity. The possibility to modulate the gut microbiota composition by dietary interventions, or by the use of probiotics or lastly by a fecal microbiota transplantation, may offer new possibility to treat obesity.

GUT MICROBIOTA FUNCTIONAL PROFILES IN SARDINIAN CENTENARIANS

L. Wu^1,2, T. Zeng², A. Zinellu¹, S. Sotgia¹, S. Rubino¹, D.J. Kelvin², C. Carru¹

¹Department of Biomedical Sciences, University of Sassari, Sassari, Italy

²Division of Immunology International Institute of Infection and Immunity, Shantou University Medical College, Shantou Guangdong, China

It’s widely recognized that Sardinia is a longevity hotspot, also the basis of longevity in this region is currently unknown. Gut microbiota may hold a clue. The change in taxonomic composition for gut microbiota with aging leads to altered metabolic activities of microbes in gut. We investigated the correlation between the gut microbiota and health status in various age groups including centenarians. We recruited 65 subjects as a part of the AKEA Study, divided into three age groups: 19 Healthy young, 24 Healthy elderly 22 Centenarians. The metagenomic sequencing was used to explore the gut microbiota composition and functional profile variation between different age groups in a cohort of Sardinians. Metagenomic sequencing data revealed that gut microbiota of the healthy elderly and young in Sardinia population share similar taxonomy composition and metabolic function profile, but a distinct composition was found for most of the centenarians.

In centenarians the proportion of dominant gut microbes and gut microbial genes was decreased, while an accumulation of sub-dominant species and genes was observed. We detected specifically high prevalence of Methanobrevibacter, Bifidobacterium, Lactobacillus and Escherichia in Sardinia centenarians.Amino acid biosynthesis potential was found to be declined, and menaquinol biosynthesis was enhanced. Interestingly healthy aging didn’t cause sharp changes of the composition and function profile of gut microbiota, rather, gut microbiota in Sardinia centenarians show a unique taxonomy and metabolic feature. Gut microbes in centenarians appear to cooperate to present a special energy harvest pattern that restricts the carbohydrate intake by the host and release beneficial metabolites. This may be a benefit that impacts the likelihood of longevity. Sardinia centenarians’ unique gut microbiota suggests that gut microbiota could be a promising target for pro-aging intervention.

ADVANTAGES AND LIMITATIONS OF DIGITALIZED MORPHOLOGY

F. Dima

GdS Diagnostica Ematologica Integrata della Società Italiana di Biochimica Clinica e Biologia Molecolare Clinica (SIBioC)

The blood count test and the differential leukocyte count is one of the most requested tests and requires considerable time commitment from the staff. After introduction of automated hematology analyzers, and development of automatic validation rules, the number of microscopic revisions has decreased considerably. In the vast majority of clinical laboratories, the largest cost is represented by the staff who, in many cases, has not increased but has often declined. The latest generation technologies of automated hematology analyzers encompasses instruments adaptable to individual realities of the various laboratories according to both the type of user and workloads. In the presence of atypical or immature cells, the analyzers generate alarms or flags which, together with other reasons, may require microscopic revision. And it is precisely the microscopic examination that includes both the preparation of the slide and the ensuing analysis which is the most expensive part of the process, also due to the need for highly qualified personnel for morphological evaluation. The current automated morphological analysis systems are capable to provide significant improvements of workflow. There are systems integrated into the hematologic chain capable of preparing and staining the slides for subsequent digitized analysis. The digitized morphology analyzers consist of an optical unit consisting of a microscope, a camera and an information system containing cell acquisition and classification software. The images are analyzed by an artificial neural network based on a database of cells and pre-classified according to the leukocyte classes. The cells are then shown on a computer screen for confirmation or possible reclassification by the operator, which also allows revision of the morphology of erythrocytes and platelets. In this study, we aimed to highlight the advantages potential and the limitations of digitalized morphology, including abnormal and immature cells. Many published studies have highlighted the good accuracy and precision of these systems for identifying normal cells and blasts, with some notable exceptions regarding inaccurate classifications of small blasts and for lymphoma cells. In general, the most recent evidence from the literature has shown a larger number of false positive results and a lower burden of false negative results in leukocyte classifications, along with a significant reduction of inter- and intra-observer variability. We hence conclude that digitized images offer many advantages compared to traditional microscopy, can be memorized for training activities, external quality evaluations and can be transmitted from remote locations for expert advice with optimization of turnaround time.

Briggs C, Longair I, Slavik M, Thwaite K, Mills R, Thavaraja V, et al. Can automated blood film analysis replace the manual differential? An evaluation of the CellaVision DM96 automated image analysis system. Int J Lab Hematol 2009;31:48–60.

CLSI. Reference Leukocyte (WBC) Differential Count (Proportional) and Evaluation of Instrumental Methods. H20-A2. Wayne,PA: Clinical and Laboratory Standards Institute; 2007

Smits SM, Leyte A. Clinical performance evaluation of the CellaVision Image Capture System in the white blood cell differential on peripheral blood smears.J Clin Pathol. 2014 Feb;67(2):168-72.

NEXT GENERATION FLOW CYTOMETRY IN THE ANALYSIS OF MINIMAL RESIDUAL DISEASE

B. Brando, A. Gatti

Western Milan Area Hospital Consortium, Legnano General Hospital, Milano, Italy

In recent years remarkable advancements have been made in the flow cytometric (FCM) analysis of rare cellular events, with the development of technical guidelines applicable to a series of clinically relevant applications (i.e. hematological malignancies, PNH, circulating tumor cells and microparticles, rare blood cell subsets). The FCM analysis of minimal residual disease (MRD) in hematological malignancies is now a standardized approach and is included in the clinical routine evaluation of therapeutic courses as well as a surrogate endpoint of outcome in clinical trials. To date FCM MRD assessments are in use with at least four hematological malignancies: B-Cell Chronic Lymphocytic Leukemia (B-CLL), Acute Lymphocytic Leukemia (ALL), Multiple Myeloma (MM), and more recently also Acute Myeloid Leukemias (AML).

The mandatory technical prerequisites to study MRD include: careful cleaning of instrument fluidics, optimized staining to ensure the maximal specificity, the concentration of samples by bulk lysis, the usage of sample pooling and the acquisition of very large cell datafiles to ensure the proper levels of sensitivity. With the advent of MRD studies, the concepts of LLOD and LLOQ, commonplace in clinical chemistry, have been adapted and translated to the peculiar FCM event analysis, and statistical tables have been specifically developed to guide operators. The calculation of MRD events (i.e. malignant cell events over the total nucleated cell denominator) is not a mere arithmetical operation, rather is a strict statistical procedure in which the level of sensitivity (LLOD) varies according to the total number of acquired cells, ideally in the 10^5 - 10^6 range. To comply with such stringent requirements, at least 30-50 MRD events must be captured, usually over several millions of ‘clean’ nucleated cell events. The concept of a detection sensitivity level that is not predefined as in other analytical fields, but is variable according to the outcome of each cell capture procedure sounds rather new, and still requires consolidation among FCM users.

Next generation FCM is therefore a new technical approach rather than a new instrumentation. Any ordinary benchtop 8-color FCM can be used, provided all the cited requirements are carefully met and the acquisition of bulky cell datafiles is ensured. It is commonly stated that event numbers count here more than color channels. Each hematological malignancy needs its special technicalities for MRD evaluation, includes different consensus MRD levels and requires disease-specific timepoints for monitoring, so ‘one size does not fit all’ in this case. Because of its crucial clinical importance, FCM MRD evaluation in hematological malignancies is today included the UKNEQAS external quality assurance programs, with the ALL scheme fully ISO17043 accredited and the others at present still in the accreditation pipeline.

REFERENCES

1. Arroz M. et al. Cytometry Part B (Clinical Cytometry) 2016; 90B: 31-39.

2. Flores-Montero J. et al. Leukemia 2017; 31: 2094-2103.

3. Burgos L and Paiva B. Methods Mol Biol 2018; 1792: 15-34.

4. Shumilov E. et al. Blood Rev 2018; doi: 10.1016/j.blre.2018.04.008.

PRELIMINARY DATA OF SURVEY ON THE QUALITY OF THE COMPLETE BLOOD COUNT REPORTING Working Group on Diagnostic Hematology (WGDH) and Working Group on Extra-Analytical Variability (WGEAV) of the Italian Society of Clinical Chemistry and Clinical Mol

R. Pajola¹, B. Manenti², D. Avino³, M.L. Ciardelli⁴, B. Cremonesi⁵, G. Da Rin⁶, A.M. Di Fabio⁷, F. Dima⁸, A. Fanelli⁹, F. Fiorini¹⁰, S. Francione¹¹, L. Germagnoli¹², G. Introcaso¹³, M. Gioia¹⁴, A. Marini¹⁵, A. Papa¹⁶, S. Pipitone¹⁷, R. Rolla¹⁸, F. Tosato¹⁹, D. Giavarina²⁰, C. Ortolani²¹, S. Buoro²

¹U.O.C. Laboratorio Analisi Ospedali Riuniti Padova Sud Schiavonia AULSS6 Euganea Veneto

²UOC SMeL 2 Analisi chimico-cliniche ASST Papa Giovanni XXIII Bergamo

³U.O. Diagnostica Ematologica P.O.“A. Tortora”-Pagani

⁴Servizio Analisi Chimico Cliniche Fondazione IRCCS Policlinico San Matteo Pavia

⁵Laboratorio Analisi, Policlinico S. Marco, Zingonia (BG) Istituti Ospedalieri Bergamaschi S.r.l.

⁶Laboratorio Medicina ASL n. 3 Bassano del Grappa

⁷UOC Patologia Clinica Ospedale Civile di Avezzano

⁸UOC di Laboratorio Analisi A.O.U.I Verona

⁹Laboratorio Analisi A.O. Careggi Firenze

¹⁰Laboratorio Analisi USL 15 Toscana

¹¹Laboratorio Analisi ASL NO Borgomanero (Novara) Piemonte

¹²Synlab Italia, Laboratorio Centrale Castenedolo (Brescia)

¹³Servizio di Medicina di Laboratorio, IRCCS-Centro Cardiologico Monzino Milano

¹⁴Laboratorio Analisi Presidio Ospedaliero “Abele Ajello” di Mazara del Vallo

¹⁵Laboratorio Analisi ASL 12 Versilia

¹⁶Medicina di Laboratorio Fondazione CNR/Regione Toscana “ G. Monasterio” Pisa

¹⁷U.O. Diagnostica Ematochimica Azienda Ospedaliera Universitaria di Parma

¹⁸S.C.D.U Laboratorio di Ricerche Chimico Cliniche - A.O.U “Maggiore della Carità” Novara

¹⁹Dipartimento di Medicina di Laboratorio, Azienda Ospedaliera Università di Padova

²⁰U.O.C. Laboratorio Analisi AULSS8 Berica, Vicenza, Veneto

²¹Università di Urbino

The Working Groups on Diagnostic Hematology (WGDH) and on Extra-Analytical Variability promoted a survey about quality of the complete blood count reporting.The survey include 36 questions and it was sent to all SIBioC members by mail and published on the website for 2 months.Twelve days after the publication,125 laboratories (Labs) representing 90% of the Italian regions responded. About 60% of Labs are basic with specialized sections and with inpatient and outpatient patients. Microscopic revision by digitalized morphology

was carried out only 4% of Labs.In the report among the quantitative parameters, there is a high degree of concordance for leukocytes, erythrocytes, hemoglobin, hematocrit, MCH, MCHC, RDW, Platelet and leukocytes differential count, while MPV and PDW are reported by 70% of Labs. International units of measure are used on 60% of Labs. The presence of blasts and immature granulocytes are not reported by 20% of Labs and the presence of plasma cells, prolymphocytes and erythroblasts are not reported by 30%. About 10% of Labs do not perform reticulocyte counting. The erythrocyte and leukocyte news parameters are not reported by 70% of Labs and reticulocyte parameters, mainly the immature fraction, are reported by 38%.Specific reference intervals for gender and age were adopted by 68% of Labs, but only 50% have instrument-specific intervals. The qualitative comments were included in the report by 83% of Labs, but only in 45% the comments are harmonized. The SIBioC-document was adopted by 66% of Labs, with adaptive modifications in 50%. Only

60% of Labs defined the criteria for choise of qualitative comments; they are often shared only verbally and only in 6% shared with the clinician.For the management of critical values 83% of Labs have a shared document. Critical values are communicated to the requesting physician by 90% of Labs and 63% have received the SIBioC indications.Preliminary data show that the activities promoted by the WGDH to harmonize the report have been effective on the “classic” parameters with an improvement compared to the 2014 survey, but much still needs to be done, especially for the new parameters. The management of comments still shows considerable heterogeneity, while the management of critical values is generally good.

RECENT ADVANCES IN LABORATORY MEDICINE FOR PRECISION MEDICINE IN NEONATOLOGY AND PEDIATRICS

M.J. Bennett

Department of Pathology and laboratory Medicine, University of Pennsylvania and Children’s Hospital of Philadelphia, USA

Clinical mass spectrometry is a rapidly expanding area of laboratory medicine. It is a particularly valuable technology for the diagnosis of biochemical genetic inherited disorders. In this presentation, I will describe the use of targeted and untargeted metabolomic analysis which identifies metabolic disorders in the newborn and in all age-groups.

Targeted metabolite analysis for metabolic disorders was first initiated in the 1950’s with an agar plate-based bacterial growth assay for measuring high levels of phenylalanine in newborn blood spots for the diagnosis of phenylketonuria. This process was initiated by Robert Guthrie and Dr Guthrie’s name is still frequently associated with the newborn screening process (the Guthrie test). The success of early diagnosis of phenylketonuria is well established.

Throughout the latter part of the 20th century more than 700 single gene defects were identified which could be diagnosed by metabolite analysis. This process was particularly enhanced by the development of clinical mass spectrometry and the ability to measure the abnormal biomarkers associated with these diseases. One of the most valuable diagnostic tools available was the use of gas chromatography-single quadrupole mass spectrometry to measure the organic acid content of patient urine samples. This represented an untargeted approach to diagnosis where the biomarkers of potential interest were mostly unknown prior to the testing process. Urine organic acid analysis can help in the diagnosis of around 100 single gene defects and is a very early example of the omics revolution in laboratory medicine. Interestingly, this process also measures many biomarkers of the gastrointestinal microbiome. There are ongoing studies of microbiome influence on conditions such as Inflammatory Bowel Disease for which pathogenesis has not yet been identified nor have good early diagnostic biomarkers.

Tandem mass spectrometry has also enhanced our ability to increase the number of metabolic disorders that can be diagnosed by a blood spot in the newborn period. David Millington and coworkers at Duke University identified the role of acylcarnitines as diagnostic biomarkers for multiple nuclear encoded mitochondrial disorders of organic acid and fatty acid metabolic pathways, in particular medium-chain acyl-CoA dehydrogenase which in many populations was found to be as common as phenylketonuria. Using multiple reaction monitoring for up to 30 acylcarnitine species and many amino acids it is possible now in a single blood spot to identify the biomarkers for about 30-40 of the known single gene defects resulting in a biochemical genetic disease. Additional biomarkers are being added for other conditions that could not be screened for previously. Clinical data on outcomes from early diagnosis of many of the screened disorders is very positive

Enhanced targeted and untargeted metabolomics processes are used to assist in the diagnosis of many of the non-screened conditions. There is particular growth in the area of disorders of post-translational protein glycosylation for which in recent years over 130 new metabolic disorders have been identified by the measurement of glycosylation profiles on circulating proteins such as transferrin and also for both n-linked and O-linked glyco-profiles using accurate mass time of flight mass spectrometry.

Most biomarkers for disease diagnosis have been identified in body fluids, yet metabolism itself is taking place primarily within cells. I will present some biomarker data for cellular metabolism as proof of principle. It is likely that intracellular metabolic testing will give better insight into the metabolic processes and disease. To this end I will present a novel assay that simultaneously measures all acyl-CoA species in a tissue sample and demonstrate a proof of principle that the assay has potential value.

EXTENDED NEWBORN SCREENING OF INHERITED DISEASE

M. Ruoppolo, F. Salvatore

Napoli

The advent of tandem mass spectrometry allowed to enlarge consistently the spectrum of metabolic diseases that might be easily and quickly detected. The main advantage of this technology is that it can detect several disorders by a single injection: LC-MS/MS, applied to newborn screening programs, can detect more than 70 different metabolic diseases on a single DBS collected within 48–72 hours of birth. Currently, many of the countries around the world have MS/MS-based NBS Programs to detect a variable range of genetic disorders. In Italy, only in recent years some regions issued legislative acts to promote expanded newborn screening with MS/MS. In May 2007 we initiated an expanded newborn screening for inborn errors of metabolism in Campania region by analyzing amino acids and acylcarnitines in DBS samples using tandem mass spectrometry. Here we report the most recent results of our experience from this program.

1. Scolamiero E, Cozzolino C, Albano L, Ansalone A, Caterino M, Corbo G, di Girolamo MG, Di Stefano C, Durante A, Franzese G, Franzese I, Gallo G, Giliberti P, Ingenito L, Ippolito G, Malamisura B, Mazzeo P, Norma A, Ombrone D, Parenti G, Pellecchia S, Pecce R, Pierucci I, Romanelli R, Rossi A, Siano M, Stoduto T, Villani GR, Andria G, Salvatore F, Frisso G, Ruoppolo M.

Targeted metabolomics in the expanded newborn screening for inborn errors of metabolism.

Mol Biosyst. 2015 Jun;11(6):1525-35 DOI: 10.1039/C4MB00729H

2. Imperlini Esther, Santorelli Lucia, Orrù Stefania, Scolamiero Emanuela, Ruoppolo Margherita, Caterino Marianna

“Mass Spectrometry-Based Metabolomic and Proteomic Strategies in Organic Acidemias,” BioMed Research International, 2016;2016:9210408. doi: 10.1155/2016/9210408. Epub 2016 Jun 14.

3. “Classical organic acidurias”: diagnosis and pathogenesis.

Villani GR, Gallo G, Scolamiero E, Salvatore F, Ruoppolo M.

Clin Exp Med. 2017 Aug;17(3):305-323. doi: 10.1007/s10238-016-0435-0. Epub 2016 Sep 9.

4. Integration of Proteomics and Metabolomics in Exploring Genetic and Rare Metabolic Diseases

Costanzo M, Zacchia M, Bruno G, Crisci D, Caterino M, Ruoppolo M.

Kidney Dis (Basel). 2017 Jul;3(2):66-77. doi: 10.1159/000477493. Epub 2017 Jun 30. Review.

NEONATAL SEPSIS IN NEONATOLOGY: CONVENTIONAL AND EMERGING BIOMARKERS

V. Fanos¹, M. Mussap²

¹Neonatal Intensive Care Unit, Neonatal Pathology, and Neonatal Section, AOU and University of Cagliari, Cagliari, Italy

²Department of Surgery, University of Cagliari, Cagliari, Italy

The assessment of the early diagnosis, the clinical course and the long-term outcomes of neonatal sepsis remain notable laboratory medicine challenges. Commonly used non-culture based diagnostic tests include the total and differential white blood cell (WBC) count, the absolute and immature neutrophil counts, and the ratio of immature to total neutrophils. Tests for evaluating the inflammatory response include C-reactive protein (CRP), procalcitonin (PCT), fibrinogen, haptoglobin, inflammatory cytokines, proteomic markers in amniotic fluid, and cell surface biomarkers, such as neutrophil CD64 and soluble CD14 subtype (presepsin). Recently, the potential role of metabolites has emerged as key issue for the early diagnosis and prediction of neonatal sepsis. Metabolomics – the omic approach investigating in biofluids and tissues the molecular phenotype and its changes over time in health and disease - seems to provide an early etiological diagnosis together with the severity and the progression of the infection. Metabolomics represents the new frontier for evaluating the individual response to the therapy, including any organ damage due to drug toxicity, and the role of microbiome in the early stage (attack of pathobionts to the organism) and in the final phase of sepsis (collapse of the microbiome). Until today, nearly 2,000 patients (including adults, children and babys) with sepsis have been evaluated by the metabolomic approach. Their biofluids (serum, plasma, blood, urine, BALF) were investigated by using either nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry coupled with gas or liquid chromatography (GC-MS and LC-MS, respectively). Data from the literature showed that a number of metabolic pathways may be altered in neonatal sepsis, including those involved in the acute phase response, hypoxia, oxidative stress, energy supply. In particular, it was found a reduced level of ATP, balanced by an increase in fatty acids oxidation, an increase glucose turnover and a switch of glucose consumption from the mitochondrial oxidative phosphorylation to other metabolic pathways such as lactate and pentose phosphate pathways. By comparing the urine metabolome of septic newborns 48 - 72 hours before the onset of clinical signs and symptoms with that of healthy newborns, we found a significant increase of glucose, lactate and acetate and a significant decrease of tamarind 4-Hydroxybenzoic acid (THBA), ribitol, ribonic acid and citrate. In a study of individualized medicine, we monitored the urinary metabolome of a very low birth weight (VLBW) newborn with a fungal infection; interestingly, at the end of the antifungal therapy the metabolome was far from that of non-infected babies. Metabolomic studies in newborns born from mothers affected by chorioamnionitis and in babies with necrotizing enterocolitis showed that gluconic acid is strongly involved, being significantly increased. Indeed, gut microbiota dysbiosis due the overgrowth of certain destructive bacterial species like Str. faecalis, Pseudomonas spp. and E. coli, led to the Entner-Doudoroff bacterial pathway, an overlooked glycolytic route. With the analysis of urine metabolites on admission in neonatal intensive care unit, metabolomics seems to promise impressive improvements in managing sepsis by: characterizing, measuring and distinguishing human metabolites from microbial metabolites (Rosetta Stone of microbiomics); monitoring the effectiveness and the toxicity of the antibiotic treatment; predicting the clinical outcome and the mortality of septic babies.

REFERENCES

Fanos V. Metabolomics and microbiomics: individualized medicine from the fetus to the adult. San Diego, CA USA: Academic Press (Elsevier), 2016.

Fanos V, Caboni P, Corsello G, Stronati M, Gazzolo D, Noto A, Lussu M, Dessì A, Giuffrè M, Lacerenza S, Serraino F, Garofoli F, Serpero LD, Liori B, Carboni R, Atzori L. Urinary (1)H-NMR and GC-MS metabolomics predicts early and late onset neonatal sepsis. Early Hum Dev 2014;90(Suppl 1):S78-83

Dessì A, Corsello G, Stronati M, Gazzolo D, Caboni P, Carboni R, Fanos V. New diagnostic possibilities in systemic neonatal infections: metabolomics. Early Hum Dev 2014;90(Suppl 1):S19-21

Dessì A, Liori B, Caboni P, Corsello G, Giuffrè M, Noto A, Serraino F, Stronati M, Zaffanello M, Fanos V. Monitoring neonatal fungal infection with metabolomics. J Matern Fetal Neonatal Med 2014;27(Suppl 2):34-8

Fattuoni C, Pietrasanta C, Pugni L, Ronchi A, Palmas F, Barberini L, Dessì A, Pintus R, Fanos V, Noto A, Mosca F. Urinary metabolomic analysis to identify preterm neonates exposed to histological chorioamnionitis: A pilot study. PLoS One 2017;12(12):e0189120

EARLY DIAGNOSIS OF AUTISM SPECTRUM DISORDER: BEYOND THE GENETIC

L. Mazzone¹, M. Siracusano^2,3, P. Curatolo¹

¹Child Neurology and Psychiatry Unit, System Medicine Department, Tor Vergata University Hospital of Rome, Italy.

²Department of Biomedicine and Prevention. University of Rome Tor Vergata, Rome (Italy).

³Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, Italy

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by impairment in social-communication skills, restricted and repetitive interests. Epidemiological studies show a recent increase in worldwide prevalence rates of ASD, currently estimated over 1% with a male to female ratio 4:1. Despite the progress in understanding the neurobiology of ASD, the causes remain still unknown. A complex relationship between genetic, epigenetic and environmental factors contributes to ASD etiopathogenesis and is responsible of the clinical phenotypic heterogeneity1. Actually, the diagnosis is still clinical, based on standardized neuropsychological assessment (Autism Diagnostic Observation Schedule –ADOS; Autism Diagnostic Interview Revised-ADI). No valid and specific biomarkers for ASD have been identified yet.

Metabolomics explores the molecular complexity of ASD and the relationships among phenotypes related to external agents. As an emerging tool of network medicine, metabolomics provides a direct functional read-out of the phenotype by the detection, identification, and quantification of metabolites in biological fluids in order to recognize metabolic alteration between comparative samples. Recent evidences show a different urinary metabolomic profile between ASD children and their unaffected siblings2-5. More in detail, a high level of mammalian-microbial cometabolites, an alteration in nicotinic-acid metabolism, a mitochondrial dysfunction, and increased oxidative stress and amino acid metabolism have been observed in ASD individuals. These findings suggest a potential role of gastrointestinal (GI) dysbiosis, perturbation of antioxidant status, excitatory/inhibitory imbalance in the etiopathogenesis of ASD comorbidities such as GI disorders, epilepsy and sleep problems. Identifying a specific ASD “metabolomic signature” related with the clinical phenotype (severity of core symptoms, developmental trajectory) represents the main future goal in assisting clinicians with earlier diagnoses.

REFERENCES

1. Benvenuto A, Manzi B, Alessandrelli R, Galasso C, Curatolo P. Recent advances in the pathogenesis of syndromic autisms. Int J Pediatr. 2009;2009:198736. doi: 10.1155/2009/198736. Epub 2009 Jun 21.

2. Noto A, Fanos V, Barberini L et al. The urinary metabolomics profile of an Italian autistic children population and their unaffected siblings. J Matern Fetal Neonatal Med. 2014;27 Suppl 2:46-52.

3. Gevi F, Zolla L, Gabriele S, Persico A. Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism. Molecular Autism. 2016.

4. Mussap M, Noto A, Fanos V. Metabolomics of autism spectrum disorders: early insights regarding mammalian-microbial cometabolites. Expert Rev Mol Diagn. 2016.

5. Yap IK, Angley M, Veselkov K et al. Urinary Metabolic Phenotyping Differentiates Children with Autism from Their Unaffected Siblings and Age-Matched Controls. J Proteome Res. 2010;9(6):2996-3004.