INTRODUCTION AND STUDY OBJECTIVE: Multiple sclerosis (MS) is an inflammatory-demyelinating pathology of the central nervous system (CNS) with a relapsing / progressive pattern. 90% of patients present an intrathecal production of oligoclonal IgG which are arranged in 'oligoclonal bands' on liquor analysis with electrophoretic methods.The IgG oligoclonal liquor cells are the biological correlates of an intrathecal expansion of clones of B lymphocytes and plasma cells - and, from the point of view of clinical practice, they represent a useful diagnostic biomarker. Cause of the continuous innovations in the therapeutic field for the treatment of MS, the need to validate prognostic markers to promptly identify the most aggressive forms of the disease is a priority. Literature data indicate that oligoclonal bands IgM have been associated to a more aggressive phenotype of disease. The method for identifying these bands, however, presents some analytical problems mainly related to the pentameric structure of IgM. The lack of appropriate standardizations also limits their use in clinical routine. The main objectives of the study were: a) to implement an accurate and reliable method for the identification of oligoclonal IgM bands, b) to obtain preliminary results on patients with MS and patients with other CNS neurological diseases. METHODS: We analyzed paired serum and CSF samples from 41 MS patients and 18 control patients with other CNS neurological diseases. Clinical-demographic data were retrospectively collected from discharge papers and follow-up visits. The IgM assays were obtained by nephelometry (serum) or ELISA (liquor). The IgM oligoclonal bands were determined by isoelectrofocusing, a high-resolution electrophoretic technique that exploits the pH gradient as a separation element. The biological samples were treated with dithiothreitol to reduce the structure of the IgM from pentameric to monomeric, allowing its entry into the mesh of the gel used for the isoelectrophoretic run. Serum and liquor protein focusing was performed on agarose gel, home-made preparation with pH range of 3-10 and expansion of the zone within the range of pH 5-8. The migration conditions have been optimized by appropriate pH standards. The focused IgM were transferred on nitrocellulose membrane, incubated with anti-human IgM antibody conjugated with alkaline phosphatase and detected with a chromogenic enzymatic reaction. The serum of a patient with monoclonal IgM gammopathy (positive control) was used to optimize the method and as an internal control in every race. RESULTS AND CONCLUSIONS: Keeping in mind the limitations of the study related to the small sample size, our results seem to confirm the few literature data that associate the IgM oligoclonal bands with an aggressive phenotype of MS. The method implemented for the evaluation of IgM bands showed good performance and was ready to test a large number of samples, preferably within a multicentre study.
INTRODUZIONE E OBIETTIVO DELLO STUDIO: la sclerosi multipla (SM) è una patologia infiammatorio-demielinizzante del sistema nervoso centrale (SNC) ad andamento recidivante/progressivo. Il 90% dei pazienti presenta una produzione intratecale di IgG oligoclonali che si dispongono in‘bande oligoclonali’ all’analisi del liquor con metodiche elettroforetiche. Le IgG oligoclonali liquorali costituiscono il correlato biologico di un’espansione intratecale di cloni di linfociti B e di plasmacellule - e, dal punto di vista della pratica clinica, rappresentano un utile biomarker diagnostico. Alla luce delle continue innovazioni in ambito terapeutico per il trattamento della SM, la necessità di validare marcatori prognostici per identificare tempestivamente le forme più aggressive di malattia è prioritaria. Dati di letteratura segnalano che le bande oligoclonali IgM si associano ad un fenotipo più aggressivo di malattia. La metodica per l’identificazione di tali bande tuttavia presenta alcune problematiche analitiche principalmente correlate alla struttura pentamerica delle IgM., La mancanza di appropriate standardizzazioni, inoltre, ne limita l’utilizzo nella routine clinica. I principali obiettivi della tesi sono stati: a) implementare una metodica accurata e affidabile per l’identificazione delle bande oligoclonali IgM, b) ottenere risultati preliminari su pazienticon SM e pazienti con altre malattie neurologiche del SNC. METODI: Abbiamo analizzato campioni appaiati di siero e liquor di 41 pazienti affetti da SM e di 18 pazienti di controllo affetti da altre malattie neurologiche del SNC. I dati clinico-demografici sono stati raccolti retrospettivamente dai documenti di dimissione e dalle visite di follow-up. I dosaggi delle IgM sono stati ottenuti tramite nefelometria (siero) o ELISA (liquor). Le bande oligoclonali IgM sono state determinate mediante isoelettrofocusing, tecnica elettroforetica a elevata risoluzione che sfrutta il gradiente di pH come elemento di separazione. I campioni biologici sono stati trattati con ditiotreitolo per ridurre la struttura dell’IgM da pentamerica a monomerica, consentendone l’entrata nelle maglie del gel utilizzato per la corsa isoelettroforetica. La focalizzazione delle proteine dei sieri e dei liquor è stata effettuata su gel di agarosio, preparato home-made con range di pH 3-10 ed espansione della zona compresa nel range di pH 5-8. Le condizioni di migrazione sono state ottimizzate mediante appropriati standard di pH. Le IgM focalizzate sono state trasferite su membrana di nitrocellulosa, incubate con anticorpo secondario anti-IgM umane coniugato con fosfatasi alcalina e rivelate con reazione enzimatica cromogenica.Il siero di un paziente con gammopatia monoclonale IgM (controllo positivo) è stato utilizzato per ottimizzare la metodica e come controllo interno in ogni corsa. RISULTATI E CONCLUSIONI: Tenendo ben presente i limiti dello studio legati alla ristretta dimensione campionaria, i nostri risultati sembrano confermare i pochi dati di letteratura che associano le bande oligoclonali IgM a un fenotipo aggressivo di SM. La metodica implementata per la valutazione delle bande IgMha mostrato buone performanceanalitiche ed è pronta per testare un numero elevato di campioni, preferibilmente all’interno di uno studio multicentrico.
BANDE OLIGOCLONALI IGM: OTTIMIZZAZIONE DI UNA METODICA DI LABORATORIO E RILEVANZA PROGNOSTICA IN SCLEROSI MULTIPLA
MARUCCIO, ALESSIO
2017/2018
Abstract
INTRODUCTION AND STUDY OBJECTIVE: Multiple sclerosis (MS) is an inflammatory-demyelinating pathology of the central nervous system (CNS) with a relapsing / progressive pattern. 90% of patients present an intrathecal production of oligoclonal IgG which are arranged in 'oligoclonal bands' on liquor analysis with electrophoretic methods.The IgG oligoclonal liquor cells are the biological correlates of an intrathecal expansion of clones of B lymphocytes and plasma cells - and, from the point of view of clinical practice, they represent a useful diagnostic biomarker. Cause of the continuous innovations in the therapeutic field for the treatment of MS, the need to validate prognostic markers to promptly identify the most aggressive forms of the disease is a priority. Literature data indicate that oligoclonal bands IgM have been associated to a more aggressive phenotype of disease. The method for identifying these bands, however, presents some analytical problems mainly related to the pentameric structure of IgM. The lack of appropriate standardizations also limits their use in clinical routine. The main objectives of the study were: a) to implement an accurate and reliable method for the identification of oligoclonal IgM bands, b) to obtain preliminary results on patients with MS and patients with other CNS neurological diseases. METHODS: We analyzed paired serum and CSF samples from 41 MS patients and 18 control patients with other CNS neurological diseases. Clinical-demographic data were retrospectively collected from discharge papers and follow-up visits. The IgM assays were obtained by nephelometry (serum) or ELISA (liquor). The IgM oligoclonal bands were determined by isoelectrofocusing, a high-resolution electrophoretic technique that exploits the pH gradient as a separation element. The biological samples were treated with dithiothreitol to reduce the structure of the IgM from pentameric to monomeric, allowing its entry into the mesh of the gel used for the isoelectrophoretic run. Serum and liquor protein focusing was performed on agarose gel, home-made preparation with pH range of 3-10 and expansion of the zone within the range of pH 5-8. The migration conditions have been optimized by appropriate pH standards. The focused IgM were transferred on nitrocellulose membrane, incubated with anti-human IgM antibody conjugated with alkaline phosphatase and detected with a chromogenic enzymatic reaction. The serum of a patient with monoclonal IgM gammopathy (positive control) was used to optimize the method and as an internal control in every race. RESULTS AND CONCLUSIONS: Keeping in mind the limitations of the study related to the small sample size, our results seem to confirm the few literature data that associate the IgM oligoclonal bands with an aggressive phenotype of MS. The method implemented for the evaluation of IgM bands showed good performance and was ready to test a large number of samples, preferably within a multicentre study.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/20123