Validazione dei metodi quantitativi bioanalitici in spettrometria di massa: regole e protocolli sperimentali
Bioanalytical method validation of quantitative mass spectrometry based assay: experimental protocols and regulations
Liquid chromatography-mass spectrometry (LC-MS) technique has rapidly extended the spectrum of clinical chemistry methods during the past decade and it has became a standard tool for endogenous and exogenous substances in research laboratories as well as in many clinical laboratories. To date, many guidelines for bioanalytical method validation have been published, but there is still the need to have specific guidelines and experimental protocols. In particular, we need to further focus and investigate the overall clinical aspects of spectrometry method validation, for both exogenous and endogenous molecules. The aim of the present document is to suggest specific experimental regulations and protocols for bioanalytical LC-MS method validation for exogenous and endogenous molecules, inspired by actual international guidelines and integrated with practical tips to obtain better performance on methods used in routine clinical practice. Here we have introduced new concepts such as “normalized matrix effect”, and protocols for method validation using “pathological samples” and “altered samples”, as these are the most commonly samples encountered in laboratory routine. In conclusion, the new rules and protocols reported in this work do not intend to replace international guidelines; we believe that these rules have to be considered besides the available guidelines with the aim to help the LC-MS users by recommending a number of experimental steps to be performed during each method/kit validation.
Pubblicato online il: 19.01.2018
Indagini genetiche di nuova generazione e terapia personalizzata: l’esempio vincente della Fibrosi Cistica
Next generation genetic studies and personalized therapy: the successful model of Cystic Fibrosis
Cystic Fibrosis (CF) is a complex genetic disease. The causative gene is the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Although monogenic, this disease has a complex genotype – phenotype relationship. Several factors originate this complexity. The most important are the high number of CFTR mutations, the difficulty of a full mutational analysis, the incomplete knowledge of the functional effect of mutations and their variable outcome, as well as the existence of modifier genes (different from CFTR) that modulate the clinical severity. This complexity impairs the diagnostic, prognostic and therapeutic ability. Next generation sequencing approaches represent a revolution in genetic studies, because of their rapidity, low-cost and high-throughput. They allow a near complete mutational characterization of CFTR mutated genotypes, with the potentiality of studying several other genes involved in CF clinical modulation. These methods are a perfect precondition for personalized therapeutic approaches of CF. In fact, a full genetic characterization appears to be crucial to applying mutation-specific therapies. Drugs specific for some CFTR mutations are already in clinical practice or in phase 3 trials. The enlargement of the CF personalized therapy to an increasing number of mutated genotypes needs a growing knowledge of structural and functional defects of CFTR. The synergy between next generation genetic approaches, the enhancement of the comprehension of molecular mechanisms of CFTR mutations and the spreading of personalized therapies, are revolutionizing the cure of CF.
Pubblicato online il: 19.01.2018
Determinazione della concentrazione dell’inibitore C1 esterasi nel siero: un invito alla standardizzazione
Determination of C1-esterase inhibitor concentration in serum: a call for standardization
Pubblicato online il: 15.01.2018
Lettere all'Editore -
Valutazione del sistema Optilite™ per la misura delle catene leggere libere delle immunoglobuline nel siero
Evaluation of the Optilite™ system for the determination of immunoglobulin free light chains in serum (sFLC)
The measurement of sFLCk and l and k/l ratio calculation is recommended for evaluation and management of plasma cell disorders. However, some analytical issues persist in their measurements, among which a too large long-term imprecision seems to be the main challenge. We evaluated the new Optilite system (The Binding Site) for sFLC determination by comparing its performance with specifications for bias, imprecision (as CV) and total error derived from biological variation of sFLC. We collected data during one year of routine use by employing three reagent lots. The system alignment was checked using the two-level (L and H) Optilite control material by comparing the obtained long-term experimental mean (n=233, both levels) with the manufacturer’s assigned values. The protocol for CV evaluation employed the liquid-frozen BioRad Liquichek Control and a frozen serum pool tested for 125 and 79 runs, respectively. Inaccuracy was evaluated by results of four UK-NEQAS exercises [system-specific (Optilite) consensus value as reference]. Average cumulative bias [1.1% (L) and -2.0% (H) for sFLCk; 5.4% (L) and 0.1% (H) for sFLCl, respectively] fulfilled the desirable goals. CVs for sFLCk (7.1% for Liquichek and 6.6% for the pool, respectively), sFLCl (7.8% for both Liquichek and the pool) and k/l ratio (8.9% for Liquichek and 10.2% for the pool, respectively) failed however to reach minimum quality goals. In EQAS evaluation, all sFLC k and l and 3 out of 4 k/l ratio results were within the allowable total error. In our experience, the Optilite system shows a good method alignment suitable for sFLC interpretation using fixed cut-offs. However, the assay reproducibility is probably not suitable for optimal long-term monitoring of individual patients.
Pubblicato online il: 15.01.2018
Contributi scientifici -
Mieloma multiplo IgD lambda: “switch” isotipico immunoglobulinico dopo trapianto autologo
IgD lambda multiple myeloma: immunoglobulin isotype switch after autologous stem cell transplantation
IgD multiple myeloma (MM) is a rare disease affecting less than 2% of patients with MM, and it is frequently characterized by an aggressive course. It is usually associated with low monoclonal protein levels, so adequate diagnostic procedures have to be performed in order to identify the involved monoclonal component (MC). We present a case of a 38-year-old man with acute kidney disease caused by an IgD lambda MM. Diagnosis was achieved by serum protein electrophoresis and immunofixation with anti IgD and IgE antisera. After autologous stem cell transplantations (ASCT) the patient developed a MC different from the original isotype, followed by an oligoclonal bands (OB) pattern. Recently, the occurrence of MC and OB unrelated to the original clone has been proven to be an important favorable prognostic factor in patients with MM who undergo ASCT. The role of the protein laboratory at diagnosis and during follow up of MM patients is highlighted.
Pubblicato online il: 02.08.2017
Casi clinici -