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Biochimica Clinica: VOL.37 N.5

Catene leggere libere delle immunoglobuline nel siero: un marcatore a metà del guado
Immunoglobulin free light chains in serum: a midstream biomarker
Biochimica Clinica 2013; 37(5) 344-346
Editoriale - Editorial
Le catene leggere libere circolanti nella gestione del paziente con amiloidosi AL
Free light chain (FLC) assays in the management of patients with AL amyloidosis

In light chain (AL) amyloidosis monoclonal FLC are not only a marker of the disease, but the toxic agent responsible for the organ damage. The possibility of measuring FLC provides an extraordinary means for managing AL amyloidosis. The quantitation of FLC, together with immunofixation of both serum and urine, allows optimal (98%-100%) sensitivity in detecting the amyloidogenic monoclonal protein. In combination with cardiac troponins and amino-terminal pronatriuretic type B peptide, FLC measurement grants an accurate prognostic stratification, which is important both in tailoring the therapeutic approach in individual patients and in designing and interpreting clinical trials. Chemotherapy targeting the amyloidogenic plasma cell clone succeeds in restoring organ function and prolonging survival only if it obtains a marked and persistent reduction of FLC, so much so that current response criteria are based on FLC

Biochimica Clinica 2013; 37(5) 347-351
Rassegne - Reviews
Utilizzo della determinazione delle catene leggere libere nel siero in ambito nefrologico
Use of serum free light chain testing in nephrology
P. Fabbrini, S. Sirtori, A. Stella

Renal disease is one of the most common sequelae of monoclonal gammopathies and, once established, has a great influence on patient’s prognosis. The kidney may be damaged both by intact immunoglobulin or, more often, by serum free light chains (sFLC). These molecules are physiologically filtered and reabsorbed by the kidney, but the interactions between host characteristics and sFLC biochemical structure or overproduction can lead to a wide variety of renal diseases. Diagnosis of renal disease due to monoclonal proteins is sometimes difficult, especially when the main clinical manifestation is chronic progressive nephropathy with minor urinary alterations, such as in light-chain deposition disease. The introduction of sFLC assays has improved the diagnostic capacity and offered an important tool to guide nephrologic treatments. Aim of this paper is to review current available indications for sFLC determination in nephrology and to offer a critical appraisal of their use in clinical practice.

Biochimica Clinica 2013; 37(5) 352-356
Rassegne - Reviews
Razionale e prospettive della determinazione delle catene leggere libere del siero nelle patologie infiammatorie croniche
Rationale and prospects of serum immunoglobulin free light chain (FLC) determination in chronic inflammatory disease
U. Basile  |  M. Mussap  | 

The development of serum assays for free FLC k and l has opened the door to new applications increasing their clinical importance beyond monoclonal gammopathies and plasma cell dyscrasias. In particular, the availability of these tests may open new prospects for diagnosing and managing various diseases characterized by chronic inflammation, namely autoimmune disease, allergic disease, viral infections, inflammatory disorders of the central nervous system and others. For most of these diseases, serum polyclonal FLC concentrations correlate with disease activity, being a potential useful index for adjusting therapeutic regimens. The aim of this review is to summarize current available data from the literature and to analyze the possible role of FLC in improving the clinical management of patients with chronic inflammatory disorders.

Biochimica Clinica 2013; 37(5) 357-364
Rassegne - Reviews
Intact immunoglobulin heavy/light chain paired assays
S. Harding  |  P. Young  |  M. Di Fazio  |  R. Hughes  | 

International guidelines recommend serum protein electrophoresis (SPEP) and immunofixation as laboratory tests to improve monoclonal gammopathy patient management. In patients with multiple myeloma (MM) these measurements are required to assess patient response to therapy, which help guide treatment and may influence clinical decisions. Here we discuss the potential role of new immunoassays that quantify the intact immunoglobulin Ig’k and Ig’l serum concentrations for IgG, IgA, IgM and IgD isotypes. The Freelite assay revolutionised patient management, particularly in the case of AL amyloidosis, and, due to the polyclonal nature of the antisera used, had a broad recognition for the highly heterogeneous free light chains (FLC). Similarly, the Ig’k and Ig’l assays utilise polyclonal antisera, identifying all MM patient sera tested to date, including those not readily identifiable by SPEP. Furthermore, as with the assessment of FLC k/l ratios by Freelite, Ig’k/Ig’l ratios have prognostic value at presentation and maximum response and may represent a hitherto unidentified MM cell biology that selectively suppresses isotype matched immunoglobulin production. Finally, we propose an algorithm utilising Ig’k and Ig’l and FLC measurements that may improve the detection of clonal evolution in MM patients.

Biochimica Clinica 2013; 37(5) 365-369
Rassegne - Reviews
Valutazione delle prestazioni del sistema analitico SPAPLUS per la misura delle catene leggere libere del siero
Evaluation of the performance of SPAPLUS system for the measurement of free light chains in serum

Measurements of serum immunoglobulin k and l free light chains (FLC) and FLC ratio calculation are recommended for the evaluation of plasma cell disorders. In this study we evaluated the performance of SPAPLUS analyzer using FreeliteTM reagents (both from The Binding Site) for FLC determination. Particularly, we compared the system performance with allowable goals for bias, imprecision and total error derived from biological variation of FLC. We evaluated the SPAPLUS FLC using data collected during a 10-month period of routine use, employing three different reagent lots. The two-level (N and H) SPAPLUS control material was used for bias estimate by comparing the obtained long-term experimental means (n=54, both levels) to the corresponding manufacturer’s assigned values. The protocol for CV evaluation employed the liquid-frozen Bio-Rad Liquichek unassayed chemistry control, measured in each performed run (n=48). Inaccuracy was checked by results from five UK-NEQAS exercises [system-specific (SPAPLUS) consensus value as reference]. Average cumulative bias was -1.5% (control N) and -1.4% (control H) for k FLC, and +6.6% (N) and +6.3% (H) for l FLC, respectively. Overall CV at physiological concentrations resulted in 10.6% for k FLC, 8.0% for l FLC and 9.9% for FLC ratio. On EQAS evaluation, all l FLC, four k FLC and three FLC ratio results were within the minimum allowable total error. Considering our previous experience with other analytical systems, the SPAPLUS solution undoubtedly represents a significant step forward. However, a further improvement in measurement imprecision is probably needed to fulfill the stringent analytical goals derived from FLC biological variation.

Biochimica Clinica 2013; 37(5) 370-375
Contributi Scientifici - Scientific Papers
Variabilità biologica delle catene leggere libere delle immunoglobuline nel siero
Biologic variation of immunoglobulin free light chains in serum

The measurement of serum k and l free light chains (FLC) and k/l FLC ratio calculation are recommended for screening, prognostic evaluation and monitoring of multiple myeloma and related plasma cell disorders. Given the lack of reliable data available in literature, in this study we assessed biologic variability components of FLCs and FLC ratio by an accurately designed protocol. We collected five blood specimens from each of 21 healthy volunteers (9 men and 12 women; age range, 23-54 years) on the same day, every two weeks for two months. Serum specimens were stored at -80 °C until analysis and analyzed in a single run in duplicate using a SPAPLuS immunoturbidimetric platform and Freelite reagents (The Binding Site). Data were analyzed by ANOVA. Serum l FLC concentrations were significantly (P <0.01) higher in men. The inter-individual variance of l FLC was higher than that of k FLC. Within- and between-subject CVs were 8.1% and 14.1% for k FLC, 7.0% and 27.5% for l FLC, and 4.5% and 15.3% for FLC ratio. All parameters had marked individuality showing that the use of population-based reference intervals could be inadequate for test interpretation. The reference change value was between 20% and 30% depending from the assay imprecision. Desirable analytical goals for imprecision (CV), bias and total error were <4.0%, ±4.1% and ±10.7% for k FLC, <3.5%, ±7.1% and ±12.9% for l FLC, and <2.3%, ±4.0% and ±7.7% for FLC ratio.

Biochimica Clinica 2013; 37(5) 376-382
Contributi Scientifici - Scientific Papers
Analisi combinata dei saggi Hevylite e Freelite nella valutazione precoce della recidiva di mieloma multiplo
Simultaneous evaluation of serum Hevylite and Freelite assays for relapse prediction in multiple myeloma

In multiple myeloma (MM), an accurate quantification of monoclonal immunoglobulin (M-Ig) and its variations during the course of the disease is important for prognosis, response evaluation and treatment selection. International guidelines recommend serum free light chain (sFLC) assays and free light chain ratio (sFLCr) for monoclonal gammopathy monitoring. Recently, a new immunoassay (Hevylite assay, sHLC), based on the analysis of k and l chains within intact immunoglobulins, has been developed. Similarly to sFLCr, a Hevylite assay ratio (sHLCr) can be determined. sHLC assay overcomes some technical limitations of sFLC assay, allows an accurate quantification of both the involved and uninvolved immunoglobulin in the tumour process and permits to quantify even very small monoclonal components. Aim of this work was to determine whether there is any additional advantage in predicting MM recurrence by sHLC compared to sFLC. A total of 208 serum samples, obtained from 37 MM patients from remission phase to relapse, were retrospectively analyzed by sFLC and sHLC assays; moreover, sFLCr and sHLCr results were compared in terms of relapse prediction. In 23 out of 37 patients (62%) sHLCr became abnormal before sFLCr and the median advantage of sHLCr vs. sFLCr in terms of time of relapse prediction was 3 months. Our analysis suggests that sHLCr is an earlier predictor of relapse than sFLCr. However, in a small group of MM cases, sFLCr became abnormal earlier than sHLCr. The combined use of sFLCr and sHLCr is, therefore, suggested to increase the predictive power of laboratory tests.

Biochimica Clinica 2013; 37(5) 383-388
Contributi Scientifici - Scientific Papers
La determinazione delle catene leggere libere nel liquido cefalorachidiano: l’esperienza di due laboratori italiani
Quantitation of immunoglobulin free light chains in cerebrospinal fluid: the experience of two Italian laboratories

The detection of oligoclonal IgG bands in cerebrospinal fluid (CSF) by isoelectricfocusing and immunodetection is the current “gold standard” method to detect an inflammatory process in central nervous system. However, as this test is time consuming and subjective, some authors have tested the measurement of free light chains (FLC) in CSF using a specific automated polyclonal antibody-based assay (Freelite, The Binding Site) with promising results. Recently, another automated nephelometric monoclonal antibody-based assay for FLC has been made available (N Latex FLC, Siemens). In our laboratories, we tested FLCk and l in CSF and serum using both assays. To test sensitivity and specificity, multiple sclerosis (MS) patients and non inflammatory neurological disease (NIND) patients as controls were selected. Both laboratories found statistically significant (P <0.05) difference between results in two groups. Using Freelite, the first laboratory defined the best cut-offs to discriminate between MS and NIND by ROC curves: i.e., 0.56 mg/L for FLCk, 7.82 for FLCk index, 0.31 mg/L for FLCl and 4.36 for FLCl index. Using N Latex FLC, the second laboratory estimated cut-offs by means of the NIND patients highest interquartile value, resulting in 0.22 mg/L for FLCk, 2.72 for FLCk index, 0.15 mg/L for FLCl and 2.07 for FLCl index. Sensitivities found with Freelite assay were 95% for FLCk index, 83% for FLCl index and 100% when both tests were considered. With N Latex FLC assay sensitivities were 100% for FLCk index and 93% for FLCl index. In both centers, isoelectricfocusing had 97% global sensitivity for MS. Our results show that, with both evaluated methods, CSF FLC can support or even replace isoelectricfocusing in clinical laboratories.

Biochimica Clinica 2013; 37(5) 389-394
Contributi Scientifici - Scientific Papers
Towards improved measurement of serum free light chains: clinical and laboratory issues
J. Tate  |  P. Mollee  | 

Serum free light chain (FLC) assays are now well established in the diagnosis and monitoring of plasma cell disorders. Nonetheless, the introduction of FLC measurement into clinical laboratories has not been without difficulty. In this paper we ask several questions about FLC measurements and describe some of the limitations and possible solutions. Main analytical issues include difficulties in defining the measurand and specificity requirements for anti- FLC antibodies, inadequate imprecision and bias and a lack of harmonised results across platforms and between different assays with the need for standardised sample dilution procedures to detect antigen excess, nonlinearity and interferences in FLC assays. Among the clinical issues a consistent approach is required to FLC reporting including standardised units, commenting and interpretation, and assay validation is required in specific disease rather than general patient populations. Collaboration is required between assay developers, laboratory scientists and clinicians to overcome these limitations in the next generation of assays.

Biochimica Clinica 2013; 37(5) 395-404
Opinioni - Opinions
La determinazione delle catene leggere libere nel siero può sostituire la ricerca e quantificazione della proteinuria di Bence Jones nella pratica clinica?
Can serum free light chain determination replace detection and quantitation of Bence Jones proteinuria in clinical practice?

Serum free light chain (FLC) determination became available for clinical laboratories 12 years ago. Since its introduction, it has been postulated that the urine study (i.e. Bence Jones proteinuria detection and quantitation) could have been abandoned due to the higher sensitivity and better practicability of the serum FLC assay. This paper investigates this hypothesis, presenting the evidence derived from the literature so far. Primary studies show some results in favour of the urine study discontinuation; however, a correct evaluation of this evidence is hampered by the lack of information about the sensitivity of the immunofixation method used as comparison. A number of guidelines and recommendations has been published on plasma cell dyscrasias, examining different clinical settings where the two tests can be used (screening, diagnosis, risk stratification, monitoring and response to treatment). Serum FLC measurements should be preferred in the screening and risk stratification subsets [apart from amyloidosis and POEMS (Polineuropathy, Organomegaly, Endocrinopathy, Monoclonal protein and Skin changes) syndrome]; on the contrary, determination of Bence Jones proteinuria should be used in the assessment of the response to therapy, when disease is quantifiable. At the moment, the FLC determination cannot replace the urine study in any circumstance and the two tests should be considered complementary until other evidences will be provided. Considering the dependence of both tests upon renal function, studies examining this issue could help elucidating their role in the wide scenario of plasma cell dyscrasias.

Biochimica Clinica 2013; 37(5) 405-418
Opinioni - Opinions
Metabolomica e scoperta di biomarcatori cardiovascolari
Metabolomics and Cardiovascular Biomarker Discovery

Metabolomics, the systematic analysis of low MW biochemical compounds in a biological specimen, has been increasingly applied to biomarker discovery. Because no single analytical method can accommodate the chemical diversity of the entire metabolome, various methods such as nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) have been employed, with the latter coupled to an array of separation techniques including gas and liquid chromatography. Whereas NMR can provide structural information and absolute quantification for select metabolites without the use of exogenous standards, MS tends to have much higher analytical sensitivity, enabling broader surveys of the metabolome. Both NMR and MS can be used to characterize metabolite data either in a targeted manner or in a nontargeted, pattern-recognition manner. In addition to technical considerations, careful sample selection and study design are important to minimize potential confounding influences on the metabolome, including diet, medications, and comorbitidies. To this end, metabolite profiling has been applied to human biomarker discovery in small-scale interventions, in which individuals are extremely well phenotyped and able to serve as their own biological controls, as well as in larger epidemiological cohorts. Understanding how metabolites relate to each other and to established risk markers for diseases such as diabetes and renal failure will be important in evaluating the potential value of these metabolites as clinically useful biomarkers. Applied to both experimental and epidemiological study designs, metabolite profiling has begun to highlight the breadth metabolic disturbances that accompany human disease. Experimental work in model systems and integration with other functional genomic approaches will be required to establish a causal link between select biomarkers and disease pathogenesis.

Biochimica Clinica 2013; 37(5) 419-427
Il meglio di Clinical Chemistry - Clinical Chemistry highlights
Un caso particolare di mieloma micromolecolare lambda
A particular case of lambda chain multiple myeloma

The measurement of serum free light chains (FLC) is recommended for diagnosis, monitoring and prognosis of monoclonal gammopathies and specific assays on automated nephelometers and turbidimeters are available. We reported a case study about a 47-years old woman with bone pain, admitted to the Orthopedic Day Surgery. Serum protein electrophoresis (SPE) both on agarose gel (AGE) and capillary zone electrophoresis (CZE) did not show abnormalities and nephelometric assays of intact immunoglobulins and total light chains were within the respective reference intervals. A relevant difference between the l FLC results was obtained using two serum FLC assays, resulting highly abnormal by the Freelite (The Binding Site) and within the reference interval by the N Latex FLC (Siemens Healthcare Diagnostics). A home-made high resolution AGE, performed both in serum and urine samples, detected a monoclonal component (MC) in the g-region and serum and urine immunofixation electrophoresis (IFE) showed a free l MC. Immunoblotting and SDS-PAGE confirmed the presence of a FLC and showed that the MC consisted of l FLC with MW of 22,000 and 44,000 Da. Bone biopsy confirmed the diagnosis of l chain multiple myeloma. The woman was admitted to the Hematology- Oncology Unit and one month after the start of the therapy, serum and urine IFE showed the MC disappearance and the serum FLC results by Freelite assays returned within the reference interval.

Biochimica Clinica 2013; 37(5) 428-430
Casi clinici - Case Report
Componenti monoclonali piccole ma dannose
Small but harmful monoclonal components

A small B-cell clone can synthesize a toxic monoclonal protein that causes severe organ damage. Diseases caused by these toxic immunoglobulins range from AL amyloidosis, light chain deposition disease and cryoglobulinemia to the POEMS (Polineuropathy, Organomegaly, Endocrinopathy, Monoclonal protein and Skin changes) syndrome. The serum monoclonal components are, in these cases, usually small and difficult to be detected by the commercially available methods. However, an early detection and a correct characterization of the monoclonal component is essential, because misdiagnosis or a delay in diagnosis can prevent the proper treatment of the patient worsening his outcome. Here we present two cases of amyloidosis AL, where the correct diagnosis was facilitated by specific and sensitive techniques. This does not mean that every clinical laboratory should be competent in this field and use these methods routinely, but simply that people should be aware of the problem, so that these clinical conditions can be properly managed.

Biochimica Clinica 2013; 37(5) 431-434
Casi clinici - Case Report
La determinazione delle catene leggere libere nel siero nella valutazione del rischio di progressione della gammopatia monoclonale di incerto significato (MGUS): proposta di un protocollo di monitoraggio clinico differenziato
Serum free light chain measurement in the assessment of risk of progression in monoclonal gammopathy of undetermined significance (MGUS): proposal for a clinical follow-up protocol
Biochimica Clinica 2013; 37(5) 435-437
Lettere all'Editore - Letters to the Editor