The dark side of current analytic methods for Bence Jones Proteinuria.

OBJECTIVE: Bence Jones proteinuria (BJP) refers to monoclonal free immunoglobulin light chains detected in urine, deriving from the clonal expansion of plasma cells in the bone marrow in patients with plasma cell dyscrasias, associated with monoclonal gammopathies of uncertain origin. This review summarizes routinely diagnostic procedures to assess BJP highlighting critical steps of pre-analytical, analytical, and post-analytical phases. QUALITATIVE AND QUANTITATIVE METHODS: The best option for BJP detection is the first morning void urine sample and immunofixation electrophoresis detection technique (IFE) the recommended method, with the employment of specific polyvalent antisera. Other qualitative tests for a quick evaluation of BJP are currently available. Densitometric analysis performed on the 24-hour urine is the recommended method to quantify BJP. To overcome the 24-hour collection, it is possible to use morning urine sample and correlate the assessed value of BJP to creatininuria. In addition to the traditional ones, we here reviewed screening methods currently used to avoid false negatives and reduce the time around test (TAT), together with immunochemical quantification methods for increased sensitivity, after checking BJP by IFE. Mass spectrometry emerges as a new challenge in the determination of BJP. CONCLUSIONS: The employment of different based-assays methods may be useful for diagnostic purposes to improve the accuracy of BJP monitoring in monoclonal gammopathies.

Evaluation of immunoglobulins subclasses and free-light chains in non-obese patients with polycystic ovary syndrome and correlations with hormonal and metabolic parameters: preliminary data.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and hyperinsulinemia that contribute to create a state of chronic low-grade inflammation. We performed an observational case-control study to investigate inflammatory and immunological parameters, such as IgG subclasses and free light chains (FLCs) and hemolytic complement activity (CH50) in non-obese PCOS, evaluating their relations with metabolic and hormonal parameters. PATIENTS AND METHODS: 36 subjects were studied: 16 PCOS patients (mean±SEM 27.13±1.82 age; BMI 24.1±0.9 kg/m2); 20 controls (aged 26.05±0.73; BMI 20.8 ± 0.4 kg/m2). The blood sample was collected for metabolic and hormonal parameters, IgG subclasses, k and λ FLCs, CH50. Hormones were measured by immunochemiluminometric assays; metabolic parameters by enzymatic assays; subclasses of IgG, FLCs, and CH50 were evaluated by the turbidimetric method. RESULTS: PCOS patients showed vs. controls lower IgG1, IgG2, IgG3 (mean±SEM 3.76±0.29 g/l, 2.63±0.20, 0.62±0.06, 0.34±0.08 vs. 6.49±0.35, 4.28±0.25, 0.84±0.07, 0.33±0.04, respectively) and higher levels of FLCs (k 12.22±0.71 vs. 6.03±0.30, λ 10.10±0.79 vs. 8.04±0.48 g/l) and CH50 (48.64±2.65 vs. 36.51±1.38 U/ml); we found correlation between IgG2 and free-testosterone (r=0.72, p=0.005) and CH50 and vitamin D (r=0.54, p=0.04); an inverse correlation was found between IgG1 and, respectively, ACTH (r=-0.57, p=0.02) and cortisol (r=0.78, p=0.001) in PCOS. CONCLUSIONS: In the complex scenario of low-grade inflammation in non-obese PCOS, we showed lower levels of main subclasses of IgG and higher CH50 levels, suggesting the involvement of other mechanisms other than the "classical" pathway of complement activation; FLCs could be attractive to monitor inflammation degree, disease activity and influence on hormonal status.

The diagnostic performance of PIVKA-II in metabolic and viral hepatocellular carcinoma: a pilot study.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a primary liver tumor derived from metabolic or viral chronic hepatitis, with few treatment options in advanced cases. New biomarkers that allow improving diagnosis and staging are widely desired. Here, we aim to evaluate the performance of Protein Induced by Vitamin K Absence or Antagonist-II (PIVKA-II) in combination with α-fetoprotein (AFP), in the diagnosis of HCC in patients with metabolic or viral hepatitis. PATIENTS AND METHODS: We enrolled 60 HCC patients (20 metabolic and 40 viral) and 20 healthy subjects (HS) as negative controls. PIVKA-II, AFP, Matrix metalloproteinase-9 (MMP-9) and Fibroblast growth factor (FGF) serum levels were assessed by immunoassays. RESULTS: AFP and PIVKA-II levels were obviously higher in patients than in HS. AFP displayed a better diagnostic performance than PIVKA-II for viral HCC while PIVKA-II was better for metabolic HCC. The combination of the two biomarkers did not improve the discriminating ability. CONCLUSIONS: PIVKA-II may be considered an independent predictor of macrovascular invasion from HCC cells and it can be used to better stratify HCC patients and should be evaluated in prospective studies for early detection of advanced HCC in metabolic subjects.

Biomarkers in HCV-related mixed cryoglobulinemia patients withnon-Hodgkin lymphoma.

OBJECTIVE: Chronic Hepatitis C virus (HCV) infection can cause severe extrahepatic manifestations, such as mixed cryoglobulins (MC), up to the development of B cell nonHodgkin's lymphoma (B-NHL). Mechanisms transforming of HCV infection into lymphoproliferative and/or autoimmune disorders are still poorly understood. In course of HCV infection, the sustained virus-driven antigenic stimulation may probably induce a B-cell clonal expansion. Measurements of serum free light chains (FLCs) levels, considered as a direct marker of B cell activity, are analyzed with increasing interest in clinical practice, for diagnosis, monitoring and follow-up of plasma cell dyscrasia. Syndecan-1 (CD138) is a transmembrane heparan sulfate proteoglycan expressed and actively shed by most myeloma cells. Membrane CD138 represents the major receptor protein for HCV attachment to the hepatocyte surface and high levels of circulating sCD138 levels are detected in patients at early stage of B-cell chronic lymphocytic leukemia. This study is aimed to evaluate sCD138 and FLC levels as diagnostic biomarkers of HCV-related MC with B-NHL. PATIENTS AND METHODS: We enrolled 35 HCV-MC-NHL patients, characterized for the specific type of cryoglobulins, and 25 healthy blood donors (HBD) as negative control. Serum sCD138 levels were determined using ELISA kits specific for human sCD138. Serum FLCs were assessed by means of the turbidimetric assay. RESULTS: We found that serum levels of sCD138, as well as FLCs, were significantly higher in patients than in HBD (p<0.001). CONCLUSIONS: In agreement with the definition of HCV-driven lymphoproliferative disorders as the consequence of a multifactorial and multistep pathogenetic process, we suggest that sCD138 and FLCs could be considered putative independent markers of worsening progression of the disease.

Serum immunoglobulin free light chain levels in systemic autoimmune rheumatic diseases.

Several reports have highlighted the abnormal increments of serum immunoglobulin free light chains (FLCs) in the course of systemic autoimmune rheumatic diseases (SARD), but a comparative analysis among different conditions is still lacking. A strong association between elevated FLC and hepatitis C virus (HCV)-related mixed cryoglobulinaemia (HCVMC) has been well established. Here, we aimed to analyse serum FLC levels in patients with four different SARD in comparison with HCVMC. Using a turbidimetric assay, free κ and λ chains were quantified in sera from 198 SARD patients (37 rheumatoid arthritis, RA; 47 systemic lupus erythematosus, SLE; 52 anti-phospholipid syndrome, APS; 62 primary Sjogren's syndrome, pSS), 62 HCVMC and 50 healthy blood donors (HD). All patient groups showed increased κ levels when compared to HD: 33·5 ± 2·6 mg/l in HCVMC, 26·7 ± 2·3 mg/l in RA, 29·7 ± 1·9 mg/l in SLE, 23·8 ± 1·1 mg/l in APS, 24·2 ± 1·1 mg/l in pSS; 10·1 ± 0·6 mg/l in HD. Free λ levels displayed a significant increase only for HCVMC (20·4 ± 1·4 mg/l) and SLE (18·4 ± 1·0 mg/l) compared to HD (13·6 ± 0·9 mg/l). The increase of κ compared to λ takes into account a κ /λ ratio of 1·6 for all groups. Our results substantially analyse and strengthen the association between FLC and SARD focusing the questions regarding their role in the pathogenesis and diagnosis of human diseases. Unfortunately, the biochemical differences distinguishing normal from pathological FLC have not been identified. Production of different isotypes is probably connected to still-unknown pathways.

Lack of association between Vitamin D status and free light chains profile with different chronic HCV-related liver and extrahepatic disorders.

OBJECTIVE: A still uncertain association between vitamin D levels and HCV chronic liver diseases has been reported. Increased levels of serum-free light chains (FLCs) and an altered k/λ FLC ratio correlate with Mixed Cryoglobulinemia (MC) vasculitis and/or B-cell non-Hodgkin's lymphoma in HCV-positive patients. We aimed to investigate the possible role of vitamin D, vitamin D Binding Protein (DBP), and FLCs levels as a tool for discriminating different stages of HCV- related MC and chronic liver diseases. PATIENTS AND METHODS: Sixty-five untreated patients were retrospectively enrolled and 21 healthy blood donors (HBD) were used as controls. Vitamin D, DBP, FLCs, and cryoglobulins levels were measured. Based on cryoglobulins, patients were divided in three subgroups (without cryoglobulins, type II, and type III). RESULTS: We didn't find any significant differences in vitamin D and DBP levels between HCV patients' main groups and HBD. Serum FLCs levels were significantly higher in HCV patients than in HBD. FLCs ratio among patients' subgroups did not reveal differences. CONCLUSIONS: Our results confirm the presence of an increased serum level of FLCs in HCV patients and suggest that nor vitamin D and DBP or FLC levels can be considered reliable biomarkers for discriminating different stages of HCV-associated chronic liver diseases and/or HCV-associated extrahepatic manifestation. We confirm that serological FLCs levels are significantly higher in patients than in HBD as a signature of B cell activation in course of HCV infection.

Serum biomarkers for sepsis in children with febrile neutropenia and cancer.

The National Institute for Health and Care Excellence (NICE) defines febrile neutropenia or "neutropenic sepsis" as a patient with an absolute neutrophil count (ANC) less than 0.5 x 109/L and temperature >38°C or signs and symptoms of sepsis.

A novel biomarker score for the screening and management of patients with plasma cell proliferative disorders.

OBJECTIVE: Monoclonal plasma cell proliferative disorders comprise a wide spectrum of diseases associated to clonal B-cell expansion. Serum protein electrophoretic profile (SPEP) and circulating free light chains (FLCs) levels are the mainstay of diseases management. Recently, soluble (s) Syndecan-1 (SDC1, CD138) produced by myeloma plasma cells has been suggested in the monitoring and follow-up of patients with myeloma. The aim of our study is to evaluate sCD138 in addition with FLCs and SPEP for the screening of patients with different evolutive disease pathways. PATIENTS AND METHODS: Sera from 73 patients with monoclonal gammopathy of undetermined significance (MGUS), 120 smoldering and 42 multiple myeloma (SMM and MM, respectively), 70 HCV-related mixed cryoglobulinemia (MC), 35 B-cell non-Hodgkin's lymphoma (B-NHL) and sera from 50 healthy donors (HD), were tested for sCD138, FLCs (assessed by means of ELISA and turbidimetric assay, respectively) and electrophoresis pattern (performed on Capillarys system) for the generation of a novel biomarker score (BS). RESULTS: Our results were grouped according to the two main lines of disease progression (vs. MM or B-NHL): in one group we found BS mean values of 0.2, 3.4, 5.3, 7.1 for HD, MGUS, SMM and MM, respectively; in the other group of 0.2, 4.4, 6.7 for HD, MC and B-NHL. CONCLUSIONS: We showed that BS mean values follow the ingravescence disease status towards the two main lines of progression to cancerous conditions; it could represent an additional useful tool in the management of screening and/or follow-up.

Free light chains a novel biomarker of cardiovascular disease. A pilot study.

OBJECTIVE: Atherosclerosis and ischemic heart disease (IHD) are the major cause of morbidity and mortality but their inflammatory pathogenesis is still unclear. In this scenario, the role of serum free light chains (sFLC) has never been fully evaluated. The aim of the present study is to assess the clinical and pathogenetic role of sFLC in patients with IHD and to propose their use as a new biomarker for cardiovascular disease. PATIENTS AND METHODS: We enrolled 117 patients, divided into 5 cohorts: 15 healthy controls, non-diabetic and without ischemic heart disease; 19 patients with type 2 diabetes (T2DM), without ischemic heart disease at recruitment; 39 patients with stable chronic angina; 27 patients with NSTEMI, 17 patients with acute STEMI. Serum sFLC and high-sensitive C-reactive protein (hs-CRP) were measured. Patients also underwent a transthoracic echocardiographic study. RESULTS: sFLC were higher in patients with IHD and T2DM. However, we did not find statistically significant differences in sFLC concentration among subgroups. No correlation resulted between sFLC and hs-CRP levels. The median value of the sFLC κ/λ ratio in the population was 0.63, therefore stratifying it into two groups according to their levels. We found that an increase in left ventricular ejection fraction at 12 months was detected in 77% of patients with κ/λ ratio higher than 0.63 and 25% of patients with κ/λ ratio lower of 0.63 (p=0.016, OR=10.0 [1.8-55.6]). CONCLUSIONS: Our study suggests that the sFLC, produced by the B-lymphocytes in the context of generalized immune activation, could play a pathogenetic role in acute coronary syndromes and that they could represent a novel risk biomarker of cardiovascular disease.

IgG cryoglobulinemia.

OBJECTIVE: Mixed Cryoglobulinemia is the most well-known Hepatitis C Virus (HCV)-associated extrahepatic manifestation. MC is both an autoimmune and B-lymphoproliferative disorder. Cryoglobulins (CGs) are classified into three groups according to immunoglobulin (Ig) composition: type I is composed of one isotype or Ig class. Type II and type III mixed CGs are immune complexes composed of polyclonal IgGs acting as autoantigens and mono, polyclonal or oligoclonal IgM with rheumatoid factor activity. IgG1 and IgG3 are the predominant subclasses involved. This study shows the simultaneous presence of IgG-RF and IgG3, supporting the hypothesis of an involvement of this subclass in the initiation of early stages of CGs. PATIENTS AND METHODS: We describe a case series of six HCV-positive patients, all of whom had peripheral neuropathy and transient ischemic attacks, presenting cryoprecipitates formed by IgG3 and IgG1. Cryoprecipitate IgG subclass research was carried out by immunofixation electrophoresis by using antisera against IgG1, IgG2, IgG3, and IgG4. RESULTS: Our six patients presented with an immunochemical pattern characterized by the mere presence of IgG1 and IgG3 subclasses with probable RF activity and one of these six patients exhibited monoclonal IgG3 in his cerebrospinal fluid. CONCLUSIONS: We can hypothesize that the IgG passage through the blood-brain barrier could have contributed to the cause of TIAs, through a mechanism involving the precipitation of circulating immune complexes formed by the two subclasses in the intrathecal vessels.

Circulating endothelial cell levels in psoriatic patients and their modification after an anti-TNF-alpha (Etanercept) treatment.

BACKGROUND: Endothelial function in psoriatic patients has been mainly evaluated through a high-resolution ultrasound measurement of flow-mediated vasodilation in the brachial artery, which is an operator-dependent and technically demanding technique: this characteristic, together with different patient selection criteria, could account for the conflicting results emerging from different studies. Recently, Circulating Endothelial Cells (CECs) level has been suggested as a novel biomarker of vascular injury. METHODS: The number of CECs was determined by a semi-automated immunomagnetic system (CellSearch system) in peripheral blood of psoriatic patients (n = 48) and healthy subjects (n = 50). In 15 patients, CEC level was also evaluated after 6 months of treatment with an anti-TNF-alpha agent, Etanercept. The plasma levels of high-sensitivity C-reactive Protein (CRP), E-selectin, VEGF and PAI-1 were measured by ELISA. The psoriasis severity was assessed by PASI score. RESULTS: A statistically significant difference (P = 0.001) was found between CEC level in psoriatic patients (10.6 ± 9.4 cells/mL) vs. the control group (3.9 ± 0.9 cells/mL). This count inversely correlated with sE-selectin levels (r(2) = 0.16; P = 0.03). After 6 months of therapy, patients experienced a significant (P < 0.05) decrease in CEC levels (3.4 ± 1.3 cells/mL) and in PASI score (from 11.7 ± 8.1 to 2.1 ± 4.0). CONCLUSIONS: The elevated CECs level that we found in a sample of high selected psoriatic patients could be expression of endothelial damage. Lowering of CECs count after treatment with Etanercept support the hypothesis that an effective systemic therapy of psoriasis may also improve the endothelial function.