Krizia Pocino

Description of an Automated Method for Urea Nitrogen Determination in Bronchoalveolar Lavage Fluid (BALF) of Neonates and Infants.

Bronchoalveolar lavage (BAL) partially recovers both the instilled saline and the alveolar fluid, so-called epithelial lining fluid (ELF), but a correction for the dilution due to the BAL technique itself is needed to know the amount of recovered ELF. In this regard, urea nitrogen may be useful and has been proposed to calculate ELF. The aim of the present study was to develop and validate a new method to measure urea nitrogen in BAL fluid (BALF). We used 19 BALF samples obtained from neonates and infants with different respiratory conditions. The urea nitrogen assay was carried out on Cobas c311 analyzer (Roche Diagnostics). A validation study shows that the method is perfectly linear (R2 = 0.999), sensitive (limit of detection = 0.055 mg/dL; limit of quantification = 0.16 mg/dL), repeatable (low = 0.15 ± 0.02, 13.3%; high = 1.80 ± 0.02, 1.1%), reproducible (low = 0.14 ± 0.02, 14.2 %; high = 1.76 ± 0.04, 2.2 %) with accuracy ranging between 93–96%. Our results support the robustness of validated procedure since the described method appears simple, precise, rapid, and suitable for routine analysis. Thus, it may be used to correct concentration of various noncellular BAL components and calculate their ELF amounts in neonates and infants.

Serum free light chain quantitative assays: Dilemma of a biomarker

Serum free light chains detection assays are consistently meeting greater interest for the diagnosis and monitoring of monoclonal gammopathies and plasma cell dyscrasias. Nowadays, there are neither standardized methods nor reference material for the determination of free light chains; for this reason, it is important to compare two different assays used in clinical laboratory.

Heavy chain disease: our experience

Heavy chain disease (HCD) is a rare lymphoproliferative disorder of plasma cells, described for the first time in 1964 by Franklin [1], characterized by production of incomplete monoclonal immunoglobulin heavy chains without associated light chains [2]. HCDs involve the three main immunoglobulin classes, the most common α-HCD, γand μ-HCD. Originally, Franklin’s disease (γ-HCD) was considered to be a lymphoproliferative disease, however, HCDs have variable clinical presentations and different histopathologic features. γ-HCD has been reported to occur equally in men and women, but in a recently reported series there was a clear female predominance [3]. The diagnosis of γ-HCD requires the evidence of a deleted immunoglobulin heavy chain without a bound light chain in the serum or urine [3]. These alterations typically result in loss of a large portion of the constant-1 (CH1) domain of the immunoglobulin heavy chain molecule responsible for light chain binding. In the absence of an associated light chain, the CH1 domain of the normal heavy chain binds to heat-shock protein 78 (hsp78) and undergoes degradation in the proteasome compartment of cells; normal heavy chains unassociated with light chains are therefore never detected in serum or urine. In the HCDs, the altered structure of the CH1 domain prevents the heavy chain from binding both the light chain and hsp78, thereby allowing it to bypass degradation by the proteasome and be secreted into the serum or urine [4]. In addition, the altered heavy chain may function as part of the transmembrane B-cell receptor and facilitate antigen-independent aggregation and downstream signaling by the receptor, thereby conferring a growth advantage to the heavy-chain expressing cells [5]. The clinical course of γ-HCD is extremely variable and ranges from an asymptomatic benign, or stable process to a rapidly progressive neoplasm leading to mortality within a few weeks. Prognosis is variable and the mean survival time has been reported to be 7.4 years (range, 1 month to >21 years) [3, 6]. We describe three cases of γ-HCD associated with chronic lymphocytic leukemia, peripheral T-cell lymphoma, not otherwise specified and small cell lung carcinoma, respectively. All three patients were male. The Ethics Committee of the “Policlinico Universitario A. Gemelli, Roma” approved this study. The first case is a 75-year-old man, who was admitted to the hospital for a 2-months history of progressive worsening of neurological symptoms, i.e. vision changes, dysphagia and amnesia. He had surgery for adenocarcinoma of the rectum 6 years before, with local recurrence after 5 years. In the last hospital stay before admission, he was treated with radiotherapy and chemotherapy with 5-fluorouracil. Total-body computed tomography scanning was negative for local, nodal or other organ involvement. Franklin’s disease was diagnosed 2 years before with no underlying lymphoproliferative or autoimmune disorder. Laboratory tests, including blood leukocyte count and CD4+ to CD8+ cells ratio, were within normal range, but a severe deficiency of serum IgA (0.39 g/L, 0.70–4.00) and IgM (<0.19 g/L, 0.40–2.30) concentrations and a mild IgG deficiency (6.45 g/L, 7.00–16.00) were found. Serum electrophoresis was negative but serum immunofixation electrophoresis (IFE) showed two γ-heavy chain monoclonal components (MCs) migrating in β, not associated with corresponding light chain, and one MC in the γ-region (small MC IgMλ); urine IFE interestingly showed a typical pattern with a broad band of incomplete γ-heavy chain (Figure 1) [7]. *Corresponding author: Umberto Basile, School of Medicine, Department of Laboratory Medicine, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy, E-mail: umberto.basile@policlinicogemelli.it. http://orcid.org/0000-0002-8328-2570 Francesca Gulli, Cecilia Napodano and Krizia Pocino: Department of Laboratory Medicine, Catholic University of the Sacred Heart, Rome, Italy Annarosa Cuccaro and Stefan Hohaus: Institute of Hematology, Catholic University of the Sacred Heart, Rome, Italy

Analytical assessment of bone serum markers in patients suffering from spina bifida

Neural tube defects are serious abnormalities that occur early in embryonic development and result from failure of the neural tube to close properly in the developing brain and/or lower spine. These defects include anencephaly, myelomeningocele (MMC) (commonly called spina bifida [SB]) and encephalocele. In SB patients there is also a spectrum of impairments, such as lower limb paralysis and sensory loss, bladder and bowel dysfunction, cognitive damaging and is often associated with Arnold-Chiari type II malformation; hydrocephalus; and are at increased risk for fractures [1, 2]. In the US, each year, about 1500 babies are born with SB and across Europe, and an estimated 4500 pregnancies are affected by NTDs [3, 4]. Etiology is multifactorial and is the result of a combination of genetic and environmental factors, such as use of certain anticonvulsants, maternal diabetes, and maternal folic acid deficiency. The aim of the present study is to assess bone formation and resorption in patients with SB and risk factors related to this pathology. Since the regulation of plasma calcium levels is crucial for neural transmission, normal cell function, bone structure and intracellular signaling, and due to the interactions among parathyroid glands, thyroid gland and vitamin D, we planned to evaluate blood calcium and phosphorus content along with: 1) parathyroid hormone (PTH), that increases bone resorption and the release of plasma Ca2+ into the circulation and stimulates the hydroxylation of vitamin D; 2) osteocalcin, which is a marker of skeletal bone turnover; 3) vitamin D, that regulates bone resorption and formation and suppresses the synthesis and release of PTH, also acting as an important mediator of innate and adaptive immune responses. The awareness of a role for vitamin D in the regulation of immune system was triggered by the discovery of vitamin D receptors (VDRs) in almost all immune cells. Moreover, it inhibits the expression of inflammatory cytokines in monocytes [5]; thyroid stimulating hormone (TSH), triiodothyronine (FT3) and tetraiodothyronine (FT4) essential for bone growth and development through activation of osteoclast and osteoblast activities. For these reasons, Vitamin D status could be very important as factor reducing co-morbidities in SB patients. In the Spina Bifida Centre of Catholic University, Rome, a team of physicians collected detailed medical history and performed a careful clinical examination. Medullar and brain magnetic resonance imaging were acquired. Written informed consent was obtained from each subject before inclusion. The study included 78 patients, 33 male and 45 female, aged 1–29 years, and 34 healthy subjects, 13 male and 21 female, aged 1–31 years. Patients were grouped on the basis of the type of lesions, as follows: 1) not ulcerated MMC; 2) ulcerated MMC; and 3) lipomeningomyelocele (LMC). Moreover, the presence of hydrocephalous and Arnold-Chiari II syndrome was pointed out. Neurological impairment was classified in three categories: patients having dorsal, lumbosacral, and sacral involvement. The ambulatory status of patients was clustered into three groups, defined as follows: 1) Individuals who did not *Corresponding author: Dr. Krizia Pocino, Institute of Biochemistry and Clinical Biochemistry, Largo A. Gemelli 8, 00168 Rome, Italy, E-mail: krizia.pocino@gmail.com Claudia Rendeli, Emanuele Ausili and Valentina Paolucci: Spina Bifida Center, Department of Pediatrics, Policlinico Universitario A. Gemelli, Rome, Italy Costantino Romagnoli: Division of Neonatology, Department of Pediatrics, Policlinico Universitario A. Gemelli, Rome, Italy Cinzia Carrozza and Ettore Capoluongo: Institute of Biochemistry and Clinical Biochemistry, Policlinico Universitario A. Gemelli, Rome, Italy

Analytical evaluation of a new liquid immunoturbidimetric assay for the determination of ferritin in serum

*Corresponding author: Dr. Rossana Molinario, Institute of Biochemistry and Clinical Biochemistry, Catholic University of the Sacred Heart, Largo A. Gemelli 8, 00168 Rome, Italy, Phone: +39 0630154250, Fax: +39 0630156783, E-mail: rossanamolinario@libero.it Chiara Autilio, Krizia Pocino, Pio Dante Daloiso, Salvatore Di Leva, Cecilia Zuppi and Mirca Antenucci: Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy Letter to the Editor

Free light chains of immunoglobulins in patients with systemic sclerosis: correlations with lung involvement and inflammatory milieu

Aim Humoral immunity and B cells are thought to play an important role in the pathophysiology of the systemic sclerosis (SSc). The production of free light chains (FLC) of immunoglobulins is abnormally high in several pathological autoimmune conditions and reflects B cell activation. Furthermore, FLCs demonstrated different biological activities including their capability to modulate the immune system, proteolytic activity and complement cascade activation. The aims of this study are to determine the FLC levels in patients with SSc compared with healthy controls (HC) and to study their possible association with organ involvement and disease characteristics. Methods Sixty-five patients with SSc and 20 HC were studied. Clinical and immunological inflammatory characteristics were assessed for all the patients with SSc. κ-FLC and λ-FLC, interleukin 6 (IL-6) and B cell activating factor levels were measured. Results The mean serum κ-FLC levels and FLC ratio were significantly higher in patients with SSc compared with HC, while the serum λ-FLC levels were comparable. The levels of FLC were comparable in patients with diffuse skin disease and limited skin involvement, while κ-FLC levels were increased in patients with restrictive lung (forced vital capacity (FVC) <80%) disease (26.4±7.4 mg/L) when compared with patients with FVC ≥80% (19.6±7.3 mg/L, P=0.009). In patients with SSc, the levels of serum κ-FLC level directly correlated with the IL-6 levels (R=0.3, P=0.001) and disease activity (R=0.4, P=0.003). Conclusions FLC levels are elevated in SSc and high levels are associated with lung involvement and with a higher degree of inflammation, supporting a possible role of B cell activation in the pathophysiology of the disease.

Different biochemical patterns in type II and type III mixed cryoglobulinemia in HCV positive patients

BACKGROUND Reversible cryoprecipitability of proteins is observed as a concomitant feature of immune complex formation. Mixed cryoglobulinemia (MC) is systemic vasculitis, associated with mixed IgM and IgG cryoglobulins (CGs) showing rheumatoid factor (RF) activity. It is frequently associated with hepatitis C virus (HCV). This study investigates the presence of IgG RF and anti-nuclear antibodies (ANA) in cryoprecipitates of patients with type III and type II MC, to understand the biochemical patterns associated with different types of MC to a greater degree. METHODS Sera from 70 HCV untreated patients with type III or type II MC were tested by immunofixation for IgG3 and through ELISA for IgG RF. Cryoprecipitates were analysed for ANA by indirect immunofluorescence to identify specific patterns. RESULTS After stratification according to MC type, the ANA patterns between type II and type III MC were statistically different. IgG3 levels and IgG-RF positivity were significantly higher in type III cryoprecipitate. We observed a higher positivity of IgG3 and a significant difference between the liver fibrosis stage, ANA and IgG-RF in the cryoprecipitate. CONCLUSION Results show a combination of biochemical markers and autoantibodies associated to mixed cryoglobulinemia; these findings could be further investigated in order to ascertain their usefulness in assessing the risk for the development of mixed cryoglobulinemia.

Serological Immunoglobulin-Free Light Chain Profile in Myasthenia Gravis Patients

Background Serological levels of free immunoglobulin light chains (FLCs), produced in excess of heavy chains during synthesis of immunoglobulins by plasma cells, can be considered a direct marker of B cell activity in different systemic inflammatory-autoimmune conditions and may represent a useful predictor of rituximab (RTX) therapeutic efficacy, as reported for rheumatoid arthritis and systemic lupus erythematosus. Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction with antibodies (abs) targeting the acetylcholine receptor (AChR) or the muscle-specific tyrosine kinase (MuSK), inducing muscle weakness and excessive fatigability. As MG course may be remarkably variable, we evaluated the possible use of FLCs as biomarkers of disease activity. Subjects and Methods We assessed FLC levels in 34 sera from 17 AChR-MG and from 13 MuSK-MG patients, in comparison with 20 sera from patients with systemic autoimmune rheumatic diseases and 18 from healthy blood donors, along with titers of specific auto-abs and IgG subclass distribution. Results We found a statistically significant increase in free κ chains in both AChR- and MuSK-MG patients, while free λ chain levels were increased only in AChR-MG. We also observed a significant reduction of both free κ and λ chains in 1/4 MuSK-MG patients along with specific abs titer, two months after RTX treatment. Conclusions From our data, FLCs appear to be a sensitive marker of B cell activation in MG. Further investigations are necessary to exploit their potential as reliable biomarkers of disease activity.

Plasmatic free light chains as inflammatory marker in insulin resistance: comparison of metabolic syndrome with adult growth hormone deficiency: Plasmatic free light chains as inflammatory marker in insulin resistance

Biological functions of immunoglobulin-free light chains (FLCs), other than in chronic inflammatory diseases, are still poorly defined; the field of insulin resistance (IR) has not been investigated, despite the strict relationships with oxidative stress (OS) and inflammation. Therefore, we evaluated FLCs levels and their relationships with metabolic parameters in adult growth hormone deficiency (GHD) and metabolic syndrome (MetS), both characterized by IR. One hundred subjects were enrolled: group A, patients with GHD [n =31, 24-69 years, mean ± SEM body mass index (BMI) 26.8 ± 1.5 kg/m2 ]; group B, patients with MetS (n = 29, 21-70 years, BMI 31.9 ± 1.3); group C, controls (N = 40, 21-62 years, BMI 21.6 ± 1.1). Groups were matched by age range and, for patients, by BMI. Morning blood sample was collected for metabolic parameters and FLCs, assessed by turbidimetric assay. GHD patients show levels of FLCs significantly higher than MetS and controls (mean ± SEM κ 37.21 ± 6.97, 15.27 ± 0.86, 12.34 ± 0.85 mg/l; λ 19.44 ± 2.61, 11.78 ± 0.72 and 11.67 ± 0.77 mg/l; κ/λ ratio 1.77 ± 0.13, 1.38 ± 0.09; and 1.10 ± 0.06, respectively); only κ were higher in MetS versus controls. Therefore, the ratio showed progressive declining values in GHD versus MetS versus controls. Our data show increased FLCs levels in GHD and MetS, with the highest values in the former. Both conditions show OS, but with different molecular patterns. FLCs may contribute to chronic inflammation, leading to OS, and cardiovascular complications of GHD. Prognostic and therapeutic implications require further investigation.

PAX3d mRNA over 2.76 copies/μL in the bloodstream predicts cutaneous malignant melanoma relapse

Objective The aim of this study was to evaluate if our molecular algorithm, based on tumor circulating transcripts, may predict relapse risk in cutaneous malignant melanoma (CMM). Results The multi-marker panel was able to differentiate patients with CMM from HC with high diagnostic sensitivity and specificity, especially for MITF-m and TGFB2 (91–100%) whose levels decreased during follow-up of recurrence-free patients, and remained stable in the case of relapse. PAX3d higher than 2.76 copies/µL emerged as a promising biomarker [specificity = 75–93% and negative predictive value = 75–98%] to stratify subjects at high risk of CMM recurrence independently of age, gender and AJCC staging [OD = 9.5(3.2–28.0), p < 0.001]. The survival analysis confirmed PAX3d performance in relapse prediction with significant differences in recurrence risk 12 months after the basal time-point (p = 0.008). Materials and Methods Peripheral blood was collected from 111 CMM patients and from 87 healthy controls (HC) randomly selected. Each specimen was examined by qRT-PCR analysis for the expression of 3 tumor-related transcripts (PAX3d, MITF-m and TGFB2) at diagnosis, and at the following 6 and 12 months during clinical monitoring. Conclusions We demonstrated the usefulness of our molecular algorithm to indirectly detect circulating melanoma cells in blood, along with PAX3d capability to assess patients’ progression and relapse prediction.