Diagnostic specificity of autoantibodies to M-type phospholipase A2 receptor (PLA2R) in differentiating idiopathic membranous nephropathy (IMN) from secondary forms and other glomerular diseases.

Autoantibody against phospholipase A2 receptor (anti-PLA2R) is a sensitive and specific biomarker of idiopathic membranous nephropathy (iMN), being found in approximately 70% of iMN patients and only occasionally in other glomerular diseases. However, whereas its diagnostic specificity vs. normal controls and other glomerulonephritides (GN) has been firmly established, its specificity vs. membranous nephropathy associated with various diseases (sMN) has given inconsistent results. The aim of our study was to evaluate the prevalence of anti-PLA2R antibodies in iMN in comparison with various control groups, including sMN. A total of 252 consecutive iMN patients, 184 pathological and 43 healthy controls were tested for anti-PLA2R antibody using indirect immunofluorescence (PLA2R IIFT, Euroimmun). Anti-PLA2R autoantibodies were detectable in 178/252 iMN patients, 1/80 primary GN, 0/72 secondary GN, 9/32 sMN and 0/43 healthy controls, with a diagnostic sensitivity of 70.6%. The diagnostic specificity of anti-PLA2R antibody vs. normal and pathological controls was 100 and 94.6% respectively. However, when the diagnostic specificity was calculated only vs. secondary forms of MN, it decreased considerably to 71.9%. Interestingly enough, 9 out of 10 anti-PLA2R positive patients in the disease control groups had membranous nephropathy associated with various diseases (7 cancer, 1 Crohn's disease, 1 scleroderma). In conclusion, anti-PLA2R positivity in a patient with MN, should not be considered sufficient to abstain from seeking a secondary cause, especially in patients with risk factors for neoplasia. The causal relationship between tumors and anti-PLA2R-induced MN remains to be established, as well as the possible mechanisms through which malignancies provoke autoimmunity.

Obstetric and vascular APS: same autoantibodies but different diseases?

Beta2 glycoprotein I (ß2GPI)-dependent antiphospholipid antibodies (aPLs) are the main pathogenic autoantibody population and at the same time the laboratory diagnostic tool for the antiphospholipid syndrome (APS). These antibodies are responsible for both the vascular and the obstetric manifestations of the syndrome but the pathogenic mechanisms behind these manifestations are not the same. For example, thrombotic events do not appear to play a major role in APS miscarriages and a direct reactivity of ß2GPI-dependent aPLs on decidual and trophoblast cells was reported. A local expression of ß2GPI on these tissues was reported both in physiological conditions and in APS women, thus explaining the local tropism of the autoantibodies. The two hit hypothesis was suggested to explain why the vascular manifestations of APS may occur only occasionally in spite of the persistent presence of aPLs. This is not apparently the case for the obstetric variant of the syndrome, making the difference even more striking. A different pathogenesis may also provide the rationale for the well-known fact that the vascular and the obstetric manifestations may occur independently although in a minority of cases.

[Primary antiphospholipid syndrome evolving into systemic lupus erythematosus: may antinucleosome antibodies be predictive?]. / L'evoluzione da sindrome da anticorpi antifosfolipidi primaria a lupus eritematoso sistemico: possono gli anticorpi anti-nucleosomi essere predittivi?

OBJECTIVE: It was reported by several groups that patients diagnosed as primary antiphospholipid syndrome (PAPS) had developed a full-blown systemic lupus erythematosus (SLE) even after many years of follow-up. Little is known about clinical and/or serological factors that may help predict such evolution. Antinucleosome antibodies (anti-NCS) were described to appear in early stages of SLE, in particular before anti-dsDNA antibodies. The aim of the study is to evaluate the prevalence of anti-NCS in a large cohort of PAPS patients. METHODS: IgG and IgM anti-NCS antibodies were detected using a home made assay with H1-stripped chromatin as antigen. Sera from 106 PAPS patients were tested; 52 of them were also tested during the follow-up, at least 2 years apart form the basal sample. RESULTS: Medium-high titre anti-NCS were found in nearly half of the patients (49/106, 46%), more frequently in those presenting features of "lupus like disease". Most of patients displayed an unchanged pattern of anti-NCS over time. We describe three cases of PAPS patients that developed SLE after many years of follow-up; high titre and low titre anti-NCS were present in two and one of them respectively several years before evolving into SLE. CONCLUSIONS: A significant proportion of PAPS patients displayed medium-high titre anti-NCS, suggesting that the autoimmune response against chromatin may be a relevant event not only in patients with SLE. Further studies are warranted to explore the predictive value of anti-NCS with respect to the evolution from PAPS to SLE.

Antiprothrombin antibodies: a comparative analysis of homemade and commercial methods. A collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni (FIRMA).

OBJECTIVE: Prothrombin (PT) is a target for antibodies with lupus anticoagulant (LA) activity, suggesting the possible application of anti-prothrombin antibody (aPT) assays in patients with antiphospholipid syndrome (APS). Different methods - both homemade and commercial - for the detection of aPT are available, but they seem to produce conflicting results. The purpose of this study was to compare the performance of different assays on a set of well-characterized serum samples. PATIENTS AND METHODS: Sera were gathered from 4 FIRMA institutions, and distributed to 15 participating centres. Forty-five samples were from patients positive for LA and/or anticardiolipin antibodies (aCL) with or without APS, and 15 were from rheumatoid arthritis (RA) patients negative for antiphospholipid antibodies. The samples were evaluated for IgG and IgM antibodies using a homemade direct aPT assay (method 1), a homemade phosphatidylserine-dependent aPT assay (aPS/PT, method 2), and two different commercial kits (methods 3 and 4). In addition, a commercial kit for the detection of IgG-A-M aPT (method 5) was used. RESULTS: Inter-laboratory results for the 5 methods were not always comparable when different methods were used. Good inter-assay concordance was found for IgG antibodies evaluated using methods 1, 3, and 4 (Cohen k > 0.4), while the IgM results were discordant between assays. In patients with thrombosis and pregnancy losses, method 5 performed better than the others. CONCLUSION: While aPT and aPS/PT assays could be of interest from a clinical perspective, their routine performance cannot yet be recommended because of problems connected with the reproducibility and interpretation of the results.

Evaluation of the influence of open and closed-ampoule technologies on particulate matter in small-volume parenterals.

SVP in glass ampoules are manufactured using two main different technological production processes: the open-ampoule process (O) and the closed-ampoule process (C). In principle, the open-ampoule production technology should lead to better controlled production process. To test this hypothesis and quantify the possible qualitative differences in the manufactured ampoules, a suitable experimental design was set up. The two ampoule production processes have been compared on the basis of the visible particulate burden. Two batches of ampoules filled with water for injections were produced for each type of process, following conventional industrial procedures. Two samples of 20,000 units were taken from each batch and inspected with different automatic inspection systems: two Brevetti CEA machines (S1, S2--light scattering) and two EISAI machines (S3, S4--light absorbtion). The comparison between the processes was based on the rejection percentage. On both inspection machines the open-ampoule production samples present rejection percentages (ranging from 0.154% to 1.248% rejection percentages) which, on average, are lower than those detected in closed-ampoule production (ranging from 1.434% to 3.86% rejection percentages). The difference between the two processes is even more marked if we also consider the data obtained using inspection machines S3 and S4. The substantial differences in performance of the four inspection machines stress the need to provide for adequate validation procedures.

Water treatment systems for pharmaceutical purposes.

Water is the key component in pharmaceutical production and in related activities. Different types of water having different standards are used in pharmaceutical production plants. An accurate definition of the uses, of the chemical-physical and microbiological specifications, of sampling and testing programs is needed for the correct design and management of water production and distribution systems. The types of water taken into consideration are purified water and water for injection, being the most critical ones. The water production systems are described analog with the principles of function and the instrumental controls specifying the key features which guarantee the high standards of chemical-physical and biological quality of the water produced. A trouble-free use of water in the plant is related to the holding and distribution system. An overview view of the design criteria and management of such system is considered. Description of the aspects regarding the validation and monitoring of water treatment systems is given.

Molecular species analysis of phospholipids by negative ion fast atom bombardment mass spectrometry: application of surface precipitation technique.

Eight phospholipidic classes from bovine brain cortex and soybean were prepared and purified by preparative high-performance and liquid chromatography, and their molecular species were identified by negative ion fast atom bombardment mass spectrometry using the 'surface precipitation' method. Its main advantages are: (i) clear-cut and abundant diagnostic ions for structural elucidation of the species; (ii) fragments characteristic of the main fatty acids, the polar head-group and the molecule frame in phospholipids; (iii) less background caused by the liquid matrix.

Structural properties of hyaluronic acid in moderately concentrated solutions.

The solution properties of hyaluronic acid having various d.p. values have been examined at physiological pH and ionic strength by light-scattering techniques at various concentrations. The angular dependence of Kc/R0 was linear at low concentrations, whereas substantial curvature was present at small angles on increasing the concentration. This phenomenon was affected by the conditions of centrifugation. Moreover, the function Kc/R0 exhibited a maximum value at concentrations of polymer inversely related to the molecular weight. The maximum concentration was always higher than the critical concentration. Ageing, molecular-weight distribution, and polymer purity influenced the light-scattering response. A model is proposed in which the polysaccharide chains intertwine at relatively low concentrations of polymer and become entangled at high concentration, eventually leading to a non-homogeneous polymer network.