Reference intervals for thyrotropin in an area of Northern Italy: the Pordenone thyroid study (TRIPP).

PURPOSE: Thyrotropin (TSH) is the most accurate marker of thyroid dysfunction in the absence of pituitary or hypothalamic disease. Studies on TSH reference intervals (RIs) showed wide inter-individual variability and prompted an intense debate about the best estimation of TSH RIs. DESIGN: We performed a population study on TSH RIs, using current data stored in the laboratory information system (LIS), at the Hospital Department of Laboratory Medicine, Pordenone (Italy), historically an area of mild-moderate iodine deficiency with a relatively high goiter prevalence. METHODS: 136,650 individuals constituted the final sample. A TSH immunoassay was performed on fasting serum samples with the Dimension Vista 1500 analyzer (Siemens Healthineers). We adopted the Kairisto's procedure to analyze TSH data downloaded by the LIS, applying the indirect strategy for deriving RIs. RESULTS: TSH RIs of the entire population were 0.32-3.36 mIU/L with a distribution skewed towards higher values. RIs were 0.26-3.61 mIU/L for females, and 0.32-3.01 mIU/L for males. Unlike other studies, TSH median levels progressively decreased from 0-4 to 85-104 years in the overall population, both in male and in female subgroups, showing an inverse correlation between TSH and age in all groups. CONCLUSIONS: This study is the first to analyze a high percentage (40%) of individuals from an ethnically homogenous Caucasian population. The results obtained emphasize the opportunity to define the TSH RIs according to age, gender and race, in addition to assay methods, and provide further insight about the possible role of iodine status.

Pathogenic Escherichia coli and enteric viruses in biosolids and related top soil improvers in Italy.

AIMS: To investigate the presence of genomic traits associated with a set of enteric viruses as well as pathogenic Escherichia coli in top soil improvers (TSI) from Italy. METHODS AND RESULTS: Twenty-four TSI samples originating from municipal sewage sludges, pig manure, green and household wastes were analysed by real time PCR for the presence of hepatitis E virus (HEV), porcine and human adenovirus (HuAdV), norovirus, rotavirus and diarrhoeagenic E. coli. None of the samples was found positive for HEV or rotavirus. Four samples were positive for the presence of nucleic acids from human norovirus, two of them being also positive for HuAdV. Real time PCR screening gave positive results for many of the virulence genes characteristic of diarrhoeagenic E. coli in 21 samples. These included the verocytotoxin-coding genes, in some cases associated with intimin-coding gene, and markers of enteroaggregative, enterotoxigenic and enteroinvasive E. coli. CONCLUSIONS: These results provide evidence that enteric viruses and pathogenic E. coli may be released into the environment through the use of sludge-derived TSI. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlight that the TSI-related environmental risk for the food chain should be more deeply assessed.

Image-guided fine-needle aspiration cytology and flow cytometry phenotyping of neck lymphadenopathy for the diagnosis of recurrent lymphoma.

OBJECTIVE: In patients with a history of lymphoma, each lymphadenopathy should be carefully evaluated. The aims of this study were to evaluate (i) the usefulness of high-resolution ultrasonography (US), US-guided fine-needle aspiration cytology (FNAC) and flow cytometry phenotyping (FCP) together in the diagnosis of recurrent lymphoma and (ii) whether these tools were independent predictors of correct results. DESIGN: Retrospective cohort study with stepwise forward logistic regression analysis of results. SETTING: Tertiary referral centre. PARTICIPANTS: A total of 151 patients with a history of lymphoma who developed a cervical mass during follow-up. METHODS: On neck US, a lymphadenopathy was shown in 129 (85.4%) patients (median age 57 years, range 18-78 years), and US-guided FNAC combined with FCP were immediately performed. All patients had surgical excision and subsequent histological examination of the enlarged node(s), to establish lymphoma subclassification. RESULTS: Final histology confirmed recurrence in 82 (63.6%) patients. According to the logistic regression analysis, FNAC and FCP were independent predictors of correct results (P = 0.009 and 0.028, respectively) and did not interfere with each other. The sensitivity, specificity and accuracy of the combination of all of the tools were 98.8%, 100% and 99.2%, respectively, and the area under the receiver operating characteristic curve was 0.902 (95% CI: 0.797-0.986). CONCLUSION: This minimally invasive procedure is easily performed and should be recommended for all patients with cervical lymphadenopathy and a history of lymphoma, avoiding the need of core-biopsy or surgical excision if recurrence was excluded.

Short communication: Isolation of Shiga toxin-producing Escherichia coli in raw milk and mozzarella cheese in southern Italy.

Shiga toxin-producing Escherichia coli (STEC) are a significant food-borne public health hazard in Europe, where most human infections are associated with 5 serogroups (O157, O26, O103, O145, and O111). In 2015, 95 food and environmental samples were examined for the presence of Shiga toxin genes (stx1 and stx2). The STEC were isolated from 2 raw milk and 1 mozzarella cheese samples that were collected in the period between June and September. To the best of our knowledge, this finding represents the first report of STEC isolation from mozzarella cheese produced in Italy, and it suggests that both the quality of raw milk used to produce mozzarella and the thermal inactivation treatment associated with the curd-stretching step should be carefully monitored.

A severe foodborne outbreak of diarrhoea linked to a canteen in Italy caused by enteroinvasive Escherichia coli, an uncommon agent.

We describe a foodborne outbreak in Italy caused by enteroinvasive Escherichia coli (EIEC), an enteric pathogen uncommon in industrialized countries. On 14 April 2012 a number of employees of the city of Milan Fire Brigade (FB) were admitted to hospital with severe diarrhoea after attending their canteen. Thirty-two patients were hospitalized and a total of 109 cases were identified. A case-control study conducted on 83 cases and 32 controls attending the canteen without having symptoms identified cooked vegetables to be significantly associated with the disease. Stool samples collected from 62 subjects were screened for enteric pathogens using PCR-based commercial kits: 17 cases and two asymptomatic kitchen-workers were positive for the Shigella marker gene ipaH; an ipaH-positive EIEC strain O96:H19 was isolated from six cases. EIEC may cause serious dysentery-like outbreaks even in Western European countries. Microbiologists should be aware of microbiological procedures to detect EIEC, to be applied especially when no common enteric pathogens are identified.

Use of virulence determinants and seropathotypes to distinguish high- and low-risk Escherichia coli O157 and non-O157 isolates from Europe.

The presence of 10 virulence genes was examined using polymerase chain reaction (PCR) in 365 European O157 and non-O157 Escherichia coli isolates associated with verotoxin production. Strain-specific PCR data were analysed using hierarchical clustering. The resulting dendrogram clearly separated O157 from non-O157 strains. The former clustered typical high-risk seropathotype (SPT) A strains from all regions, including Sweden and Spain, which were homogenous by Cramer's V statistic, and strains with less typical O157 features mostly from Hungary. The non-O157 strains divided into a high-risk SPTB harbouring O26, O111 and O103 strains, a group pathogenic to pigs, and a group with few virulence genes other than for verotoxin. The data demonstrate SPT designation and selected PCR separated verotoxigenic E. coli of high and low risk to humans; although more virulence genes or pulsed-field gel electrophoresis will need to be included to separate high-risk strains further for epidemiological tracing.

Plasmonic Metasurfaces for Specific SERS Detection of Shiga Toxins.

The interest in the development of nanoscale plasmonic technologies has dramatically increased in recent years. The photonic properties of plasmonic nanopatterns can be controlled and tuned via their size, shape, or the arrangement of their constituents. In this work, we propose a 2D hybrid metallic polymeric nanostructure based on the octupolar framework with enhanced sensing property. We analyze its plasmonic features both numerically and experimentally, demonstrating the higher values of their relevant figures of merit: we estimated a surface-enhanced Raman spectroscopy (SERS) enhancement factor of 9 × 107 and a SPR bulk sensitivity of 430 nm/RIU. In addition, our nanostructure exhibits a dual resonance in the visible and near-infrared region, enabling our system toward multispectral plasmonic analysis. Finally, we illustrate our design engineering strategy as enabled by electron beam lithography by the outstanding performance of a SERS-based biosensor that targets the Shiga toxin 2a, a clinically relevant bacterial toxin. To the best of our knowledge, this is the first time that a SERS fingerprint of this toxin has been evidenced.

Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011.

The Escherichia coli strain causing a large outbreak of haemolytic uraemic syndrome and bloody diarrhoea in Germany in May and June 2011 possesses an unusual combination of pathogenic features typical of enteroaggregative E. coli together with the capacity to produce Shiga toxin. Through rapid national and international exchange of information and strains the known occurrence in humans was quickly assessed.We describe simple diagnostic screening tools to detect the outbreak strain in clinical specimens and a novel real-time PCR for its detection in foods.

Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsDNA antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test.

The aim of this study was to evaluate the diagnostic performance of four new enzyme immunoassays (EIAs) for anti-double-stranded-DNA (anti-dsDNA) antibodies, in comparison with the Farr assay and the Crithidia luciliae immunofluorescence test (CLIFT). To this purpose, sera from four patient groups were collected: 52 sera from patients with systemic lupus erythematosus (SLE); 28 from patients with other connective tissue diseases (CTD); 36 from patients with hepatitis C virus (HCV) infection; and 24 from those with acute viral infection. All sera were tested for anti-dsDNA antibodies by four EIA methods using a different antigenic DNA source [synthetic oligonucleotide (Method A), circular plasmid (Method B), recombinant (Method C), and purified extracted (Method D)], and by CLIFT and Farr assays. The diagnostic sensitivity of the assays was as follows: 84.6% (Method A), 73% (B), 82.7% (C), 84.6% (D), 55.8% (CLIFT), and 78.8% (Farr). Specificity was 82.9% (A), 97.7% (B), 96.5% (C), 94.3% (D), 96.5% (CLIFT), and 90.9% (Farr). From these data, we can conclude that the new-generation EIA methods evaluated in this study have higher sensitivity than the CLIFT and Farr assays and, with the exception of Method A, have specificity similar to the CLIFT and slightly higher than the Farr assay. These findings suggest that EIA tests may replace CLIFT as a screening test and the Farr assay as a specific test, for anti-dsDNA antibody detection.

Anti-prothrombin antibodies predict thrombosis in patients with systemic lupus erythematosus: a 15-year longitudinal study.

OBJECTIVE: To evaluate the role of anti-prothrombin (anti-PT) antibodies in predicting thrombosis in patients with systemic lupus erythematosus (SLE). METHODS: An inception cohort of 101 SLE patients (12 males, 89 females; mean age 30 +/- 8 years), was considered. Clinical and laboratory evaluations were regularly performed during a 15-year follow-up (median 108 months) with a special focus on thromboembolic events. Serum samples were collected at time of diagnosis and at least once a year thereafter. IgG and IgM anti-PT, anti-cardiolipin (aCL) and anti-beta(2)glycoprotein I (beta(2)GPI) antibodies were measured by enzyme-linked immunosorbent assay (ELISA); lupus anticoagulant (LAC) was assayed by the dilute Russell's viper venom time and activated partial thromboplastin time tests. The analytical specificity of anti-PT ELISA was investigated. The timing of thrombosis occurrence was calculated using the Kaplan-Meier method. RESULTS: In the 15-year follow-up, thrombosis occurred in 14 out of the 101 patients: venous thrombosis in nine cases and arterial thrombosis in five. IgG and/or IgM anti-PT, anti-beta(2)GPI and aCL antibodies, and LAC activity were detected in ten, nine, seven, and nine cases, with sensitivity for thrombosis of 71.4%, 64.3%, 50% and 64.3%, respectively. Thrombosis-free survival was 90% at 5 years and 85.8% at 10 and 15 years, respectively. Thrombosis was predicted by anti-PT (P = 0.001), anti-beta(2)GPI antibodies (P = 0.002) and LAC activity (P = 0.001). Moreover, the risk of thrombosis progressively increased with the number of positive antiphospholipid antibody tests. The presence of four positive antibody tests was associated with a risk of thrombosis thirtyfold higher than in their absence. CONCLUSIONS: This longitudinal study shows that IgG anti-PT antibodies are predictors of thrombosis in SLE patients.

Validation of a new immunoenzymatic method to detect antibodies to RNA polymerase III in systemic sclerosis.

OBJECTIVE: To test the reliability of a new enzyme-linked immunosorbent assay (ELISA) to identify anti-RNA polymerase III (RNAP III) positive sera from Italian patients with Systemic Sclerosis (SSc) and other chronic inflammatory disorders. METHODS: A comparison between the new ELISA for anti-RNAP III and the gold standard technique, immunoprecipitation (IP), was first performed on 106 SSc patients, 16 patients with other connective tissue diseases and 10 healthy subjects. A further ELISA evaluation was performed on 224 SSc patients, 120 subjects with other rheumatic or infectious diseases, and 81 healthy controls. RESULTS: Plotting ELISA and IP data in a Receiver Operator Characteristic curve, the ELISA cut-off value providing the best specificity (99.1%) and sensibility (100%) was 28 U/ml (AUC=0.999; p<0.0001). Using this cut-off in the second analysis, anti-RNAP III positive results were found in 41 (18.3%) SSc patients, all negative for anticentromere or anti-topoisomerase I antibodies, while only 3 subjects tested positive among the 120 sera collected from other patients. All the healthy subjects were negative. CONCLUSION: This new ELISA for anti-RNAP III is highly accurate when a proper cut-off value is employed and represents a valid substitute to IP in a clinical setting.

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.

Definition of the upper reference limit for thyroglobulin antibodies according to the National Academy of Clinical Biochemistry guidelines: comparison of eleven different automated methods.

PURPOSE: In the last two decades, thyroglobulin autoantibodies (TgAb) measurement has progressively switched from marker of thyroid autoimmunity to test associated with thyroglobulin (Tg) to verify the presence or absence of TgAb interference in the follow-up of patients with differentiated thyroid cancer. Of note, TgAb measurement is cumbersome: despite standardization against the International Reference Preparation MRC 65/93, several studies demonstrated high inter-method variability and wide variation in limits of detection and in reference intervals. Taking into account the above considerations, the main aim of the present study was the determination of TgAb upper reference limit (URL), according to the National Academy of Clinical Biochemistry guidelines, through the comparison of eleven commercial automated immunoassay platforms. METHODS: The sera of 120 healthy males, selected from a population survey in the province of Verona, Italy, were tested for TgAb concentration using eleven IMA applied on as many automated analyzers: AIA-2000 (AIA) and AIA-CL2400 (CL2), Tosoh Bioscience; Architect (ARC), Abbott Diagnostics; Advia Centaur XP (CEN) and Immulite 2000 XPi (IMM), Siemens Healthineers; Cobas 6000 (COB), Roche Diagnostics; Kryptor (KRY), Thermo Fisher Scientific BRAHMS, Liaison XL (LIA), Diasorin; Lumipulse G (LUM), Fujirebio; Maglumi 2000 Plus (MAG), Snibe and Phadia 250 (PHA), Phadia AB, Thermo Fisher Scientific. All assays were performed according to manufacturers' instructions in six different laboratories in Friuli-Venezia Giulia and Veneto regions of Italy [Lab 1 (AIA), Lab 2 (CL2), Lab 3 (ARC, COB and LUM), Lab 4 (CEN, IMM, KRY and MAG), Lab 5 (LIA) and Lab 6 (PHA)]. Since TgAb values were not normally distributed, the experimental URL (e-URL) was established at 97.5 percentile according to the non-parametric method. RESULTS: TgAb e-URLs showed a significant inter-method variability. Considering the same method, e-URL was much lower than that suggested by manufacturers (m-URL), except for ARC and MAG. Correlation and linear regression were unsatisfactory. Consequently, the agreement between methods was poor, with significant bias in Bland-Altman plot. CONCLUSIONS: Despite the efforts for harmonization, TgAb methods cannot be used interchangeably. Therefore, additional effort is required to improve analytical performance taking into consideration approved protocols and guidelines. Moreover, TgAb URL should be used with caution in the management of differentiated thyroid carcinoma patients since the presence and/or the degree of TgAb interference in Tg measurement has not yet been well defined.

Characterization of an emergent clone of enteroinvasive Escherichia coli circulating in Europe.

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness indistinguishable from that caused by Shigella, mainly in developing countries. Recently an upsurge of cases of EIEC infections has been observed in Europe, with two large outbreaks occurring in Italy and in the United Kingdom. We have characterized phenotypically and genotypically the strains responsible for these epidemics together with an additional isolate from a sporadic case isolated in Spain. The three isolates belonged to the same rare serotype O96:H19 and were of sequence type ST-99, never reported before in EIEC or Shigella. The EIEC strains investigated possessed all the virulence genes harboured on the large plasmid conferring the invasive phenotype to EIEC and Shigella while showing only some of the known chromosomal virulence genes and none of the described pathoadaptative mutations. At the same time, they displayed motility abilities and biochemical requirements resembling more closely those of the non-pathogenic E. coli rather than the EIEC and Shigella strains used as reference. Our observations suggested that the O96:H19 strains belong to an emerging EIEC clone, which could be the result of a recent event of acquisition of the invasion plasmid by commensal E. coli.

A prospective study of 1038 pregnancies on the predictive value of anti-annexin V antibodies for fetal loss.

Retrospective studies have demonstrated that anti-annexin V (anti-AnxV) antibodies are linked to miscarriage. Their predictive value is, however, unknown. We have carried out a prospective study to evaluate the relationship between anti-AnxV antibodies and the pregnancy outcome. A serum sample was taken from 1038 consecutive healthy women at the beginning of pregnancy. IgG and IgM anti-AnxV antibodies were measured by an ELISA method. The cutoff value was set at 5 units for both IgG and IgM. Out of 1038 women, 116 (11.4%) had a miscarriage by the 22nd week; 10 were lost to follow-up, 10 had an induced abortion, 6 had a preterm delivery, and 896 carried their pregnancy through to term. An adverse outcome of the pregnancy proved to be directly related to the number of previous miscarriages (P = .008) and the age of the woman (P = .002). IgG and IgM anti-AnxV were present in 25% and 27% of the women who miscarried, and in 23% and 28% of those who gave birth (mean antibody concentration IgG, 4.2 vs. 4.4 U/mL; IgM, 3.7 vs. 3.5 U/mL). IgG and IgM anticardiolipin and anti-beta(2)GPI, together with antinuclear, antithyroperoxidase, and antithyroglobulin antibodies, were also measured in the 116 sera of the women with miscarriage and in an equal number of women who gave birth. Their positivity or level proved not to be useful in discriminating between the risk of miscarriage and term delivery. This large-scale prospective study demonstrates that the presence of IgG and IgM anti-AnxV antibodies, when measured in healthy women, does not give a positive predictive lead towards the possibility of a miscarriage, and it is not useful in evaluating the risk of miscarriage at the beginning of pregnancy.

Accuracy of semiquantitative immunoenzymatic methods in quantitation of anti-topoisomerase I (Scl-70) antibodies.

Reports of a possible correlation between anti-Scl-70 antibody concentration and clinical manifestations in systemic sclerosis patients have recently appeared in the scientific literature. The goal of our study was to evaluate, by means of a multicenter study, the analytical reliability of immunoassay systems in the quantitative measurement of Scl-70 antibodies. Three blind samples (H, M, L) at different anti-Scl-70 antibody concentrations, and a low concentration antibody serum (LPC) used as a common calibrator, were sent three times in a 6-month time span to 39 Italian clinical laboratories. Each laboratory was asked to calculate dosages following the enzyme-linked immunosorbent assay (ELISA) method they used and report the optical density values of each sample (ODs), of the cutoff serum provided by the manufacturer of the kit used (ODco) and of LPC (ODLPC). The overall analytical imprecision (between methods and between laboratories) of the three different determinations of the values respectively expressed in ODs, ODs/ODco and ODs/ODLPCratio was 47.1, 52.8 and 34.0% for sample H, 56.2, 47.4% and 34% for sample M and 84.6, 86.0 and 86.6% for sample L. The average intra-method analytical imprecision was, respectively, 20.7, 29.8 and 18.6% for sample H, 24.6, 26.5 and 19.3% for sample M, and 30.6, 28.1 and 20.2% for sample L. The commercial ELISA methods currently used to determine the presence of anti-Scl-70 autoantibodies show considerable differences in the quantitative determination. The best results for reproducibility analyses have been obtained when the values were expressed as a ratio between the ODs of the sample and of the common calibrator (ODs/ODLPC). Forward-looking clinical studies that can clarify the usefulness of quantitative determination of anti-Scl-70 antibodies in the monitoring of diffuse scleroderma patients can be performed only when standard serum with a known antibody concentration and calibration curves for quantitative ELISA measurements are made available.

Shiga toxin-producing Escherichia coli O157, O26 and O111 in cattle faeces and hides in Italy.

INTRODUCTION: Ruminants are regarded as the natural reservoir for Shiga toxin-producing Escherichia coli (STEC), especially of serogroup O157. MATERIALS AND METHODS: During 2011 and 2012, 320 samples (160 faecal samples from the rectum and 160 hide samples from the brisket area) were collected from 160 cattle at slaughter in Northern Italy during warm months (May to October). Cattle were reared in different farms and their age at slaughter ranged between nine months and 15 years, most of them being culled cattle (median age: six years; average age: 4.6 years). Samples were tested by immunomagnetic-separation technique for E coli O157 and O26 and by a screening PCR for stx genes followed by cultural detection of STEC. The virulence genes stx1, stx2, eae, and e-hlyA were detected and among stx2-positive isolates the presence of the stx2a and stx2c variants was investigated. RESULTS: Twenty-one of 160 cattle (13.1 per cent; 95 per cent CI 8.3 to 19.4 per cent) were found to be faecal carriers of STEC. STEC O157 was found in 10 (6.3 per cent) samples, STEC O26 in six (3.8 per cent) and STEC O111 in one (0.6 per cent). Four isolates (2.5 per cent) were O not determined (OND). Six out of 160 (3.8 per cent; 95 per cent CI 1.4 to 8.0 per cent) hide samples were positive for STEC; four hides (2.5 per cent) were contaminated by STEC O157 and two (1.3 per cent) by STEC O26. In three cattle (1.9 per cent) STEC from both faeces and hides were detected. Among STEC O157, 87.5 per cent of them carried the stx2c gene and 12.5 per cent carried both stx1 and stx2c genes. No O157 isolate harboured stx2a variant. STEC O26 and O111 carried the stx1 gene only. One OND strain carried both the stx2a and stx2c genes. CONCLUSIONS: This study shows that STEC O157 from cattle can harbour the stx2c variant, which is associated with haemolytic uraemic syndrome in humans, and that cattle hides may be a source of human pathogenic STEC O157 and O26 in the slaughterhouse environment.

Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry.

This study was performed by the Italian Society of Laboratory Medicine (SIMeL) in order to establish the variability between the different analytical systems currently used in clinical laboratories for the detection of autoantibodies diagnostic of systemic autoimmune disease. Sixteen industrial, and two university laboratories participated in this study which entailed the determination of anti-nuclear (ANA), anti-dsDNA and anti-ENA antibodies in 11 sera from patients with clinically diagnosed systemic rheumatic disease, using reagents produced by these companies and different methodologies (indirect immunofluorescence, immunoenzymatic assay, counterimmunolectrophoresis, immuno and western blotting). We found 93.5% agreement between the methods used for the detection of ANA, 85.2% for anti-dsDNA antibodies, and 86.9% for anti-ENA antibodies. Among the anti-ENA antibodies, regardless of the method used, detection percentages were excellent for anti-RNP and anti-SSB/La (100%), good for anti-SSA/Ro (93%), but unacceptable for the anti-Jo-1 (67%), anti-Scl70 and anti-Sm (47%) antibodies. This further stresses the need for rigorous standardisation of commercial reagents and analytical procedures, as well as the introduction of external quality assessment (EQA) programs, and a complete definition of operative protocols adjusted to the sensitivity and specificity of the various methods.

Analytical and diagnostic accuracy of "second generation" assays for thyrotrophin receptor antibodies with radioactive and chemiluminescent tracers.

AIMS: To investigate the analytical and diagnostic accuracy of thyrotrophin (TSH) receptor antibody assays using recombinant human TSH receptors. METHODS: Sera from 68 patients with Graves' disease, 23 patients with autoimmune thyroiditis, and 119 healthy controls were evaluated in four different laboratories using both radioactive and chemiluminescent tracers. Functional sensitivity, interlaboratory precision, optimal cutoff values for Graves' disease, and the correlation between the two methods were evaluated. RESULTS: Functional sensitivity was 0.98 IU/litre for both assays. Interlaboratory precision, expressed as per cent coefficient of variation over a wide range of antibody concentrations, varied from 5.7% to 15.1% for the radioligand, and from 6.6% to 19.9% for the chemiluminescence assay. The two methods (radioactive and chemiluminescent) were closely correlated. All the sera from untreated or relapsing patients with Graves' disease gave TSH receptor antibody values above 2.1 IU/litre, whereas in none of the healthy controls did values exceed 2.5 IU/litre. Receiver operating curve analysis allowed an optimal cutoff point to be defined at 1.99 IU/litre, according to a sensitivity of 100% and specificity of 99.1%. CONCLUSIONS: These data show the high analytical and diagnostic accuracy of the human TSH receptor assays, both with radioactive and chemiluminescent tracers, when both functional sensitivity and interlaboratory reproducibility are considered. These two methods could be proposed as first line diagnostic markers for Graves' disease.

The role of antitissue transglutaminase assay for the diagnosis and monitoring of coeliac disease: a French-Italian multicentre study.

AIMS: Tissue transglutaminase (tTG) was recently identified as the major autoantigen in coeliac disease. The aim of this multicentre study was to evaluate the impact of a new immunoenzymatic assay for the detection of IgA anti-tGT antibodies. METHODS: Seventy four Italian and French clinical laboratories participated in this study; anti-tTG IgA with an enzyme linked immunosorbent assay (ELISA) method using guinea pig liver extract as the coating antigen, anti-endomysium IgA autoantibodies (EMA), and total serum IgA were determined in 7948 patients, 1162 of whom had coeliac disease (737 untreated cases and 425 on a gluten free diet). A proportion of the sera were then sent to a reference laboratory for anti-tTG retesting with an ELISA method using recombinant human tTG antigen. RESULTS: Seven thousand four hundred and fifty eight (93.8%) sera were EMA/antiguinea pig tTG concordant (positive or negative); 490 (6.2%) were non-concordant. The sensitivity of EMA and antiguinea pig tTG in the 737 untreated patients with coeliac disease was 92.1% and 94.8%, respectively, and the specificity was 99.8% and 99.2%, respectively. Retesting of the discordant sera showed that of the 162 sera classified as EMA negative/antiguinea pig tTG positive, only 49 were positive for human recombinant anti-tTG, and that 39 of these were also EMA positive. Furthermore, of the 36 sera classified as EMA positive/antiguinea pig tTG negative, only two were confirmed as EMA positive. CONCLUSIONS: The antiguinea pig tTG assay is more sensitive but less specific than EMA, whereas the antihuman recombinant tTG assay is far more specific and just as sensitive as antiguinea pig tTG. Testing for EMA presents considerable interpretative problems and is difficult to standardise.