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Villalta D.,Allergologia e Immunologia Clinica | Alessio M.G.,Laboratorio Analisi Chimico Cliniche | Sorrentino M.C.,Laboratorio Analisi Chimico Cliniche e Microbiologiche | Tampoia M.,Laboratorio Of Patologia Clinica | And 7 more authors.
Journal of Clinical Laboratory Analysis | Year: 2016

Background: Autoimmune hepatitis (AIH) is a rare condition characterized by the presence of autoantibodies distinctive of type 1 AIH (AIH-1) and type 2 AIH (AIH-2). The aim of this study was to evaluate the autoantibody profile in a cohort of pediatric and adult AIH patients, using both indirect immunofluorescence (IIF) and a new multiplexed line-blot assay. Methods: Sera from 63 pediatric and 53 adult AIH patients were tested for antinuclear (ANA), antismooth muscle (SMA), anti-liver kidney microsome 1 (anti-LKM1), anti-liver cytosol 1 (anti-LC1) autoantibodies using IIF methods; for anti-LKM1, anti-LC1, and soluble liver antigen/liver-pancreas (anti-SLA/LP) autoantibodies using the line-blot; for anti-F-actin autoantibodies using IIF both on VSM47 cell-line and on rat intestinal epithelial cells. Results: AIH-1 was the most common type of AIH in the adult cohort (73.6%), while AIH-2 was the most common AIH in the pediatric cohort (61.9%). Both in adult and pediatric AIH-2 anti-LKM1 were the prevalent autoantibodies. In pediatric AIH-2 anti-LC1 autoantibodies were more frequent than in adult AIH-2 (59 vs. 28.6%), and in 35.9% of cases they were present alone. In 17 patients anti-LC1 autoantibodies were detected only with the line-blot assay. The levels of anti-LKM1 and of anti-LC1 were not different between adult and pediatric AIH, and the overall agreement between the results obtained with the two IIF methods for F-actin detection was 98.8% (CI 95%: 94.4-99.7%). Conclusions: The line-blot assay showed a higher sensitivity than IIF for anti-LC1 detection. Anti-LKM1 and anti-LC1 autoantibody levels are not different in adults and children. An almost perfect agreement between the two IIF methods for anti-F-actin detection has been observed. © 2016 Wiley Periodicals, Inc.


Villalta D.,Allergologia e Immunologia Clinica | Sorrentino M.C.,Laboratorio Analisi Chimico Cliniche e Microbiologiche | Tampoia M.,Patologia Clinica | Alessio M.G.,Laboratorio Analisi Chimico Cliniche | And 5 more authors.
Clinica Chimica Acta | Year: 2015

Objective: To evaluate the autoantibody profile in patients with primary biliary cirrhosis (PBC) using a new multiplexed line-blot assay specifically designed for the diagnosis of autoimmune liver diseases. Methods: Sera of 58 consecutive PBC patients and 191 disease controls (144 with autoimmune liver diseases other than PBC, and 67 with non-autoimmune chronic liver diseases) were tested by both the multiplexed line-blot Autoimmune Liver Disease Profile 2 (ALD2) and by IIF on HEp-2 cells and on rat kidney/liver/stomach tissues. ALD2 contains the following PBC-associated antigens: AMA-M2, natively purified from bovine heart; M2-E3, a recombinant fusion protein including the E2 subunits of PDC, BCOADC and OGDC; sp100, PML and gp210 recombinant proteins. Results: With the ALD2 assay, a positive reaction to AMA-M2, M2-E3, sp100, PML and gp210 in PBC patients was observed in 77.6%, 84.5%, 34.5%, 15.1% and 18.9%, respectively, of the PBC sera. The overall sensitivity and specificity for PBC were 98.3% and 93.7%. Using IIF, positivity rates to AMA, and to antinuclear autoantibodies with membranous/rim-like and multiple nuclear dot patterns were 86.2%, 8.6% and 22.4%, respectively. The overall sensitivity and specificity for PBC of the IIF method were 86.2% and 97.9%, respectively. Conclusions: The ALD2 line-blot showed a good diagnostic accuracy for PBC and a higher sensitivity than the IIF method to detect sp100 and gp210 autoantibodies. © 2014 Elsevier B.V.


PubMed | Laboratorio Analisi Chimico Cliniche, Patologia Clinica, Laboratorio Analisi Chimico Cliniche e Microbiologiche, Laboratorio Centrale and 2 more.
Type: | Journal: Clinica chimica acta; international journal of clinical chemistry | Year: 2014

To evaluate the autoantibody profile in patients with primary biliary cirrhosis (PBC) using a new multiplexed line-blot assay specifically designed for the diagnosis of autoimmune liver diseases.Sera of 58 consecutive PBC patients and 191 disease controls (144 with autoimmune liver diseases other than PBC, and 67 with non-autoimmune chronic liver diseases) were tested by both the multiplexed line-blot Autoimmune Liver Disease Profile 2 (ALD2) and by IIF on HEp-2 cells and on rat kidney/liver/stomach tissues. ALD2 contains the following PBC-associated antigens: AMA-M2, natively purified from bovine heart; M2-E3, a recombinant fusion protein including the E2 subunits of PDC, BCOADC and OGDC; sp100, PML and gp210 recombinant proteins.With the ALD2 assay, a positive reaction to AMA-M2, M2-E3, sp100, PML and gp210 in PBC patients was observed in 77.6%, 84.5%, 34.5%, 15.1% and 18.9%, respectively, of the PBC sera. The overall sensitivity and specificity for PBC were 98.3% and 93.7%. Using IIF, positivity rates to AMA, and to antinuclear autoantibodies with membranous/rim-like and multiple nuclear dot patterns were 86.2%, 8.6% and 22.4%, respectively. The overall sensitivity and specificity for PBC of the IIF method were 86.2% and 97.9%, respectively.The ALD2 line-blot showed a good diagnostic accuracy for PBC and a higher sensitivity than the IIF method to detect sp100 and gp210 autoantibodies.


PubMed | Laboratorio Of Patologia Clinica, Laboratorio Analisi Chimico Cliniche, Patologia Clinica, Laboratorio Analisi Chimico Cliniche e Microbiologiche and 3 more.
Type: Journal Article | Journal: Journal of clinical laboratory analysis | Year: 2016

Autoimmune hepatitis (AIH) is a rare condition characterized by the presence of autoantibodies distinctive of type 1 AIH (AIH-1) and type 2 AIH (AIH-2). The aim of this study was to evaluate the autoantibody profile in a cohort of pediatric and adult AIH patients, using both indirect immunofluorescence (IIF) and a new multiplexed line-blot assay.Sera from 63 pediatric and 53 adult AIH patients were tested for antinuclear (ANA), antismooth muscle (SMA), anti-liver kidney microsome 1 (anti-LKM1), anti-liver cytosol 1 (anti-LC1) autoantibodies using IIF methods; for anti-LKM1, anti-LC1, and soluble liver antigen/liver-pancreas (anti-SLA/LP) autoantibodies using the line-blot; for anti-F-actin autoantibodies using IIF both on VSM47 cell-line and on rat intestinal epithelial cells.AIH-1 was the most common type of AIH in the adult cohort (73.6%), while AIH-2 was the most common AIH in the pediatric cohort (61.9%). Both in adult and pediatric AIH-2 anti-LKM1 were the prevalent autoantibodies. In pediatric AIH-2 anti-LC1 autoantibodies were more frequent than in adult AIH-2 (59 vs. 28.6%), and in 35.9% of cases they were present alone. In 17 patients anti-LC1 autoantibodies were detected only with the line-blot assay. The levels of anti-LKM1 and of anti-LC1 were not different between adult and pediatric AIH, and the overall agreement between the results obtained with the two IIF methods for F-actin detection was 98.8% (CI 95%: 94.4-99.7%).The line-blot assay showed a higher sensitivity than IIF for anti-LC1 detection. Anti-LKM1 and anti-LC1 autoantibody levels are not different in adults and children. An almost perfect agreement between the two IIF methods for anti-F-actin detection has been observed.


Caldini A.,Laboratorio Generale | Graziani M.S.,Azienda Ospedaliera Universitaria Integrata di Verona | Basile U.,Catholic University of the Sacred Heart | Cataldo I.,Laboratorio Of Patologia Clinica | And 6 more authors.
Biochimica Clinica | Year: 2014

This document examines laboratory tests to be used for the management of monoclonal gammopathies in different clinical scenarios, from screening to monitoring and assessment of the response to therapy. The content is based on international recommendations and guidelines currently available. It includes sections on the analytical aspects of different tests (serum protein electrophoresis, typing and quantification of monoclonal components, Bence Jones protein determination and free light chain measurement) and on their clinical significance as well. Different clinical settings are examined: screening, diagnosis, risk stratification, monitoring and response assessment. For each of those, laboratory tests to be used are indicated. Aim of the document is to help clinical laboratories avoiding unnecessary tests, ensuring in the meantime that all the investigations required for a optimal patient management are carried out.


Bizzaro N.,Laboratorio Of Patologia Clinica | Tozzoli R.,Laboratorio Of Patologia Clinica | Morozzi G.,Patologia Clinica | Antico A.,Laboratorio Analisi | And 6 more authors.
Rivista Italiana della Medicina di Laboratorio | Year: 2014

Background.: The Study Group on Autoimmune Diseases of the Italian Society of Laboratory Medicine (SIMeL) carried out a national survey to detect structural characteristics, the technologies used, the volume of diagnostic activity and aspects of clinical governance of Italian autoimmunology laboratories in 2012.Methods.: The data were collected using a questionnaire distributed in electronic form in January 2013 to 315 Italian autoimmunology laboratories. The questionnaire included 38 questions related to the characteristics of the laboratory, the size of the referring area, the number of tests carried out, the analytical phase (instrumentation, antibody tests and methods), the definition of reference intervals. The last series of questions intended to explore ways of reporting, the use of interpretive comments, and the relationship with the clinicians, both hospital specialists and general practioners.Results.: 235 (74.6%) laboratories responded in whole or in part to the questions. The laboratories operating in the field of autoimmunity in Italy are 87% placed in public hospitals and 13% in private facilities. About half (48%) run between 10,000 and 50,000 tests / year, and 30% more than 50,000. Only 7% of the laboratories perform fewer than 5,000 exams and these are mostly private. Among the analytical methods, indirect immunofluorescence is used in 97%, ELISA methods are used in 96% and immunochemiluminescence in 62% of the laboratories. 63% of participants declared to add an interpretive comment in the report: 5% using only canned comments, 9% using only narrative comments, and 49% using predefined or customized comments depending on the result. In general, there are good working relationships with hospitals clinicians and with general practitioners; counseling is provided by 57% of laboratories, both public and private.Conclusions.: The high number of responses we received suggests that the data collected and analyzed are quite representative of the organizational reality of the Italian laboratories and are an excellent picture of the current state of the autoimmunology diagnostics in our country. © 2014, Springer-Verlag Italia.


Bizzaro N.,Laboratorio Of Patologia Clinica | Antico A.,Laboratorio Analisi | Platzgummer S.,Laboratorio Centrale | Tonutti E.,Immunopatologia e Allergologia | And 5 more authors.
Autoimmunity Reviews | Year: 2014

Background: Indirect immunofluorescence (IIF) plays an important role in immunological assays for detecting and measuring autoantibodies. However, the method is burdened by some unfavorable features: the need for expert morphologists, the subjectivity of interpretation, and a low degree of standardization and automation. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of anti-nuclear antibodies (ANA), the biomedical industry has developed technological solutions which might significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. Methods: We collected 104 ANA-positive sera from patients with a confirmed clinical diagnosis of autoimmune disease and 40 ANA-negative sera from healthy blood donors. One aliquot of each serum, without information about pattern and titer, was sent to six laboratories of our group, where the sera were tested with the IIF manual method provided by each of the six manufacturers of automatic systems. Assignment of result (pos/neg), of pattern and titer was made by consensus at a meeting attended by all members of the research team. Result was assigned if consensus for pos/neg was reached by at least four of six certifiers, while for the pattern and for the titer, the value observed with higher frequency (mode) was adopted. Seventeen ANA-positive sera and six ANA-negative sera were excluded. Therefore, the study with the following automatic instrumentation was conducted on 92 ANA-positive sera and on 34 ANA-negative sera: Aklides, EUROPattern, G-Sight (I-Sight-IFA), Helios, Image Navigator, and Nova View. Analytical imprecision was measured in five aliquots of the same serum, randomly added to the sample series. Results: Overall sensitivity of the six automated systems was 96.7% and overall specificity was 89.2%. Most false negatives were recorded for cytoplasmic patterns, whereas among nuclear patterns those with a low level of fluorescence (i.e., multiple nuclear dots, midbody, nuclear rim) were sometimes missed.The intensity values of the light signal of various instruments showed a good correlation with the titer obtained by manual reading (Spearman's rho between 0.672 and 0.839; P. <. 0.0001 for all the systems). Imprecision ranged from 1.99% to 25.2% and, for all the systems, it was lower than that obtained by the manual IIF test (39.1%). The accuracy of pattern recognition, which is for now restricted to the most typical patterns (homogeneous, speckled, nucleolar, centromere, multiple nuclear dots and cytoplasmic) was limited, ranging from 52% to 79%. Conclusions: This study, which is the first to compare the diagnostic accuracy of six systems for automated ANA-IIF reading on the same series of sera, showed that all systems are able to perform very well the task for which they were created. Indeed, cumulative automatic discrimination between positive and negative samples had 95% accuracy. All the manufacturers are actively continuing the development of new and more sophisticated software for a better definition in automatic recognition of patterns and light signal conversion in end-point titer. In the future, this may avert the need for serum dilution for titration, which will be a great advantage in economic terms and time-saving. © 2013 Elsevier B.V.


Bizzaro N.,Laboratorio Of Patologia Clinica | Covini G.,IRCCS Instituto Clinico Humanitas | Rosina F.,Presidio | Muratori P.,Policlinico Universitario SantOrsola Malpighi | And 21 more authors.
Clinical Reviews in Allergy and Immunology | Year: 2012

Serum anti-mitochondrial antibodies (AMA) are the serological hallmark of primary biliary cirrhosis (PBC), yet up to 15% of PBC sera are AMA negative at routine indirect immunofluorescence (IIF) while being referred to as "probable" cases. The diagnostic role of PBC-specific antinuclear antibodies (ANA) remains to be determined. We will report herein data on the accuracy of new laboratory tools for AMA and PBC-specific ANA in a large series of PBC sera that were AMA-negative at IIF. We will also provide a discussion of the history and current status of AMA detection methods. We included IIF AMA-negative PBC sera (n=100) and sera from patients with other chronic liver diseases (n=104) that had been independently tested for IIF AMA and ANA; sera were blindly tested with an ELISA PBC screening test including two ANA (gp210, sp100) and a triple (pMIT3) AMA recombinant antigens. Among IIF AMA-negative sera, 43/100 (43%) manifested reactivity using the PBC screening test. The same test was positive for 6/104 (5.8%) control sera. IIF AMA-negative/PBC screen-positive sera reacted against pMIT3 (11/43), gp210 (8/43), Sp100 (17/43), both pMIT3 and gp210 (1/43), or both pMIT3 and Sp100 (6/43). Concordance rates between the ANA pattern on HEp-2 cells and specific Sp100 and gp210 ELISA results in AMA-negative subjects were 92% for nuclear dots and Sp100 and 99% for nuclear rim and gp210. Our data confirm the hypothesis that a substantial part of IIF AMA-negative (formerly coined "probable") PBC cases manifest disease-specific autoantibodies when tested using newly available tools and thus overcome the previously suggested diagnostic classification. As suggested by the recent literature, we are convinced that the proportion of AMA-negative PBC cases will be significantly minimized by the use of new laboratory methods and recombinant antigens. © Springer Science+Business Media, LLC 2010.


PubMed | Laboratorio Of Patologia Clinica, Laboratorio Analisi, Immunopatologia e Allergologia, Microbiologia e Virologia and 2 more.
Type: Comparative Study | Journal: Autoimmunity reviews | Year: 2014

Indirect immunofluorescence (IIF) plays an important role in immunological assays for detecting and measuring autoantibodies. However, the method is burdened by some unfavorable features: the need for expert morphologists, the subjectivity of interpretation, and a low degree of standardization and automation. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of anti-nuclear antibodies (ANA), the biomedical industry has developed technological solutions which might significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading.We collected 104 ANA-positive sera from patients with a confirmed clinical diagnosis of autoimmune disease and 40 ANA-negative sera from healthy blood donors. One aliquot of each serum, without information about pattern and titer, was sent to six laboratories of our group, where the sera were tested with the IIF manual method provided by each of the six manufacturers of automatic systems. Assignment of result (pos/neg), of pattern and titer was made by consensus at a meeting attended by all members of the research team. Result was assigned if consensus for pos/neg was reached by at least four of six certifiers, while for the pattern and for the titer, the value observed with higher frequency (mode) was adopted. Seventeen ANA-positive sera and six ANA-negative sera were excluded. Therefore, the study with the following automatic instrumentation was conducted on 92 ANA-positive sera and on 34 ANA-negative sera: Aklides, EUROPattern, G-Sight (I-Sight-IFA), Helios, Image Navigator, and Nova View. Analytical imprecision was measured in five aliquots of the same serum, randomly added to the sample series.Overall sensitivity of the six automated systems was 96.7% and overall specificity was 89.2%. Most false negatives were recorded for cytoplasmic patterns, whereas among nuclear patterns those with a low level of fluorescence (i.e., multiple nuclear dots, midbody, nuclear rim) were sometimes missed. The intensity values of the light signal of various instruments showed a good correlation with the titer obtained by manual reading (Spearmans rho between 0.672 and 0.839; P<0.0001 for all the systems). Imprecision ranged from 1.99% to 25.2% and, for all the systems, it was lower than that obtained by the manual IIF test (39.1%). The accuracy of pattern recognition, which is for now restricted to the most typical patterns (homogeneous, speckled, nucleolar, centromere, multiple nuclear dots and cytoplasmic) was limited, ranging from 52% to 79%.This study, which is the first to compare the diagnostic accuracy of six systems for automated ANA-IIF reading on the same series of sera, showed that all systems are able to perform very well the task for which they were created. Indeed, cumulative automatic discrimination between positive and negative samples had 95% accuracy. All the manufacturers are actively continuing the development of new and more sophisticated software for a better definition in automatic recognition of patterns and light signal conversion in end-point titer. In the future, this may avert the need for serum dilution for titration, which will be a great advantage in economic terms and time-saving.


Mosca A.,University of Milan | Basilico N.,University of Milan | Grande R.,Laboratorio Centrale | Taramelli D.,University of Milan
Biochimica Clinica | Year: 2011

This overview covers several aspects of malaria, from basic information on the biology of the genus Plasmodium to the pathogenesis and diagnosis of the species infecting humans. Current treatment regimens and drug-resistance problems are also discussed. Particular attention is given to the various aspects of malaria epidemiology and to the cellular and immune mechanisms that may sustain the protection against malaria in subjects heterozygotes for various hemoglobin disorders. A short critical review of actual tools for the diagnosis and monitoring of malaria is also provided. Finally, a presentation of the Italian Malaria Network is outlined.

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