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Introduction: Serological assays are alternative laboratory tools for the diagnosis of parasitic infections. The aim of this work was to evaluate the performance of the indirect fluorescent antibody test (IFAT) and Western blotting (WB) for the detection of IgG anti-Giardia antibodies in human sera. Methodology: Sera from individuals infected with Giardia duodenalis, other parasites or non-parasitized were selected for serological assays. Ninety-seven sera were tested by IFAT at 1:20 and 1:40 dilutions and of these, 40 samples were also analyzed by WB. Results: The sensitivity and specificity of the IFAT was 97% and 46.9% at 1:20 sera dilution, and 39.4% and 59.4% at 1:40 sera dilution. The low molecular weight polypeptides fractions of 25 kDa, 27–31 kDa and 45–55 kDa were the most frequently identified by the sera of individuals infected with G. duodenalis, along with low cross-reactivity, presenting an individual sensitivity of 42.8%, 50.0% and 57.1%, and specificity of 83.3%, 83.3% and 91.7%, respectively. The highest overall sensitivity of WB (85.7%) was based on the immunoreactivity of sera with at least one of those proteins. The concordance between the detection of G. duodenalis in feces by microscopy and the WB results was considered substantial (Kappa = 0.61). Conclusion: Constant exposure to Giardia infection throughout a lifetime can maintain high levels of specific antibodies in serum, even without active infection. Moreover, proteins found in intestinal amoebas may hinder the serological diagnosis of giardiasis in endemic areas due to cross-reactivity, which can be partially solved using Giardia low molecular weight proteins. [ABSTRACT FROM AUTHOR]

Despite the advantages of automated systems for antinuclear antibody (ANA) analysis, the prediction of end-point titers avoiding serial dilutions is still in progress. The aims of this study were to set a conversion table providing discriminant ranges of fluorescence signal intensity values (FI) corresponding to the end-point titers and validate this tool in a real-life laboratory setting. Eight hundred ninety-four serum samples were analyzed for ANA using Image Navigator System. In order to classify FI into non-overlapping groups corresponding to conventional end-point titers, statistical discriminant analysis was used. Validation study was performed calculating agreement and error rates between visual readings and conversion table of 1119 routine ANA positive samples. Setting of FI ranges corresponding to the end-point titers for different staining patterns was computed. For samples showing single pattern, the overall agreement between visual readings and conversion table was 98.4% for all titers ranging from 1:160 to 1:2560, of which 68.0% had the same titer and 30.4% were within ± one titer difference. Concordance rates according to ANA patterns were as follows: (1) nuclear 98.4%, of which 67.0% had the same titer and 31.4% ± one titer; (2) cytoplasmic 100%, of which 72.7% had the same titer and 27.3% than ± one titer; (3) mitotic 66.6%, of which 33.3% had more ± one titer. Our study developed a quantification method for autoantibodies titers assessment based on just one single sample dilution instead of traditional serial dilution approach, providing significant advantages in routine laboratory in terms of reduction in hand-on time and harmonization of results. [ABSTRACT FROM AUTHOR]

Human visceral leishmaniasis (HVL) is a neglected disease that occurs in 98 countries on five continents, and it is endemic in tropical and subtropical regions. In South America, the etiological agent of HVL is Leishmania infantum (syn. Leishmania chagasi), mainly transmitted through the bite of an infected sandfly female from the genus Lutzomyia. In American HVL endemic areas, the occurrence of asymptomatic infection is common, which contributes to the possibility of L. infantum transmission during a blood transfusion. To know the prevalence of L. infantum asymptomatic infection in blood donors from the microregion of Adamantina, we investigated 324 peripheral blood samples from donors through immunofluorescence (IFAT) and PCR–RFLP techniques. Seven blood samples (2.16%) tested positive for Leishmania by IFAT, and from those, six presented positive results by PCR (85.71%), which were later identified as L. infantum by RFLP. The presence of L. infantum in the peripheral blood of blood donors supported the hypothesis of transmission by blood transfusion and points to the need to include tests for visceral leishmaniasis in blood bank screening tests and pre-storage measures, especially in endemic areas to prevent the exponential increase of HVL by blood transfusion. [ABSTRACT FROM AUTHOR]

Circulating autoantibodies are a key diagnostic tool in autoimmune hepatitis (AIH), being positive in 95% of the cases if tested according to dedicated guidelines issued by the International Autoimmune Hepatitis Group. They also allow the distinction between type 1 AIH, characterized by positive anti-nuclear and/or anti-smooth muscle antibody, and type 2 AIH, characterized by positive anti-liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibody. Anti-soluble liver antigen is the only AIH-specific autoantibody, and is found in 20–30% of both type 1 and type 2 AIH. Anti-neutrophil cytoplasmic antibody is frequently positive in type 1 AIH, being associated also with inflammatory bowel disease and with primary/autoimmune sclerosing cholangitis. The reference method for autoantibody testing remains indirect immunofluorescence on triple tissue (rodent liver, kidney and stomach), allowing both the detection of the majority of liver-relevant reactivities, including those autoantibodies whose molecular target antigens are unknown. Of note, the current knowledge of the clinical significance of autoantibodies relies on studies based on this technique. However, immunofluorescence requires trained laboratory personnel, is observer-dependent, and lacks standardization, leading to ongoing attempts at replacing this method with automated assays, the sensitivity, and specificity of which, however, require further studies before they can be used as a reliable alternative to immunofluorescence; currently, they may be used as complementary to immunofluorescence. [ABSTRACT FROM AUTHOR]

Parasites of the genus Sarcocystis can infect several species of animals and cause multiple diseases such as equine protozoal myeloencephalitis. Felines are considered hosts of this protozoa; therefore, the present study aimed to detect anti-Sarcocystis spp.–specific antibodies in domestic cats that were under clinical evaluation, using the indirect immunofluorescence antibody test. Anti-Sarcocystis-specific immunoglobulin Gs were detected in 24 out of 497 (4.82%) cat serum samples. These findings support the fact that natural Sarcocystis infections do occur in cats. Furthermore, it highlights the importance of domestic cats as both intermediate and definitive hosts in the Sarcocystis life cycle, maintaining the parasite and serving as a source of infection for various other animals. To the best of our knowledge, this is the first study to identify antibodies against the genus Sarcocystis in cats from a region in southern Brazil. [ABSTRACT FROM AUTHOR]

Background: To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) using HEp-2 substrates based on common practice and guidance by the International Consensus on ANA patterns (ICAP). Method: Participants included two groups [16 clinical laboratories (CL) and 8 in vitro diagnostic manufacturers (IVD)] recruited via an email sent to the Association of Medical Laboratory Immunologists (AMLI) membership. Twelve (n = 12) pre-qualified specimens were distributed to participants for testing, interpretation and reporting HEp-2 IFA. Results obtained were analyzed for accuracy with the intended and consensus response for three main categorical patterns (nuclear, cytoplasmic and mitotic), common patterns and ICAP report nomenclatures. The distributions of antibody titers of specimens were also compared. Results: Laboratories differed in the categorical patterns reported; 8 reporting all patterns, 3 reporting only nuclear patterns and 5 reporting nuclear patterns with various combinations of other patterns. For all participants, accuracy with the intended response for the categorical nuclear pattern was excellent at 99% [95% confidence interval (CI): 97–100%] compared to 78% [95% CI 67–88%] for the cytoplasmic, and 93% [95% CI 86%–100%] for mitotic patterns. The accuracy was 13% greater for the common nomenclature [87%, 95% CI 82–90%] compared to the ICAP nomenclature [74%, 95% CI 68–79%] for all participants. Participants reporting all three main categories demonstrated better performances compared to those reporting 2 or less categorical patterns. The average accuracies varied between participant groups, however, with the lowest and most variable performances for cytoplasmic pattern specimens. The reported titers for all specimens varied, with the least variability for nuclear patterns and most titer variability associated with cytoplasmic patterns. Conclusions: Our study demonstrated significant accuracy for all participants in identifying the categorical nuclear staining as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cytoplasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel. [ABSTRACT FROM AUTHOR]

Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting these rare events. Here, we describe a protocol to induce formation of bi-species mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies in vitro. This procedure can be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming. [ABSTRACT FROM AUTHOR]

The computer aided analysis of Indirect-Immunofluorescence (IIF) images is important for the differential diagnosis of several autoimmune diseases. A fully automatic approach consists in segmentation of individual cells in IIF images and subsequently its classification into various pattern types. This paper explores the segmentation of HEp2 cell in IIF images through the use of a filtering based approach. Our algorithm is based on a local convergence filter named as Sliding Band Filter (SBF). We propose a modified SBF that is capable of handling the low contrast, noise and illumination variations peculiar to IIF images. In addition, we follow a simple algorithmic pipeline and achieve better accuracy as compared to several state of the art segmentation algorithms on standard HEp2 image dataset. [ABSTRACT FROM AUTHOR]

Introduction: Dense fine-speckled 70 (DFS70) antibody is defined as an antinuclear antibody (ANA) pattern in indirect immunofluorescence (IIF). The presence of anti-DFS70 antibody has been shown as a potential marker for the exclusion of systemic autoimmune rheumatic diseases (SARD) (without any other SARD-associated autoantibodies). We aimed to investigate the frequency of anti-DFS70 antibodies in patients with SARD and in the blood bank donors (BD). Materials and methods: The study group consists of 418 rheumatoid arthritis (RA), 101 systemic lupus erythematosus (SLE), 71 Sjogren's syndrome (SS), 43 ankylosing spondylitis (AS), 36 systemic sclerosis-scleroderma (SSc), 2555 undifferentiated connective tissue disease (UCTD), and 507 BD. All samples were tested on the HEp-2 IIF-ANA assay. Samples that showed DFS70 pattern in IIF were confirmed by a specific DFS70 antibody enzyme-linked immunosorbent assay (ELISA). Results: The DFS70 pattern was detected in 43 (1.33%) in SARD and four (0.78%) in BD. The anti-DFS70 antibody was detected in three (0.59%) in BD, six (1.43%) in RA, three (2.97%) in SLE, one (1.40%) in SS, and 25 (0.97%) in UCTD, however, it was not detected in AS and SSc by ELISA. There was no significant difference between BD and SARD (p = 0.28). Distinctly, the frequency of anti-DFS70 was significantly different for SLE in SARD (p = 0.02). Conclusion: Anti-DFS70 antibody was more prevalent in the subsets of SARD than BD. This result may be related to the demographic formation of study groups and individual immunological status. More comprehensive studies are needed to investigate the importance of the anti-DFS70 antibody for SARD. Key Points • This study draws attention to the importance of anti-DFS70 antibodies in the diagnostic algorithm in systemic autoimmune rheumatic diseases. • This study emphasizes the further investigation of anti-DFS70 antibodies in undifferentiated connective tissue diseases. • This study emphasizes the need to verify the DFS70 pattern detected in IIF-ANA test for definitive diagnosis with additional confirmation methods. [ABSTRACT FROM AUTHOR]

The dense fine speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine anti-nuclear antibody (ANA) screening. There is a negative association between anti-DFS70 status and systemic autoimmune rheumatic disease (SARD), especially in the absence of concomitant SARD-specific autoantibodies. The purpose of this study was to determine the need for confirming anti-DFS70 status when a DFS pattern is observed in IIF-ANA. The frequency of anti-DFS70 detection on Western blot and the positive rate of connective tissue disease (CTD)-related autoantibody screening with a fluorescence-based enzyme immunoassay was evaluated in DFS (n = 182) and non-DFS (n = 359) groups. Specific autoantibodies against 15 autoantigens were identified by line immunoassay. We evaluated the frequency of cases of DFS mistaken for non-DFS and non-DFS cases mistaken for DFS, as well as the clinical impacts of these misinterpretations. Among cases of IIF-ANA with an observable DFS pattern, 68.1% had only anti-DFS70 without CTD-related autoantibodies, 20.3% were false positive for IIF-ANA, and the remaining 11.5% had CTD-related autoantibodies independent of anti-DFS70 status. These results indicated that CTD-related autoantibodies may be present with or without anti-DFS70 even if a DFS pattern is observed in IIF-ANA. Among patients who are ANA negative or have a low probability of SARD, an anti-DFS70 confirmation test has no clinical benefit and cannot replace specific tests for detecting CTD-related autoantibodies. Specific tests to detect CTD-related autoantibodies should be performed instead of anti-DFS70 confirmation tests when a DFS pattern is observed in IIF-ANA. [ABSTRACT FROM AUTHOR]

A sensitive indirect immunofluorescence (IF) assay for bovine neutrophil and somatic cell counting was developed. The obtained indirect IF was compared with direct IF. The direct IF method uses a single-fluorophore-conjugated antibody directed against the target of interest. The indirect IF method uses two antibodies. The primary antibody is unconjugated. The secondary antibody is directed against the primary and has fluorescent marker. Calibration curves for somatic cells (SC) in buffer model solutions were obtained by both methods, using fluorescence spectrophotometry. The measured linear range of somatic cells by the direct method was from 2 × 104 to 3 × 106 cells and by the indirect method was from 3 × 104 to 3 × 106 cells. The signal obtained by the indirect method was higher than the direct method at low concentrations of SC (from 30,000 to 100 000 cells/mL). That signal amplification probably is due to more than one fluorescent secondary antibody coupling to bound primary antibody. The same effect is observed by fluorescence microscopy. Consequently, the indirect IF method is more sensitive than the direct IF method. Conjugated primary antibodies are more expensive than their unconjugated counterparts, and secondary antibodies are relatively inexpensive. Therefore, using the same conjugated secondary antibody to detect different primary antibodies is cost-effective. Furthermore, a two-color staining microscopic procedure was proposed for simultaneous estimation of total SC count and neutrophil cell count. [ABSTRACT FROM AUTHOR]

Occurrence of autoantibodies (autoAbs) is a hallmark of autoimmune diseases, and the analysis thereof is an essential part in the diagnosis of organ-specific autoimmune and systemic autoimmune rheumatic diseases (SARD), especially connective tissue diseases (CTDs). Due to the appearance of autoAb profiles in SARD patients and the complexity of the corresponding serological diagnosis, different diagnostic strategies have been suggested for appropriate autoAb testing. Thus, evolving assay techniques and the continuous discovery of novel autoantigens have greatly influenced the development of these strategies. Antinuclear antibody (ANA) analysis by indirect immunofluorescence (IIF) on tissue and later cellular substrates was one of the first tests introduced into clinical routine and is still an indispensable tool for CTD serology. Thus, screening for ANA by IIF is recommended to be followed by confirmatory testing of positive findings employing different assay techniques. Given the continuous growth in the demand for autoAb testing, IIF has been challenged as the standard method for ANA and other autoAb analyses due to lacking automation, standardization, modern data management, and human bias in IIF pattern interpretation. To address these limitations of autoAb testing, the CytoBead® technique has been introduced recently which enables automated interpretation of cell-based IIF and quantitative autoAb multiplexing by addressable microbead immunoassays in one reaction environment. Thus, autoAb screening and confirmatory testing can be combined for the first time. The present review discusses the history of autoAb assay techniques in this context and gives an overview and outlook of the recent progress in emerging technologies. [ABSTRACT FROM AUTHOR]

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According to the recent recommendations of the American College of Rheumatology, ANA Task Force, IIF technique should be considered the gold standard in antinuclear antibodies (ANAs) testing. To overcome the lack of standardization, biomedical industries have developed several computer-aided diagnosis (CAD) systems. Two hundred and sixty-one consecutive samples with suspected autoimmune diseases were tested for ANA by means of IIF on routinely HEp-2 assay kit (Euroimmun AG). Assignment of result was made if consensus for positive/negative was reached by at least 2 out of 3 expert physicians. ANA-IIF was also carried out using 3 CAD systems: Zenit G-Sight ( n = 84), Helios ( n = 85) and NOVA View ( n = 92); human evaluation was repeated on the same substrate of each CAD system (Immco, Aesku and Inova HEp-2 cells, respectively). To anonymize the results, we randomly named these three systems as A, B and C. We ran a statistical analysis computing several measures of agreement between the ratings, and we also improved the evaluation by using the Wilcoxon's test for nonparametric data. Agreement between the human readings on routinely HEp-2 assay kit and human readings on CAD HEp-2 assay was substantial for A ( k = 0.82) and B ( k = 0.72), and almost perfect for C ( k = 0.89). Such readings were statistically different only in case A. Comparing experts' readings with the readings of CAD systems, when the samples were prepared using CAD HEp-2 assay kits, we found almost perfect agreement for B and C ( k = 0.86; k = 0.82) and substantial agreement for A ( k = 0.73). Again, human and CAD readings were statistically different only in A. When we compared the readings of medical experts on routinely HEp-2 assay kit with the output of the CAD systems that worked using their own slides, we found substantial agreement for all the systems (A: k = 0.62; B: k = 0.65; C: k = 0.71). Such readings were not statistically different. The change of the assay kit and/or the introduction of a CAD system affect the laboratory reporting, with an evident impact on the autoimmune laboratory workflow. The CAD systems may represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra- and inter-laboratory variability in a new vision of the diagnostic autoimmune platform. [ABSTRACT FROM AUTHOR]

Monkey esophagus (ME) is a well-accepted substrate for diagnosing pemphigus vulgaris (PV) by indirect immunofluorescence (IIF). However, its availability is sometimes limited due to ethical concerns. This study aimed to investigate the usefulness of human cervix (HC) as a substrate in the diagnosis of PV by IIF. Initially, serum from 1 PV patient was incubated with tissues from 48 HCs. Median IIF titers on HCs that had different demographic and clinical characteristics were compared. Sera from 5 PV patients were then incubated with ME and 21 HCs. For each serum, the titer of IIF on HC that was not different from ME by more than two-fold dilutions was acceptable. Last, sera from 42 PV, 14 pemphigus foliaceous, and 62 non-pemphigus patients were used to evaluate sensitivity and specificity. The results demonstrate that differences in demographic data among HCs did not affect IIF titers. Titers obtained from ME and HC were comparable (81-100 % acceptable values). Sensitivity of HC for diagnosis of PV was better than for diagnosis of pemphigus foliaceus (90.5 and 71.4 %, respectively). Specificity for PV and PF was 96.2 %. We proposed that HC substrate can be used as an alternative substrate for diagnosis of PV by IIF. [ABSTRACT FROM AUTHOR]

The presence of antinuclear antibodies (ANA) is associated with a wide range of ANA-associated autoimmune rheumatic diseases (AARD). The most commonly method used for the detection of ANA is indirect immunofluorescence (IIF) on HEp-2 cells. This method is very sensitive but unspecific. As a consequence, ANA testing on HEp-2 substrates outside a proper clinical specialist framework may lead to inappropriate referrals to tertiary care specialists and, worst case inappropriate and potentially toxic therapy for the patient. Among ANA, isolated anti-DFS70 antibodies represent a potentially important biomarker that can be clinically used to discriminate AARD from non-AARD patients in ANA IIF positive individuals. Therefore, their presence may avoid unnecessary follow-up testing and referrals. In our study, we investigated if the implementation of a new ANA workup algorithm allowing for the identification of anti-DFS70 antibodies is cost-effective through the reduction of both unnecessary follow-up testing and outpatient clinic visits generated by the clinical suspicion of a potential AARD. None of the 181 patients included with a positive monospecific anti-DFS70 antibody result developed SARD during the follow-up period of 10 years. The reduction in number of tests after ANA and anti-DFS70 positive results was significant for anti-ENA (230 vs. 114 tests; p < 0.001) and anti-dsDNA antibodies (448 vs. 114 tests; p < 0.001). In addition, the outpatient clinic visits decreased by 70 % ( p < 0.001). In total, the adoption of the new algorithm including anti-DFS70 antibody testing resulted in a cost saving of 60869.53 € for this pilot study. In conclusion, the use of anti-DFS70 antibodies was clearly cost-efficient in our setting. [ABSTRACT FROM AUTHOR]

Purpose: The dense fine speckled (DFS) pattern as detected by indirect immunofluorescence (IIF) on HEp-2 cells has been associated with several inflammatory diseases but is most commonly observed in individuals that do not have an antinuclear antibody (ANA)-associated rheumatic disease and even in apparently healthy individuals. Consequently, the accurate identification and correct reporting of this IIF pattern is of utmost importance and accordingly has been recognized by several international study groups for the detection of ANA. Furthermore, the DFS IIF pattern has recently been recommended as a competency level recognition pattern by the International Consensus on Antinuclear Antibody (ANA) Pattern (ICAP, ) Committee. The objective of this study was to use an internet-based survey to assess how accurately the DFS IIF pattern was recognized by experienced technologists. Methods: High-resolution digital IIF images were captured using the automated IIF NOVA View instrument (Inova Diagnostics, San Diego, CA). Ten images were posted in an anonymous, international, internet-based interpretive survey. Two hundred and thirty IIF technologists were invited to participate. Four of the images in the survey were from previously characterized serum samples with classical ANA IIF patterns (nucleolar, centromere, homogeneous, and speckled) and two of the images were from samples with a DFS IIF ANA pattern and isolated anti-DFS70 antibodies as determined by a chemiluminescence immunoassay. The remaining four images were from sera with the classic IIF ANA patterns referred to above and mixed with a monospecific anti-DFS70-positive sample. The survey included multiple choice selections: homogeneous, DFS, centromere, nucleolar, speckled, other, or unrecognizable. Results: 125 of the 230 participants who completed the survey had diverse levels of experience in IIF pattern recognition on HEp-2 cells ranging from <1 year to >10 years of experience (average >10 years). Participants had a high concordance in correctly classifying the classical ANA IIF patterns: ranging from 95.2 % for centromere to 74.4 % for nucleolar patterns. The unmixed DFS pattern was recognized with significantly lower accuracy (~50 %; p < 0.05). However, less than 10 % correctly identified mixed patterns derived from the sera containing both clinically relevant ANA and anti-DFS70 antibodies. Conclusions: Recognizing the DFS ANA IIF pattern and mixed IIF patterns composed of DFS + clinically relevant ANA patterns poses a significant challenge. Consequently, it seems imperative that DFS-specific immunoassays should be used to confirm the presence of anti-DFS70 antibodies before definitive results are reported to physicians. [ABSTRACT FROM AUTHOR]

Anti-signal recognition particle (SRP) antibodies are important serological markers for the diagnosis and the prognosis of idiopathic inflammatory myopathy (IIM), especially to distinguish immune-mediated necrotizing myopathy (IMNM). This study was set up to investigate the phenotype associated with anti-SRP antibodies and to evaluate the methods for detecting these antibodies. Clinical and biological data were retrospectively obtained from 60 adult patients with anti-SRP antibodies detected by a dot immunoassay from 12 centers. Thirty-six (60 %) out of these 60 patients suffered from an IIM, and among them, 21 patients were diagnosed as IMNM. Among patients with a definite IIM, proximal weakness and myalgia were prominent symptoms at the time of diagnosis. Only few patients displayed severe extra-muscular symptoms such as cardiac involvement or severe myositis. Mean creatine kinase levels were high for all patients except for two of them. When testing by indirect immunofluorescence (IIF) on HEp2 cells, the fraction of patients displaying the typical anti-SRP fine speckled staining of the cytoplasm was higher in patients with IIM (30/36) (83 %) than in patients with non-IIM (3/24) (12.5 %) ( p < 0.0001). Thirty (91 %) out of 33 patients with a positive immunodot and a characteristic IIF cytoplasmic staining suffered from a clinical definite myositis, whereas only 6 (22 %) out of 27 patients with a positive immunodot but a negative cytoplasmic pattern suffered from a myositis ( p < 0.00001). This series highlights the strong heterogeneity of anti-SRP positivity that encompassed IMNM and non-IMNM and supports the necessity of considering both IIF and dot immunoassay to confirm the diagnosis of anti-SRP-associated myositis. [ABSTRACT FROM AUTHOR]

Anti-endothelial cell antibodies (AECA) may be involved in the development of heart allograft rejection. Its detection might be a cheap and noninvasive method to identify high-risk patients. An indirect immunofluorescence method on human umbilical vein endothelial cells was used to investigate the presence of AECAs in 260 pre- and post-transplant serum samples sequentially collected from 34 patients within the first year after heart transplantation (HTX). The presence of AECAs before (23.5 %) and early after HTX (14.7 %) was associated with a significantly increased risk of early acute rejection (75 and 60 %, respectively) compared to 33 % in AECA-negative patients ( p = 0.049). Moreover, rejections from AECA-positive patients were more severe ( p = 0.057) with a significantly increased incidence of multiple ( p = 0.025). The mean number of the sum of rejection episodes was significantly higher in AECA-positive patients ( p ≤ 0.05). Patients free of AECAs mainly received mycophenolate mofetil as primary immunosuppression ( p = 0.067). Nevertheless, the presence of AECAs did not affect long-term outcome and mortality of HTX patients. Despite a low number of patient samples, the detection of AECAs before and early after HTX could be used as a biomarker for an increased risk of early acute rejection in high-risk patients. This easy method might be a valuable tool to support screening procedures to improve individualized immunosuppressive therapy. [ABSTRACT FROM AUTHOR]

Indirect immunofluorescence (IIF) is the main technique for the detection of antinuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA). The fully automated IIF processor HELIOS is the first IIF processor that is able to automatically prepare slides and perform automatic reading. The objective of the present study was to determine the diagnostic performance of this system for ANA and ANCA IIF interpretation, in comparison with visual IIF. ANA detection by visual IIF or HELIOS was performed on 425 sera samples including: 218 consecutive samples submitted to a reference laboratory for routine ANA testing, 137 samples from healthy subjects and 70 ANA/ENA positive samples. For ANCA determination, 170 sera samples were collected: 40 samples for routine testing, 90 samples from healthy blood donors and 40 anti-PR3/anti-MPO positive subjects. Good correlation was found for the visual and automated ANA IIF approach regarding positive/negative discrimination of these samples (kappa = 0.633 for ANA positive samples and kappa = 0.657 for ANA negative samples, respectively). Positive/negative IIF ANCA discrimination by HELIOS and visual IIF revealed a complete agreement of 100 % in sera from healthy patients and PR3/MPO positive samples (kappa = 1.00). There was 95 % agreement between the ANCA IIF performed by automated and visual IIF on the investigation of routine samples. Based on these results, HELIOS demonstrated a high diagnostic performance for the automated ANA and ANCA IIF interpretation that was similar to a visual reading in all groups of samples. [ABSTRACT FROM AUTHOR]

The QUANTA Flash CTD Screen Plus is a chemiluminescent immunoassay (CIA) for the detection of the major antinuclear antibodies (ANA) on the BIO-FLASH platform. NOVA View is an automated fluorescence microscope that acquires digital images of indirect immunofluorescent assay (IFA) slides. Our goal was to evaluate the clinical performance of the two automated systems and compare their performance to that of traditional IFA. Sera from patients with systemic autoimmune rheumatic diseases (SARD, n = 178), along with disease and healthy controls ( n = 204), were tested with the CTD CIA and with NOVA Lite HEp-2 ANA, using both the manual method of reading the IFA slides and the NOVA View instrument. The CTD CIA showed 78.1 % sensitivity for SARD, coupled with 94.1 % specificity. Manual IFA and NOVA View showed somewhat higher sensitivity (81.5 and 84.8 % in SARD, respectively), but significantly lower specificity (79.4 and 64.7 %, respectively). Both automated systems displayed somewhat different performance, due to the different principals of ANA detection: IFA with NOVA View digital image interpretation had higher sensitivity, while the CTD CIA showed higher specificity. With the added benefits of full automation, the new CTD CIA is an attractive alternative to traditional ANA screening. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is the causative agent of serious infection such as meningoencephalitis and septicaemia with about 25 % mortality rate. The frequency is as high as 50 % in neonates. Although Listeriosis is known increasingly as a serious threat for health, there is little study in some countries, especially Asian countries. Conventional methods for detection of L. monocytogenes are laborious and time-consuming. Tests that are based on indirect immunofluorescence (IIF) method are easy and fast. This study was carried out on 120 pregnant women aged between 14 and 45 years old (mean age of 27.6) suffering from abortion and 60 healthy pregnant women with the same mean age referred to Razi and Imam Khomeini hospitals for abortion in Ahvaz, Iran. In this study, blood samples from every woman were drawn for determining of antibody titer against L. monocytogene by indirect immunofluorescence method. The results of serologic study showed that 25 cases of the case group and four cases of the control group had an antibody titer of 1:160 against this microorganism. In the case group of 25 patients who had antibody titer 1:160, only 12 patients had 1:320 titer, while of four patients in the control group, two patients had 1:320 titer (odds ratio of 3.2). Therefore, in the case group, 20.83 % had a titer of 1:160, and 10 % had a titer of 1:320. In the control group, the rates of the above antibody titers were 6.66 and 3.33 %, respectively. In conclusion, the results investigations have shown that there is a serological respond to L. monocytogenes in the women of reproductive age. The number of positive patients was greater in women who had spontaneous abortion compared with women who never had the spontaneous abortion. [ABSTRACT FROM AUTHOR]

Lately, many hospital laboratories are using for antinuclear antibody (ANA) screening the solid-phase immunoassays (ELISA-ANA) instead of the traditional immunofluorescence ANA test (IF-ANA). Results of previous studies that compare the two technologies show poor correlation between them in both juvenile idiopathic arthritis (JIA) and childhood lupus erythematous (SLE) patients. In this study, we investigated whether ELISA-ANA and traditional IF-ANA results are comparable in pediatric patients with different rheumatic diseases. A total of 156 consecutive patients were included in the study-90 children with JIA, 33 with reactive arthritis, 19 with SLE, 4 with idiopathic chronic uveitis, and 10 with other systemic rheumatic diseases. ANA determination was performed using both assays. The higher rate of discrepancies between the two methods of ANA screening appeared in the JIA population (19/90, p < 0.001). All JIA patients with false-negative results by ELISA had significant or high IF-ANA titres. The prevalence of JIA-associated uveitis was higher in the group of false-negative ELISA-ANA children than in the ELISA-positive patients but without statistical significance ( p = 0.62). On the contrary, in SLE group, the consistency rate of the two assays was 100 %, and the reactivity levels of ELISA-ANA were significantly higher than in ELISA-positive JIA patients ( p < 0.001). ELISA seems to be a reliable method for screening ANA in childhood SLE, but not in JIA. Limited by the few SLE patients, our findings need further consideration. [ABSTRACT FROM AUTHOR]

Autoimmune diseases are very serious and also invalidating illnesses. The benchmark procedure for their diagnosis is the indirect immunofluorescence (IIF) assay performed on the HEp-2 substrate. Medical doctors first determine the fluorescence intensity exhibited by HEp-2 wells and then report the staining pattern. Despite its pivotal role, IIF is affected by inter- and intra-laboratory variabilities demanding for the development of computer-aided-diagnosis tools supporting medical doctor decisions. With reference to staining pattern recognition, state-of-the-art approaches recognize five main patterns characterized by well-defined cell edges. These approaches are based on cell segmentation, a task that recent work suggests to be harder than the classification itself. In this paper, we extend the panel of detectable HEp-2 staining patterns, introducing the recognition of centromere and cytoplasmic patterns, which have a high specific match with certain autoimmune diseases, from other stainings. Since image segmentation algorithms fail on these samples, we developed a classification system integrating local descriptors and the bag of visual word approach, which represents image contents without the burden of segmentation. We tested our approach on a large dataset of HEp-2 images with high variability in both fluorescence intensity and staining patterns correctly recognizing the 97.12 % of samples. The system has also been validated in a daily routine fashion on 108 consecutive IIF analyses of hospital outpatients and inpatients, achieving an accuracy rate of 97.22 %. [ABSTRACT FROM AUTHOR]

The autoimmune disorders (AID) have since long been considered to be commoner in Western world as compared to Asian countries. This, however, may not be true as in developing countries, there are incomplete epidemiological data and lack of advanced diagnostic facilities leading to under diagnosis in many cases. In this study, we performed an 11-year retrospective analysis of medical records of all clinically suspected and immunofluorescence antinuclear antibody test (IF-ANA)-positive cases. The IF-ANA-positive cases in the year 2006-2007 were further analyzed to find out the morbidity contribution by IF-ANA-positive AID. A total of 36,310 serum samples were screened for antinuclear antibody (ANA) between the years 1996 and 2006. The mean positivity was 12.3%. A constant and statistically significant increase in AID was noticed over the last 11 years. In the year 2006-2007, out of 3,435 suspected AID cases, 18.9% were ANA positive. Of these, 86.0% were adult patients with age ranging from 2¼ to 88 years. A female preponderance was also noted with a female-to-male ratio of 3:1. Among the ANA-positive connective tissue disorders (CTD), systemic lupus erythematosus was the most common clinical diagnosis (4.6/10,000 cases) followed by scleroderma (1.2/10,000) and overlap syndrome (0.7/10,000). Rheumatic, renal and hematopoietic systems were commonly involved. The overall frequency of CTD was 21%. The report is the first and largest hospital-based study from India, highlighting the rising incidence and clinical profile of ANA-positive AID. [ABSTRACT FROM AUTHOR]

Drug-induced pemphigus has been reported in patients receiving angiotensin-converting enzyme inhibitors. The aim of this work was to study a group of hypertensive patients without skin diseases treated with angiotensin-converting enzyme (ACE) Inhibitors (I), to verify the presence of serum circulating anti-antibodies. The indirect immunofluorescence showed that 33 sera (52.38%) presented autoantibodies directed to an antigen of the cytoplasm of the superficial epidermal keratinocytes. Two of the 33 positive sera had antibodies to Dsg1 and/or 3 in ELISA. Immunoblot analyses were negative. All the 48 control sera were found to have no circulating antibodies using the three assays. Our results would confirm that ACEI drugs may trigger the production of circulating autoantibodies also in patients without clinical manifestations of pemphigus. [ABSTRACT FROM AUTHOR]

We report on 2 patients with unusual retinal manifestations of cat scratch disease (CSD), caused by Bartonella henselae. Case 1. A 42-year-old farmer presented with a 5-day history of blurred vision in his right eye. Right visual acuity was 20/25. Fundus examination of the right eye revealed mild vitreous hemorrhage and diffuse retinal hemorrhages in the mid-peripheral retina. Fluorescein angiography showed multiple vasculitic occlusions in the same area. A blood sample taken on the day of examination revealed the presence of immunoglobulin (Ig)M and IgG to B. henselae. Oral azithromycin was given for 8 days. One month later, right visual acuity was 20/20, the vitreous and retinal hemorrhages resolved, and arteriolar attenuation and sclerosis was observed in the peripheral temporal retina. Case 2. A 66-year-old craftsman with systemic hypertension and hypercholesterolemia complained of sudden visual loss (light perception) in his left eye. Fundus evaluation and fluorescein angiography revealed central retinal artery occlusion (CRAO) in the affected eye. About 2 weeks earlier, he had been bitten and scratched on his right hand by a stray cat. Serologic testing detected the presence of IgM to B. henselae. Oral azithromycin was given for 6 days. One month later, left visual acuity was hand motion. Ophthalmologists should be aware that unusual ocular complications associated with CSD include vitreous hemorrhage with retinal vasculitis and isolated CRAO. Vitreous hemorrhage and retinal vasculitis may be the only clinical manifestation of CSD. [ABSTRACT FROM AUTHOR]

Background: Clustering of autoimmune diseases is common and may be due to genetic background and exposition to environmental triggers. Objective: The aim is to carry out a laboratory and clinical study of the prevalence of gastrointestinal organ-specific autoantibodies in rheumatoid arthritis (RA) patients and their relatives. Methods: Serum samples of 156 RA patients, 200 relatives, and 100 healthy controls were studied for anti-smooth muscle antibody (ASMA), anti-mitochondrial (AMA), anti-parietal cell (APCA), anti-liver-kidney microsome (LKM), and anti-endomysium antibodies (IgA-EmA) by indirect immunofluorescence. Results: A total of eight out of the 156 (5.1%) RA patients were positive for the autoantibodies (ASMA = 1; AMA = 2, APCA = 5). In the relative group, 12/200 (6%) had at least one positive autoantibody (ASMA = 1; AMA = 2, APCA = 7, IgA-EmA = 2). In the control group, two out of the100 (2%) healthy controls were positive (ASMA = 1, APCA = 1). No statistical difference was found between RA patients, their relatives, and controls in relation to the frequency of autoantibodies evaluated. Conclusion: Although RA patients and their relatives have positivity of AMA, ASMA, and APCA without statistical difference in relation to healthy individuals, the findings may be of value for adequate clinical approach of these subjects. [ABSTRACT FROM AUTHOR]

In this study, preimplantation mouse embryos were used as a new model for the study of actin distribution in nuclei and the identification of functional forms of nuclear actin. The combination of the direct detection of actin by fluorescent-conjugated phalloidin and DNase I with indirect immunofluorecence was applied as an integrated approach to studying the localization of actin in nuclei of two-cell mouse embryos. Aggregates of monomeric actin and two oligomeric forms of actin were revealed in nuclei. Each of these forms demonstrated their own pattern of distribution. Oligomeric actin recognized by antibodies to the actin C-terminal domain was associated with condensed chromatin, as well as with metaphase chromosomes and chromatin of the second polar body. Monomeric actin and another oligomeric form recognized by antibodies to actin N-terminal domain were revealed in the area of dispersed chromatin localization. [ABSTRACT FROM AUTHOR]

Background/Aims. Anti-Saccharomyces cerevisiae-antibodies (ASCA) are used to discriminate Crohn’s disease (CD) from ulcerative colitis (UC). ASCA tests are not standardised and different methods for ASCA detection exist. This study was undertaken to compare ASCA tests and to clarify their diagnostic value. Patients/Methods. One hundred and two sera from CD patients, 53 from UC patients, and 50 sera from normal controls were examined for ASCA IgA and ASCA IgG using four different ELISA (Aesku.lab, Inova, Euroimmun, and Medipan) and indirect immunofluorescence (Euroimmun). Sensitivity, specificity, and positive and negative predictive values (PPV and NPV) for CD in this population were determined. Agreement between tests was expressed by kappa statistics. Results/Findings. The presence of either ASCA IgA or ASCA IgG had a PPV between 77 and 88%. Only the combined presence of ASCA IgA and ASCA IgG had a specificity which was constantly above 90%. Sensitivity, specificity, PPV, and NPV were then: Inova: 53, 97, 95, and 68%; Euroimmun ELISA: 46, 97, 94, and 66%; Aesku.lab: 50, 97, 94, and 66%; Medipan: 30, 98, 94, and 59%; indirect immunofluorescence: 51, 97, 94, and 66%. Agreement between ELISA test results of the Aesku.lab, Euroimmun, Medipan IgA and Inova systems was good (kappa: 0.63–0.79); between the Medipan IgG ELISA or indirect immunofluorescence and the others, it was lower (kappa: 0.33–0.6). If both ASCA IgA and ASCA IgG were detected by indirect immunofluorescence as well as ELISA, specificity for CD increased to >99% at a sensitivity of 23–38%. Interpretation/Conclusions. The combined detection of ASCA IgA and ASCA IgG by indirect immunofluorescence as well as ELISA may optimise the discrimination of CD from UC. [ABSTRACT FROM AUTHOR]

Background Methods. We studies 1714 corporate workers (median age, 48 years; range, 30 to 59 years) who had an annual health check from 1998 to 1999 at Kawasaki Social Insurance Hospital in Japan. We used an indirect immunofluorescence method for screening serum AMA. We applied the prevalence of AMA-positive people in the study group to the general population in Japan. Then the inferred AMA-positive population was compared to the actual number of patients with PBC in statistics published by the Japanese Government. Results. AMA was detected in 11 of 1714 people (0.64%; 95% confidence interval, 0.26% to 1.02%). All these 11 sera reacted with 2-oxoacid-dehydrogenase complex by immunoblotting. Of these 11 individuals, none had subjective symptoms, all had normal serum bilirubin levels, and 6 had abnormal liver function test results. Using published statistics for the Japanese population, we inferred that there were approximately 336 472 AMA-positive people in Japan from age 30 to 59 years. The number of patients with symptomatic PBC recorded by the nationwide epidemiological survey of the Japanese Government was 2459. Thus, we inferred the rate of symptomatic PBC among AMA-positive persons to be about 0.73% (2459/336 472). Conclusions. AMA is not a rare antibody in the general population, but few people develop recognizable PBC even if they have AMA. The prevalence of antimitochondrial antibody (AMA) in humans and its relationship to the development of primary biliary cirrhosis (PBC) are not well known. We have estimated the frequency of AMA in the general population, and studied its association with PBC. [ABSTRACT FROM AUTHOR]

Non-organ-specific autoantibodies (NOSAs) are frequently found in patients with hepatitis C virus (HCV) chronic infection. Genetics is likely involved in the development of autoimmune reactivities, and differences in the prevalence of HCV-related autoantibodies among populations of various geographic areas should be expected. We evaluated the prevalence and the clinical impact of NOSAs in a series of HCV-infected patients from southern Italy. We studied 283 consecutive anti-HCV positive patients (162 men, 121 women, mean age 54.5 +/- 13.5 years), 94 of whom were cirrhotics and 189 noncirrhotics. Serum from each patient and from 41 hepatitis B surface antigen (HBsAg)-positive/anti-HCV negative control subjects were tested (dilution 1:40) for autoantibodies by indirect immunofluorescence. Qualitative/quantitative HCV-RNA determinations were also performed. The prevalence of NOSAs was significantly higher in anti-HCV-positive subjects than in HBsAg-positive patients (P < 0.006). Autoantibodies were significantly associated with both cirrhosis (P < 0.0001) and older age (P < 0.05). No significant association between NOSAs and either female gender or virological parameters (HCV-RNA positivity, viral load, and genotype) was found. In conclusion, the autoantibody positivity in HCV-infected patients from southern Italy is significantly related to cirrhosis and older age, although its general prevalence is similar to that reported in populations from the north of the country. [ABSTRACT FROM AUTHOR]

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The present study was designed to identify and characterize muscarinic acetylcholine receptors in normal human melanocytes. We used subtype-specific oligonucleotide primers to localize the five genetically defined mAChR mRNAs (m1 through m5) by reverse transcription-polymerase chain reaction. These experiments showed that all five mAChR subtype mRNAs are expressed in melanocytes. The PCR products were verified by restriction analysis and Southern blotting. Receptors were visualized in cultures of normal human melanocytes and specimens of normal human skin by subtype-specific rabbit anti-receptor polyclonal antibodies. Radioligand binding assays with the lipophilic drug [3H]quinuclidinyl benzilate demonstrated approximately 9000 high affinity binding sites/cell. Micromolar concentrations of muscarine or carbachol transiently increased intracellular Ca2+, which could be attenuated by atropine, demonstrating coupling of the receptors to mobilization of intracellular free Ca2+. Lower concentrations of muscarine induced spontaneous repetitive spike-like increases of intracellular Ca2+ which is characteristic for the activation of muscarinic receptors. These results indicate that normal human skin melanocytes express the m1, m2, m3, m4, and m5 subtypes of classic muscarinic acetylcholine receptors on their cell membrane and that these receptors regulate the concentration of intracellular free Ca2+, which may play an important physiologic role in melanocyte behavior and skin pigmentation. [ABSTRACT FROM AUTHOR]

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are - at least in species with large genomes - intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence. [ABSTRACT FROM AUTHOR]

The acetylation pattern of H3 was studied on field bean chromosomes by means of indirect immunofluorescence using polyclonal antibodies recognizing H3 isoforms acetylated at lysine positions 9/18, 14 and 23. H3 was found to be hypoacetylated at lysine residues 9/18 and 14 within the heterochromatic regions composed of tandem repetitive Fok-I elements. Hyperacetylation of these residues was observed at the nucleolar organizing region (NOR) and in heterochromatic regions composed of repeats other than Fok-I elements. In contrast, H4 was underacetylated (H4.Ac5, 8, 12) or uniformly acetylated (H4.Ac16) at all heterochromatic regions, and acetylated above the average at all four lysines only within the NOR. Acetylation of lysine-23 of H3 was uniform, except for the NOR that showed no fluorescence. Inhibition of deacetylase during and after replication of heterochromatin by trichostatin A had no influence on the acetylation status of H3 but mediated an increase in acetylation of lysines 5, 12 and 16 of H4 above the average in the field bean heterochromatin. Thus, the chromosomal acetylation patterns of H4 and H3 of this species revealed common and divergent features. Whereas the acetylation level of H4 correlates well with the potential transcriptional activity and inversely with the time of replication of defined chromatin domains of Vicia faba, this is not generally true for H3. [ABSTRACT FROM AUTHOR]

α-Smooth muscle (αSM) actin of endothermic vertebrates is selectively recognized by the monoclonal antibody anti-αSM-1. Immunoreactivity to this antibody has been shown to be localized in the NH-terminal sequence Ac-EEED (Chaponnier et al. 1994). Among terrestrial ectothermic vertebrates, two amphibian ( Triturus vulgaris, Rana esculenta) and three reptilian species ( Pseudemys scripta elegans, Natrix natrix, Podarcis sicula) were screened to investigate if their vascular and visceral smooth muscles were stained by anti-αSM-1. In all the specimens tested, Western-blot analysis of tissue extracts immunodecorated with anti-αSM-1 revealed a single polypeptide chain having the same electrophoretic mobility as bovine αSM actin. The binding to amphibian and reptilian tissue extracts was inhibited by the synthetic peptide Ac-EEED, but not Ac-DEED, as occurs in mammals. αSM actin expression was found in vascular and visceral smooth muscle cells of the species tested. The media of small and large blood vessels was labelled by anti-αSM-1. In the stomach and intestine the outer longitudinal and inner circular layers of the muscularis and of the muscularis mucosae were stained. In addition, myofibroblasts of the subepithelial layer were labelled. A more restricted expression of this isoactin was detected in turtle ( P. scripta elegans) visceral smooth muscle cells, which may be related to the involvement of the digestive system in respiratory activity. These data suggest that in vertebrate evolution αSM actin arose earlier than previously proposed. [ABSTRACT FROM AUTHOR]

The monoclonal antibody A2B5 reacts with the surface membrane of most neurons in monolayer cultures of cerebellum, retina, spinal cord, and dorsal root ganglion of embryonic and early postnatal C57BL/6J mice maintained in vitro for culture periods of 2 to 10 days. A small percentage of astroglial cells also expresses A2B5 antigen in murine, chicken and rabbit cerebellum, in chicken retina, and in murine spinal cord and dorsal root ganglion. Less mature astroglial cells are stained for A2B5 antigen to a greater extent than the more mature astrocytes. Astrocytes from rat cerebellum and mouse retina were not found to express A2B5 antigen under the present culture conditions. Some of the less mature oligodendrocytes recognized by 04 antibodies express A2B5 antigen, while the more mature 01 antigen and galactocerebroside-positive oligodendrocytes were not found to be A2B5 antigen-positive. Fibroblasts or fibroblast-like cells do not express detectable levels of A2B5 antigen. After fixation of the cells with paraformaldehyde and ethanol, all cell types present in culture are labeled by the A2B5 antibody intracellularly. [ABSTRACT FROM AUTHOR]

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera ( Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia. [ABSTRACT FROM AUTHOR]

The cytoplasmic islet cell antibody patterns of sera from islet cell antibody positive non-diabetic and diabetic endocrine autoimmune patients, and newly-diagnosed Type 1 (insulin-dependent) diabetic patients were characterised using four layer immunofluorescence with monoclonal antiproinsulin or anti-glucagon antibodies. Two distinct islet cell antibody types were identified. One gave a diffuse cytoplasmic staining in both Beta and Alpha cells ('whole' islet pattern), and was not affected by pre-incubation with rat brain homogenate. The other had a granular appearance with staining restricted predominantly to Beta cells ('selective' islet pattern) and was completely inhibited by pre-incubation with rat brain homogenate. Some sera appeared to have a 'mixed' islet pattern, in which glucagon-positive cells gave a weaker cytoplasmic staining than proinsulin-positive cells. The granular 'selective' pattern was found in sera from 19 (79%) of 24 non-diabetic endocrine autoimmune patients, in two (22%) endocrine autoimmune patients who developed Type 1 diabetes ( p<0.0001 vs non-diabetic endocrine autoimmune patients), and in none of 19 newly-diagnosed diabetic patients. The 'whole' islet pattern was found only in sera from patients who had, or who subsequently progressed to, Type 1 diabetes. This study has identified a novel islet cell antibody specificity and demonstrates that in islet cell antibody positive endocrine autoimmune patients, only islet cell antibodies which stain both Beta and Alpha cells are associated with progression to Type 1 diabetes. [ABSTRACT FROM AUTHOR]

The conventional indirect immunofluorescence test of islet cell antibodies was recently improved by the development of a two-colour immunofluorescence assay using a monoclonal proinsulin antibody to detect islet B cells. The aim of this study was to test whether in this new assay the prevalence and titre of ICA were affected by the time of incubation carried out in the presence of aprotinin (Trasylol) as an inhibitor of proteolysis. The end-point titre of ICA was therefore determined in sera from 70 children aged 0.6 to 15 years with recent onset Type 1 (insulin-dependent) diabetes mellitus, 50 healthy control subjects and 97 non-diabetic siblings of Type 1 diabetic children. In the conventional twocolour assay, ICA was positive in 53/70 (76%) Type 1 diabetic patients, 1/50 control subjects and 2/97 siblings after 30 min incubation. Prolonged incubation for 18 h increased the prevalence of ICA positive samples to 62/70 (89%) in the diabetic patients and to 2/50 in the control subjects, while the prevalence among the siblings was unchanged. Of the ICA positive non-diabetic subjects, one control child has a father with Type 1 diabetes, and one of the siblings subsequently developed Type 1 diabetes. In the diabetic patients the median titre was 1:32 for the 30 min incubation, and it increased to 1:64 for the 18 h incubation ( p<0.001). A marked prozone effect was seen; 16% of the samples from the Type 1 diabetic children sera were negative at a 1:2 dilution, but were found positive at higher dilutions. In conclusion, an 18 h incubation increases the end point titres and the prevalence of ICA in the two-colour ICA assay in Type 1 diabetic children of recent onset. The prevalence and levels of ICA among these patients may be larger than hitherto expected. [ABSTRACT FROM AUTHOR]

Studying 239 Type 1 (insulin-dependent) diabetic patients and 144 of their first-degree relatives, we found a significant prevalence of autoimmune manifestations in both groups, compared with sex-and age-matched control subjects ( p< 0.001). In particular, in diabetic patients we found a high frequency of autoimmune thyroid disease and idiopathic Addison's disease and also a significant prevalence of thyroid ( p< 0.001), parietal cell ( p< 0.05) and adrenal antibodies ( p< 0.05). In the relatives a high frequency of thyroid disease, thyroid, parietal cell and adrenal antibodies and a significant prevalence of islet cell antibodies ( p< 0.05) were detected. In both groups functional glandular tests and gastric biopsies performed on the basis of autoantibody positivity revealed 13 examples of subclinical hypothyroidism, two cases of reduced adrenocortical reserve and five of atrophic gastritis. Autoantibody screening in diabetic patients and their relatives permitted the early diagnosis of the underlying endocrine disorders. [ABSTRACT FROM AUTHOR]

The sensitivity and specificity of the assay for islet cell cytoplasmic antibodies in human serum were examined using cryostat sections from fresh frozen pancreas. The specificity of the assay was close to 100% while the sensitivity was 40%-98% depending on the pancreas used. Inter-observer variation was 12-27%. End-point titres of islet cell antibodies varied with the sensitivity of each pancreas. End-point titration of the antibodies in two different laboratories using the same pancreas was significantly correlated (Spearman test p < 0.001). We conclude that a reliable determination of islet cell antibody titres in human serum requires careful characterization of the sensitivity and specificity of each pancreas used as a source of frozen sections, in the indirect immunofluorescence assay. [ABSTRACT FROM AUTHOR]

We have determined thyroid microsomal and gastric parietal cell autoantibodies in 972 patients with onset of Type 1 (insulin-dependent) diabetes before age 20 years. In a cross sectional study, the frequencies of these autoantibodies did not change significantly over the ensuing 20 years following diagnosis of diabetes. In a longitudinal study of 424 patients with Type 1 diabetes followed up to 5.3 years, few new patients with thyro-gastric autoantibodies were identified. Whereas the frequencies of these autoantibodies among the diabetic patients were more than fivefold greater than for age, sex and race matched controls, they resembled those seen in the non-diabetic general population (n=1524) for the over 60 year age group. We conclude that in patients with thyrogastric autoantibodies and Type 1 diabetes, the onset of autoimmune processes affecting the thyroid, gastric mucosa and pancreatic islets tend to be simultaneous, although the resultant diseases have different natural histories. [ABSTRACT FROM AUTHOR]

The reactivity of islet cell cytoplasmic antibodies (ICA)-positive and ICA-negative sera of recent onset type 1 diabetic patients was studied in human fetal pancreata of 12-18 weeks' gestation and compared with the reactivity of these sera in adult human control pancreata. The aims of the study were: (1) to observe the presence of ICA staining in human fetal islet cells; (2) to compare endpoint titres (in Juvenile Diabetes Foundation units) of ICA-positive patient sera in fetal pancreata and adult human control pancreata. Ten ICA-positive sera and eight ICA-negative sera from newly diagnosed diabetic patients and four sera from healthy controls were tested on three human adult and eight human fetal pancreata. As in the adult control pancreata. ICA-positive sera reacted to insulin-, glucagon-, and somatostatin-positive cells of fetal pancreata of all gestational ages. This was observed both in single cells and in cells in islet-like cell clusters. Dilution of a reference serum gave similar results in both adult and fetal pancreata. In contrast, the ICA-positive patient sera yielded a striking heterogeneity in fetal as well as in adult pancreata. However, end-point titres between adult and fetal pancreata did not differ significantly ( P>0.05). In conclusion, ICA-positive sera from recent onset diabetic patients show that the expression of molecules to which ICA react is present in all islet cells and starts before week 12 of gestation. [ABSTRACT FROM AUTHOR]

To elucidate the cellular basis of hemoglobin transition in Xenopus laevis the distribution of larval and adult hemoglobins was analyzed by indirect immunofluorescence in the circulating erythrocytes during metamorphosis. In addition, the morphological characteristics as well as the capacity for synthesis of DNA and hemoglobin in the erythrocytes were followed during the same developmental period. Our quantitative analysis on the distribution of larval and adult hemoglobins suggests that they are localized in different cells. Hemoglobin transition, therefore, most likely reflects replacement of the larval erythrocyte population by new cells which are committed to adult globin synthesis. Since hemoglobin transition is not accompanied by an increase in the abundance of immature erythroid cells with active DNA synthesis, we assume that the presumptive adult erythroid cells are released into circulation at a relatively advanced stage of maturation. The decline in the synthesis of DNA and larval hemoglobin further indicates that cessation of cell renewal in the larval erythrocyte population may represent a decisive step in hemoglobin transition. [ABSTRACT FROM AUTHOR]

Serum samples from 90 cases of rheumatoid arthritis (RA), 218 control patients and 100 healthy controls were tested by indirect immunofluorescence for the presence of the antiperinuclear factor (APF), an autoantibody to the keratohyaline granules of human buccal mucosal cells. The sensitivity and specificity of the APF test for RA were 82 and 90%, respectively, slightly higher than those obtained when the same serum samples were tested for rheumatoid factor (RF) using a latex agglutination method. The diagnostic gain of APF in the RF-negative RA cases was greater than that of RF in the APF-negative cases (54% vs. 37%). In spite of a few technical disadvantages, the APF test should be therefore performed in the serological routine examination of rheumatic diseases. As APF was also found in 11% of degenerative joint disease (DJD) cases and the estimated prevalence ratio RA/DJD is, in Italy, 1/11, the predictive value of a negative APF test proved to be much higher than that of a positive test (98% vs. 40%) for the diagnosis of RA. APF was significantly associated with other autoantibodies (RF and antinuclear antibody). [ABSTRACT FROM AUTHOR]

Protonemata of Funaria hygrometrica were exposed to the herbicidal MT inhibitor oryzalin. A reduction of the growth rate together with a disturbance of oriented polar growth is observed. Both effects are reversible. Visualization of MT by IFT reveals differential sensitivities of MT. At lower concentrations (⩽10 M) only the cytoplasmic MT are depolymerized causing impairment of the migration of the nucleus and the transport of the plastids. Close association of MT with the surface of the plastids is demonstrated. At higher concentrations of oryzalin spindle and phragmoplast MT are affected as well. They are found in unusual orientations and display a variety of aberrant forms like multipolar spindles or the occurrence of several 'mini-spindles' within one cell. The mode of action of oryzalin is discussed and the necessity of a continuous network of cytoplasmic MT between nucleus and growing tip for the maintenance of polar growth is emphasized. [ABSTRACT FROM AUTHOR]

Background: The International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) has recently proposed nomenclature in order to harmonize ANA indirect immunofluorescence (IIF) pattern reporting. ICAP distinguishes competent-level from expert-level patterns. A survey was organized to evaluate reporting, familiarity, and considered clinical value of ANA IIF patterns. Methods: Two surveys were distributed by European Autoimmunity Standardization Initiative (EASI) working groups, the International Consensus on ANA Patterns (ICAP) and UK NEQAS to laboratory professionals and clinicians. Results: 438 laboratory professionals and 248 clinicians from 67 countries responded. Except for dense fine speckled (DFS), the nuclear competent patterns were reported by > 85% of the laboratories. Except for rods and rings, the cytoplasmic competent patterns were reported by > 72% of laboratories. Cytoplasmic IIF staining was considered ANA positive by 55% of clinicians and 62% of laboratory professionals, with geographical and expertise-related differences. Quantification of fluorescence intensity was considered clinically relevant for nuclear patterns, but less so for cytoplasmic and mitotic patterns. Combining IIF with specific extractable nuclear antigens (ENA)/dsDNA antibody testing was considered most informative. Of the nuclear competent patterns, the centromere and homogeneous pattern obtained the highest scores for clinical relevance and the DFS pattern the lowest. Of the cytoplasmic patterns, the reticular/mitochondria-like pattern obtained the highest scores for clinical relevance and the polar/Golgi-like and rods and rings patterns the lowest. Conclusion: This survey confirms that the major nuclear and cytoplasmic ANA IIF patterns are considered clinically important. There is no unanimity on classifying DFS, rods and rings and polar/Golgi-like as a competent pattern and on reporting cytoplasmic patterns as ANA IIF positive. [ABSTRACT FROM AUTHOR]

The ontogeny and localization of crystallins during free lens development (i.e. lens development without the optic vesicle) were investigated in Xenopus laevis using the indirect immunofluorescence staining method with an antiserum raised against homologous total lens soluble proteins. Since the developing free lenses pass through stages similar to those of the lenses regenerated from the inner cell layer of the outer cornea following lentectomy in the same species Freeman's classification was used to identify the stages of free lens development. The first appearance of a positive reaction occurred at early stage IV in a number of cells in an area where future lens fibre cells would develop. With further differentiation of the free lens more and more cells in the fibre area started to show a positive reaction and the first positive reaction in the epithelium was observed late in stage V. Histological examination revealed that a fully differentiated free lens and a normally developed lens are similar but that the free lens is smaller. [ABSTRACT FROM AUTHOR]