Indirect Immunofluorescence Method Research Articles

Investigation of antinuclear antibodies in chronic hepatitis B patients

Antinuclear antibodies (ANA) facilitate the diagnosis and evaluation of patients in many systemic autoimmune conditions. Besides, ANA may also be detected in chronic infectious diseases. Although a number of investigations associated with autoantibody positivity in patients with chronic hepatitis C were reported, autoantibody positivity in patients with chronic hepatitis B remain rarely addressed in the literature. The aim of this study was to evaluate the antinuclear antibody (ANA), antimitocondrial antibody (AMA), anti-smooth muscle antibodies (ASMA) and anti-liver-kidney microsomal antigen (LKM) antibodies in chronic hepatitis B patients. Serum samples were obtained from adult patients with chronic hepatitis B diagnosis according to "European Association for the Study of the Liver (EASL)" criteria. Samples were taken from 47 patients (22 female, 25 male) with treatment-naive, histologically-proven chronic hepatitis B. Cases co-infected with HCV and/or HIV or that also had systemic autoimmune diseases were excluded. As a control group, 30 healthy blood donors were included in the study. Autoantibodies, including ANA, AMA, ASMA and LKM were detected with indirect immunofluorescence (IIF) method (Euroimmune, Lubeck, Germany) and evaluated by fluorescence microscope (Eurostar III plus, Germany). Positive results were graded into 4 levels ( "+", "++","+++" and "++++") from weak to strong Positive samples were studied with a immunoblotting method (ANA Profile 3, Euroimmun, AG) for the detection of extractable nuclear antigen (ENA). The positive results were detected in 8 (17%) of the HBV patients while all the samples were negative in the control group. The difference between the groups was significant (p< 0.05). Among the 47 serum samples tested, none of the patients were positive for AMA, ASMA, LKM. ANA was present in eight of the serum samples in which six were female and two were male patients. Among the IIF patterns of ANA positivity, one mixed pattern (homogeneous and nucleolar) and one cytoplasmic anti-golgi antibody pattern were detected. Positivity grade was ''++''. Other positive patterns were nucleolar (two patients), granular (two patients), ribozomal (one patient) and homogeneous (one patient) and positivity grade was ''+''. ENA was detected in three samples. Two of them was granular pattern positive samples. SS-A was borderline (±) in one and SS-B was borderline (±) in one of the samples. In the mixed pattern positive sample, histon was detected as ''+''. Autoantibody positivity between the patient and control groups were statistically significant (p< 0.05). The difference between autoantibody positivity and gender/age was not statistically significant. In conclusion, autoimmune manifestations may be detected in patients with chronic hepatitis B. Low level titer of antibodies such as ANA, AMA, ASMA or LKM may be present in such patients. An increased frequency of these autoantibodies may be associated with non-autoimmune conditions such as chronic viral infection even in treatment-naive patients.

Comparative Evaluation of Indirect Immunofluorescence and NS-1-Based ELISA to Determine Zika Virus-Specific IgM

Differential diagnosis of the Zika virus (ZIKV) is hampered by cross-reactivity with other flaviviruses, mainly dengue viruses. The aim of this study was to compare two commercial methods for detecting ZIKV immunoglobulin M (IgM), an indirect immunofluorescence (IIF) and an enzyme immunoassay (ELISA), using the non-structural (NS) 1 protein as an antigen, both from EuroImmun, Germany. In total, 255 serum samples were analyzed, 203 of which showed laboratory markers of ZIKV infections (PCR-positive in serum and/or in urine and/or positive or indeterminate specific IgM). When tested with IIF, 163 samples were IgM-positive, while 13 samples were indeterminate and 78 were negative. When IIF-positive samples were tested using ELISA, we found 61 positive results, 14 indeterminate results, and 88 negative results. Among the indeterminate cases tested with IIF, ELISA analysis found two positive, two indeterminate, and nine negative results. Finally, 74 of the 78 IIF-negative samples proved also to be negative using ELISA. For the calculations, all indeterminate results were considered to be positive. The agreement, sensitivity, and specificity between ELISA and IIF were 60.2%, 44.9%, and 94.9%, respectively. Overall, 101 samples showed discrepant results; these samples were finally classified on the basis of other ZIKV diagnostic approaches (PCR-positive in serum and/or in urine, IgG determinations using IIF or ELISA, and ZIKV Plaque Reduction Neutralization test—positive), when available. A final classification of 228 samples was possible; 126 of them were positive and 102 were negative. The corresponding values of agreement, sensitivity, and specificity of IIF were 86.0%, 96.8%, and 72.5%, respectively. The corresponding figures for ELISA were 81.1%, 65.9%, and 100%, respectively. The ELISA and IIF methods are both adequate approaches for detecting ZIKV-specific IgM. However, considering their respective weaknesses (low sensitivity in ELISA and low specificity in IIF), serological results must be considered jointly with other laboratory results.

Estimate of the Prevalence of Anti-Gastric Parietal Cell Autoantibodies in Healthy Individuals Is Method Dependent.

Anti-parietal cell antibodies (APCA) are a serologic marker of autoimmune gastritis. Their prevalence in healthy individuals is not well defined. We evaluated APCA prevalence in 515 healthy blood-donors by rat/primate tissue indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), and immunoblot. Fifty-three of 515 (10.3%) subjects were positive for APCA by at least one method: 18 only by ELISA, 10 by rodent tissue IIF, and one by primate tissue IIF; 18 were positive by ELISA and primate tissue IIF, and one by ELISA and rodent tissue IIF. Two were positive by both IIF methods, and three were triple positive. APCA positivity was confirmed by immunoblot in 100% of ELISA positive, in 95.8% of positive primate tissue IIF, and in 50% of positive rat tissue IIF. A great discrepancy in APCA prevalence detected by different methods in this cohort was apparent. Thus, the results on APCA prevalence in healthy individuals are likely method-dependent.

Human Epithelial Type-2 Cell Image Classification Using an Artificial Neural Network with Hybrid Descriptors

ABSTRACTAntinuclear antibody (ANA) testing is best performed using the indirect immunofluorescence (IIF) method with human epithelial type-2 (HEp-2) cells as the substrate. IIF is a subjective procedure in which HEp-2 patterns are analyzed manually from the microscope. Therefore, ANA test results greatly rely on the experience and expertise of pathologists. Hence, complete automation of the ANA test is required to avoid incorrect diagnoses. This paper represents an algorithm for the complex HEp-2 cell classification problem. The proposed algorithm used a small hybrid feature set that characterizes the texture and morphology of the HEp-2 cells along with artificial neural network (ANN). The hybrid features were extracted by breaking up the image into eight binary images. The proposed hybrid descriptors were more efficient than the popular co-occurrence matrix descriptor and local binary pattern descriptors for texture analysis. The proposed algorithm was evaluated on the ICPR 2016 IIF HEp-2 cell image dataset. The results indicated that the hybrid descriptor with an ANN approach achieved improved performance, with “96.8%” mean class accuracy.

The effects of melatonin on the activity of the c-fos gene in the structures of the hypothalamic paraventricular nucleus under prolonged lighting

The effect of constant illumination as a stress factor on the state of the "early response" c-fos gene in the lateral magnocellular subnuclei of the paraventricular nucleus (lmPVN) of the hypothalamus of rats at different time intervals (day and night) has been studied. To identify c-Fos in histological sections of the hypothalamus, a secondary (indirect) immunofluorescence method is used. Expression of the product of this gene, the c-Fos protein, in animals kept under normal conditions of alternation of illumination and darkness, shows a clear-cut circadian character. There was a significant decrease in the area of the immunopositive sites of structures at night by 19.4% compared to daytime measurements. The average values of the areas of such immunopositive subnuclei sites varied also in subgroups of rats under light stimulation conditions, in which samples of lmPVN were selected for study at 2 pm and 2 am, but the differences between groups did not reach the level of reliability. Under conditions of light stress, the c-Fos concentration index decreased by 29.4% during the day, and by 16.5% at night in relation to similar values in the intact group. In animals that were in the standard light mode, the c-Fos protein concentration index was significantly higher during the day than at night. In the intact group, the night value of the c-Fos concentration index averaged only 71.5% of the daily value. In rats subjected to constant illumination, the day and night values of the c-Fos concentration index did not differ reliably. To correct stress-induced changes in the activity of the "early response" c-fos gene in the rat hypothalamus, melatonin (0.5 mg/kg body weight of the animal) was used. Injections of melatonin to rats exposed to constant illumination normalized the circadian rhythm of the area of the material immunoreactive to c-Fos, compared to the group of animals that did not receive melatonin. When using melatonin against the background of constant illumination, a sharp increase of the protein concentration in the lmPVN subnuclei of hypothalamus in daytime and less pronounced at night intervals was detected. Injections of melatonin to animals were also reflected in the daily dynamics of the index of c-Fos protein content in the lmPVN subnuclei under conditions of constant illumination. In the daytime observation period, the index was almost twice the experimental data on stressed animals without the introduction of the hormone, bringing it closer to normal. Prospects for further research in this sphere will allow us to better understand the place and role of subnuclei of paraventricular nuclei of the hypothalamus in the mechanisms of the formation of circadian rhythms of the rat brain.

Comparison between direct and indirect immunofluorescence method for determination of somatic cell count

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.

Solid phase assays versus automated indirect immunofluorescence for detection of antinuclear antibodies.

Solid phase assays (SPAs) and automated microscope systems are increasingly used to screen for antinuclear antibodies (ANAs). The goal of this study was to evaluate the performance of three automated ANA screening assays; NOVA Lite HEp-2 using NOVA View® (NV, Inova Diagnostics), an automated indirect immunofluorescence method, EliA™ CTD Screen (Fluorescence Enzyme Immunoassay, FEIA; Thermo Fisher) and QUANTA Flash® CTD Screen Plus (Chemiluminescence immunoassay, CIA; Inova Diagnostics). The assays were performed on 480 diagnostic samples from patients with an ANA-associated rheumatic disease (AARD; systemic lupus erythematosus, primary Sjögren's syndrome, systemic sclerosis, inflammatory myopathy, mixed connective tissue disease) and on 767 samples from diseased and healthy controls. Using cut-offs proposed by the manufacturers, the sensitivity was 95%, 80.5% and 86% for NV, FEIA and CIA, respectively. The corresponding specificity was 61% (NV), 97.5% (FEIA) and 88% (CIA). The sensitivity associated with a specificity of ~95% was 79%, 82% and 78% for NV, FEIA, and CIA, respectively. Receiver operating characteristics (ROC) curve analysis revealed no differences in area under the curve (AUC) between the 3 assays when all diseases were grouped. For Sjögren's syndrome, the AUC was higher for SPAs than for NV, whereas for systemic sclerosis, the AUC was higher for NV than for CIA. For all assays, the likelihood ratio for AARD increased with increasing antibody levels and for double positivity of NV with SPA. In conclusion, the performance of automated SPA and IIF was assay- and disease-dependent. Taking into account antibody levels and combining IIF with SPA adds value.

Human epithelial type-2 cell categorization using hybrid descriptor with binary tree

Indirect immunofluorescence (IIF) method with human epithelial type-2 (HEp-2) cell as substrates is recommanded in anti nuclear antibody (ANA) test. These IIF slides are observed under microscope by pathologists to prepare the report. So, the ANA test is subjective and requires systematic automation. This paper proposes a novel algorithm for HEp-2 cellcategorization, which can be implanted in the ANA test automation system. A hybrid descriptor which represents the textural and morphological characters of the objects of interest was used along with Binary tree. The hybrid descriptors were generated by decomposing the image into binary images by using lower and upper thresholds. The performance of the proposed algorithm was evaluated on the ICPR 2016 IIF HEp-2 cell image dataset, the results concluded that Hybrid Descriptor with Binary Tree approach achieved the best performance with “97.5%” mean class accuracy.

Cryoglobulinemia and its correlation with clinical extrahepatic manifestations in chronic hepatitis C

Cryoglobulinemia is a condition with a confirmed relationship with HCV infection, but many other extrahepatic symptoms are also present in hepatitis C. Aim: The aim of this study was to assess the incidence and type of extrahepatic manifestations and cryoglobulinemia as well as their correlation with clinical symptoms in Polish patients. Material and methods: 246 consecutive patients with hepatitis C were studied. Clinical symptoms were assessed by anamnesis and a clinical investigation. Cryoglobulins were estimated by a method basing on their ability to reverse precipitation in temperature +4ºC. Monoclonality of cryoglobulins was estimated by agarophoresis immunotyping – capillary electrophoresis and immunofixation. The presence of classical rheumatoid factor (RF-IgM) was tested using ELISA. Anti-nuclear antibodies (ANA) were tested using the indirect immunofluorescence method. Anti-CCP, anti-SSA, anti-α-fodrin antibodies, RF IgG, IgM and IgA were assessed using commercial enzymatic tests. Statistical methods were: Fisher exact test, generalized additive models, odds ratios with 95% confidence intervals, aid of spline curve, SAS system version 9.4. Results: Cryoglobulins were found in 93 persons (37.8%). Type II cryoglobulinemia was detected in 28 persons. Patients with cryoglobulinemia were elder (p&lt;0.0004). 54 patients (21.95%) manifested clinical extrahepatic symptoms. Arthralgia was found in 27 cases (10.98%). Skin changes were found in 22 patients (8.94%). 12 persons had glomerulonephritis (4.88%). 11 patients (4.47%) had sicca syndrome. 6 patients developed peripheral polyneuropathy (2.4%). 4 persons developed B cell lymphoma (1.63%). There was no correlation between presence of symptoms and grading, staging, age, HCV genotype, and the presence of autoantibodies. Extrahepatic manifestations were present more frequently in women (p&lt;0.0008).

Development of two strains of Fusarium culmorum with a different aggressiveness in the soil and on the roots of barley of two genotypes

Fusarium culmorum can affect plants including cereals or develop saprophytically in the soil. It is unknown whether its saprophytic ability is different in strains with a different aggressiveness. This knowledge could be used for effective breeding of resistant cultivars. Here, we aimed to study the development of two F. culmorum strains with a different aggressiveness in the soil under barley, to compare their saprophytic ability and to reveal the influence, if any, of the host plant on the development of strains in the soil and on the roots. Saprophytic development of the strains was studied on membranes inoculated with macroconidia and placed into non-sterile soil under barley of two genotypes. The fungus was identified on the membranes and on barley roots by indirect immunofluorescent method. Both strains could develop saprophytically. The more aggressive strain (Fc538) showed a lesser saprophytic fitness than the less aggressive strain (Fc885): its mycelial density was lower and the number of chlamydospores was greater. Barley genotypes influenced the development of the fungal strains. Interestingly, conditions for saprophytic development of both strains were more favourable in the soil under barley of the resistant genotype. The more aggressive strain colonized barley of the resistant genotype more actively. Rot symptoms appeared earlier in the barley of the resistant genotype, but the number of diseased plants was greater in the barley of the susceptible genotype. The presence of a saprophytic stage in life cycle should slow down the accumulation of aggressive races in field populations of F. culmorum. Possible interactions between F. culmorum strains and barley plants are discussed.

ПАРАМЕТРЫ КЛЕТОЧНОГО ЗВЕНА ИММУНИТЕТА У БОЛЬНЫХ МЕХАНИЧЕСКОЙ ЖЕЛТУХОЙ ЗЛОКАЧЕСТВЕННОГО ГЕНЕЗА В ЗАВИСИМОСТИ ОТ УРОВНЯ БИЛИРУБИНА

Цель. Изучить особенности клеточного звена иммунитета у больных механической желтухой (МЖ) злокачественного генеза в зависимости от уровня билирубина.Материалы и методы. В группу изучения вошли 50 больных МЖ злокачественного генеза. Контрольная группа, состоящая из 125 практически здоровых добровольцев, была сопоставима по полу и возрасту с группой обследованных больных. При исследовании иммунного статуса использовался метод непрямой иммунофлуоресценции лимфоцитов с помощью моноклональных антител к CD3+, CD4+, CD8+, CD16+, CD19+, CD25+, CD95+, HLA-DR+. Статистическая обработка данных проводилась с помощью пакетов прикладных программ Statistica for Windows 8.0 и Microsoft Excel, 2007 Критический уровень значимости при проверки статистических гипотез принимался равным р&lt;0,05.Результаты. У больных МЖ злокачественного генеза имеются следующие особенности клеточного звена иммунитета: увеличены количество лейкоцитов, лейко-Т-клеточный, лейко-В-клеточный индексы, снижены число лимфоцитов, pan-маркеров Т-лимфоцитов, Т-хелперов, цитотоксических лимфоцитов, абсолютное количество NK-клеток, индексы HLA-DR+/CD19, CD4+/CD8+. При изучении особенностей иммунитета в зависимости от уровня билирубина выявлялись однонаправленные нарушения клеточного звена иммунитета.Заключение. У всех больных развивался вторичный Т-клеточный иммунодефицит, вероятно обусловленный сочетанным влиянием опухолевого, токсического и инфекционного факторов. Только у больных МЖ с уровнем билирубина более 200 мкмоль/л появляются дополнительные изменения в виде увеличения относительного количества В-лимфоцитов и снижение соотношения Т-хелперов к цитотоксическим лимфоцитам, за счет уменьшения первых.

Isaacs syndrome with CASPR2 antibody: A series of three cases

Isaacs syndrome is a form of peripheral nerve hyperexcitability, characterized by spontaneous muscle twitching and stiffness. Some patients are reported to be positive for CASPR2 antibody that may be one of the pathogenic autoantibodies in Isaacs syndrome. We reported a series of three patients with Isaacs syndrome, including their clinical features, electrophysiologic findings, laboratory parameters and therapeutic responses. All the three patients were positive for CASPR2 antibodies examined on transfected human embryonic kidney 293 cells by indirect immunofluorescence method. One patient had invasive thymoma. Symptomatic treatment was not sufficient for them, while immunotherapies including corticosteroids, double filtration plasmapheresis and rituximab provided favorable outcomes. The titers of CASPR2 antibody decreased after immune modulating therapy in parallel to clinical improvements in two patients.

Evaluation of an automated connective tissue disease screening assay in Korean patients with systemic rheumatic diseases.

This study aimed to evaluate the diagnostic utilities of the automated connective tissues disease screening assay, CTD screen, in patients with systemic rheumatic diseases. A total of 1093 serum samples were assayed using CTD screen and indirect immunofluorescent (IIF) methods. Among them, 162 were diagnosed with systemic rheumatic disease, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and mixed connective tissue disease (MCT). The remaining 931 with non-systemic rheumatic disease were assigned to the control group. The median ratios of CTD screen tests were significantly higher in the systemic rheumatic disease group than in the control group. The positive likelihood ratios of the CTD screen were higher than those of IIF in patients with total rheumatic diseases (4.1 vs. 1.6), including SLE (24.3 vs. 10.7). The areas under the receiver operating characteristic curves (ROC-AUCs) of the CTD screen for discriminating total rheumatic diseases, RA, SLE, and MCT from controls were 0.68, 0.56, 0.92 and 0.80, respectively. The ROC-AUCs of the combinations with IIF were significantly higher in patients with total rheumatic diseases (0.72) and MCT (0.85) than in those of the CTD screen alone. Multivariate analysis indicated that both the CTD screen and IIF were independent variables for predicting systemic rheumatic disease. CTD screen alone and in combination with IIF were a valuable diagnostic tool for predicting systemic rheumatic diseases, particularly for SLE.

IMMUNOMARKERS IN ACUTE CORONARY SYNDROME UPON ADMISSION AND ITS TRANSITION TO MYOCARDIAL INFARCTION

The aim is to study and develop some prognostic criteria for the clinical outcome of acute coronary syndrome into myocardial infarction based on immunomarkers. Materials and methods. Are presented the results of examination of 85 patients admitted to the infarction department of the Republican Clinical Hospital of the Center for Specialized Emergency Medical Care in Makhachkala. Immunomarkers were determined by the methods of enzyme immunoassay and indirect immunofluorescence. Results . Of the studied markers of cardiospecific changes in the immunity system, the indices of the autoimmune response to antigenic substances of necrotic myocardium are most pronounced. An almost 3-fold increase in thrombus aspiration to cardiolipin was determined (p &lt;0.01), and in 75% of cases, thrombus aspiration was determined for myocardial cells, mainly of the fibrillar type. The prognosis of clinical outcome of ACS in Q wave myocardial infarction at the hospital stage is associated with concentration intervals (separation points) of the following: immunomarkers: Neopterin from 20 to 28 nmol/L, TA to CL from 15 to 20 U/ml and 75% of cases there are TAs to myocardial cells. Patients who have these indicators at admission to the hospital (reference point) are a high-risk group for the clinical outcome of ACS in Q wave -MI (endpoint). After calculating the general operating characteristics of the tests, we obtained concentration intervals for a block of cardiospecific immunomarkers: Se = 67%, Sp = 83%, PVP = 57%, PVN = 89%, DE = 80%, RR = 6.8. The presented indices of immunomarkers fall into the prognostic block of the scheme. Conclusion. The identification of a high-risk group of patients with ACS allows for early hospitalization to conduct treatment measures in accordance with standards of management of patients with occlusive lesions of coronary vessels causing large-focal myocardial infarction.

Circulation autoantibody against Lamin A/C in patients with Sjögren's syndrome.

Lamin A/C proteins are major components of nuclear laminae and were encoded by the LMNA gene. Recent studies have found that in addition to provides nuclear-membrane strength; it also regulates the gene expression. Lamin A/C has been confirmed as an autoantigen in RA, SLE and vasculitis. Anti-Lamin A/C antibodies also have been found by indirect immunofluorescence method. In this study, we used various research methods to confirm Lamin A/C is an autoantigen in Han Chinese patients with confirmed Sjögren's syndrome (SS). To further investigate the relationship between the autoimmune disease antigens, we compared the amino acid sequence of Lamin A/C epitope and several common antigens' antigenic determinant. As a result, we found that Lamin A/C has similar epitopes with U1RNP. It means that the potential relationship exist between Lamin A/C and U1RNP. Clinical data we collected also showed that anti-Lamin A/C and anti-U1RNP antibodies always appear in same serum sample. Therefore, we speculated that cross-reaction may take place between antigen and potential antigen, which have similar epitope. Then, by epitope spreading, the potential antigen can be a new autoantigen. Our study provided a new thinking for further research about the relationship between autoantigens and their development mechanism in autoimmune diseases.

Analysis of Cell Cycle Progression in Saccharomyces cerevisiae Using the Budding Index and Tubulin Staining.

The budding index and the morphology of the spindle and the nucleus are excellent markers for the analysis of the progression through the different stages of the cell cycle in Saccharomyces cerevisiae. Here, we describe a protocol to evaluate the budding index in this model organism using phase contrast microscopy. We also describe an indirect immunofluorescence method designed for the visualization of microtubules and the nucleus in S. cerevisiae. Finally, we explain how both methodologies can be used in order to analyze cell cycle progression in the budding yeast.

Quantitative Immunofluorescence to Study Phagosome Maturation.

Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes, and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation. These changes can be assessed at the single-phagosome level by using immunofluorescence methods to study phagosome maturation. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Most commonly, phagosome maturation in macrophages can be determined by staining the cells for Lysosomal-Associated Membrane Protein I (LAMPI) and measuring the fluorescence intensity of LAMPI around each phagosome by microscopy or flow cytometry.

Diagnostic value of islet autoantibody assays practised in Russia. 1. Classic immunofluorescence islet cell antibody assay, immunoradiometric glutamic acid decarboxylase antibody assay, and ELISA tyrosine phosphatase antibody and insulin antibody assays.

Objective. To estimate performance characteristics and diagnostic value of immunofluorescent islet cell antibody (ICA) assay, immunoradiometric glutamic acid decarboxylase antibody (GADA) assay, and ELISA tyrosine phosphatase IA-2 antibody (IA-2A) and insulin antibody (IA) assays.Research Design and Methods. Antibodies were tested in 438 children and adolescents with newly diagnosed diabetes mellitus (DM) type 1, and in 891 subjects without DM type 1. ICA were determined by the classic indirect immunofluorescent method recommended by the Juvenile Diabetes Foundation International, GADA were determined with the Immunotech IRMA Anti-GAD kit, and IA-2A and IA were determined with Medizym Anti-IA2 and Orgentec Anti-Insulin ELISA kits, respectively. Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the tests were estimated with contingency tables. Diagnostic accuracy was estimated from areas under receiver operating curves (AUC).Results. ICA test was of the greatest diagnostic value (Se=88%, Sp=96%, PPV=96%, NPV=94%, AUC=0,94), followed by IA-2A (Se=66%, Sp=98%, PPV=98%, NPV=59%, AUC=0,82) and GADA (Se=73%, Sp=84%, PPV=75%, NPV=83%, AUC=0,79). IA test exhibited a very low Se (4,3%) and lacked diagnostic accuracy (AUC=0,5).Conclusions. We recommend to use ICA, IA-2A and GADA tests surveyed in our study for diagnosis of DM type 1 and differential diagnosis of DM. We don’t recommend IA testing with an Orgentec Anti-Insulin ELISA kit for usage in clinical practice.

Diagnostic value of islet autoantibody assays practised in Russia. 1. Classic immunofluorescence islet cell antibody assay, immunoradiometric glutamic acid decarboxylase antibody assay, and ELISA tyrosine phosphatase antibody and insulin antibody assays

Objective. To estimate performance characteristics and diagnostic value of immunofluorescent islet cell antibody (ICA) assay, immunoradiometric glutamic acid decarboxylase antibody (GADA) assay, and ELISA tyrosine phosphatase IA-2 antibody (IA-2A) and insulin antibody (IA) assays.Research Design and Methods. Antibodies were tested in 438 children and adolescents with newly diagnosed diabetes mellitus (DM) type 1, and in 891 subjects without DM type 1. ICA were determined by the classic indirect immunofluorescent method recommended by the Juvenile Diabetes Foundation International, GADA were determined with the Immunotech IRMA Anti-GAD kit, and IA-2A and IA were determined with Medizym Anti-IA2 and Orgentec Anti-Insulin ELISA kits, respectively. Sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the tests were estimated with contingency tables. Diagnostic accuracy was estimated from areas under receiver operating curves (AUC).Results. ICA test was of the greatest diagnostic value (Se=88%, Sp=96%, PPV=96%, NPV=94%, AUC=0,94), followed by IA-2A (Se=66%, Sp=98%, PPV=98%, NPV=59%, AUC=0,82) and GADA (Se=73%, Sp=84%, PPV=75%, NPV=83%, AUC=0,79). IA test exhibited a very low Se (4,3%) and lacked diagnostic accuracy (AUC=0,5).Conclusions. We recommend to use ICA, IA-2A and GADA tests surveyed in our study for diagnosis of DM type 1 and differential diagnosis of DM. We don’t recommend IA testing with an Orgentec Anti-Insulin ELISA kit for usage in clinical practice.

Toxoplasma gondii infection in dairy ewes: Vertical transmission and influence on milk production and reproductive performance

The present study aimed to evaluate the transmission of toxoplasmosis (vertical and venereal) and its influence on milk production and reproductive problems of Lacaune sheep seropositives for Toxoplasma gondii. Males and females were serologically selected using indirect immunofluorescence method in three steps of the study. Step 1: In order to evaluate the influence of toxoplasmosis on milk production, the volume of milk produced by 40 sheep (22 seronegatives and 18 seropositives for T. gondii) was weekly measured throughout the lactation period. There were no significant differences between these two groups; in other words, toxoplasmosis did not affect milk production. Step 2: In order to assess T. gondii venereal transmission, five samples of semen from seropositive rams (n = 5) were tested by endpoint and real time PCR with two days of interval; however, these semen samples were PCR negatives for T. gondii. Step 3: To evaluate reproductive problems, 12 seropositive animals out of a flock of 68 pregnant ewes showed signs of reproductive problems, such as abortion or fetal resorption. T. gondii transplacental transmission was evaluated on blood drawn from newborn lambs (n = 41), and their respective seropositive mothers (n = 30) after single, double or triple births. Serological tests showed that 65.8% of the lambs had antibodies against this protozoan, indicating a high transmission from ewe to fetus during pregnancy. Therefore, it is concluded that toxoplasmosis in sheep may impair reproduction with a high percentage of vertical transmission.