Indirect Immunofluorescence Method Research Articles

A comparative study of immunofluorescence pattern and line immune assay profile in the detection of the antinuclear antibodies

Background and Aims: To compare the efficacy of the Immunofluorescence and Line Immune Assay profile in the detection of ANA antibody. To ascertain an association between ANA patterns and the specific antibodies. To correlate the obtained ANA patterns with the Immunoassay. Settings and Design: This cross sectional study was done at a tertiary care center at Hyderabad Telangana. The samples were tested for ANA by Indirect Immunofluorescence (IIF) method and by Line immunoassay (LIA) methods. Materials and Methods: Serum samples of patients with a clinical suspicion of autoimmune disease were tested for ANA by indirect immunofluorescence (IIF) method and Line immunoassay (LIA) during January 2018-December 2018. Serum samples for indirect immunofluorescence were processed in 1:100 dilution using HEp–2010/liver/stomach/kidney biochip (EUROIMMUN AG). The serum samples were further processed for the line immunoassay in 1:100 dilution on nylon strips coated with purified and recombinant antigens as discrete lines with plastic backing coated with the antigens nRNP/Sm, Sm, SSA, Ro-52, SSB, PM-Scl, Scl-70, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, ribosomal protein-P, histones, anti-mitochondrial antibodies (AMA-M2) along with the control band. The analysis was done by the comparison of the intensity of reaction with the positive control line by image analysis. Results: Out of 236 samples tested, 77 were tested positive for Immunofluorescence and Line immuno assay, 115 were tested negative for Immunofluorescence and Immunoblot assay, only 1 was tested positive for Immunofluorescence and negative for Line immunoassay and 43 were tested positive for Line immunoassay and negative for Immunofluorescence. Conclusion: Detection of ANA by IIF method is a cost effective method. It may be used for the screening purposes for patients with or without clinical criterion for the autoimmune disease in daily clinical practice. The fluorescent pattern can also predict presence of certain specific antibodies in the sera. This may regulate one from requesting for line immunoassay, which is expensive. The Line Immuno assay may be reserved for those patients who need them for prognostic significance.

Circulation autoantibodies against C-terminus of NuMA in patients with Behçet's disease.

Circulating autoantibodies have a close association with autoimmune diseases, which may be seen even in healthy individuals. These are also considered as promising source of new biomarkers in various autoimmune diseases. However, their profile is not completely understood till now. Here, we evaluated autoantibodies against nuclear mitotic apparatus protein located at the carboxy terminus (C-NuMA)in blood circulation of Han Chinese patients, using different technical approaches to discover pathological reaction leading to Behçet’s disease (BD). In the first step, the recombinant human carboxy-terminal region of NuMA peptide (C-NuMA) was over-expressed and purified. In the second step, the indirect immunofluorescence method was used with patients’ sera, and commercial anti-NuMA antibody was used to determine the NuMA as a potential autoantigen. Results were confirmed at cell level by western blots, indicating that two of ten patients with Behçet’s disease could react with the recombinant C-NuMA,and the presence of antibodies were further verified by immunoprecipitation technique. Finally, the corresponding immunoassay (ELISA) was developed and optimized with specific recombinant C-NuMA as an in vitro method to test the confirmed patients with Behçet’s disease. Our findings demonstrated that C-terminus of NuMA is an immune target of Behçet’s disease in Han Chinese patients.

Нарушение метаболизма ретинола - важное патогенетическое звено формирования осевой близорукости

Цель исследования - определение изменений в экспрессии поверхностных рецепторов лимфоцитов, содержание малонового диальдегида и витамина А в периферической крови у пациентов с миопией по сравнению со здоровыми донорами и построение модели патогенеза. Методы. В исследовании приняли участие 38 детей с приобретенной близорукостью средней степени и отсутствием сопутствующих хронических заболеваний, 35 детей составили группу контроля. Возраст детей варьировал от 11 до 17 лет. Методом непрямой иммунофлюоресценции используя моноклональные антитела определяли количество лимфоцитов экспрессирующих CD3, CD4, CD8, CD16, CD56, CD20, CD72, CD38, CD25, CD71, HLA-DR, CD95, CD54, а также мембранные иммуноглобулины mIgM и mIgG. Концентацию малонового диальдегида (МДА) в крови и витамина А в плазме крови определяли традиционными биохимическими методами. Результаты. У пациентов с миопией обнаружено увеличение числа CD20, CD54 и CD95 позитивных лимфоцитов. Выявленные нарушения со стороны иммунной системы носят вторичный характер. Уровень малонового диальдегида в крови у больных миопией был повышен, а витамина А снижен по сравнению с контролем. На основе этих данных предложена модель патогенеза, в которой центральное место занимает нарушение процесса физиологической регенерации фоторецепторных дисков. Замедленное поглощение клетками пигментного эпителия и ослабление процесса внутриклеточного гидролиза их компонентов вызывают активацию перекисного окисления липидов, образование малонового диальдегида, повреждение сосудистого эндотелия и фибробластов склеры. Процесс ремоделирования фибробластов лимитирован метаболитами, поступающими через поврежденный эндотелий, что приводит к синтезу непрочного коллагена V типа с короткими цепями. Увеличение осевых размеров глаза при миопии - это растяжение и ослабление склеры глаза, связанное с развитием в соединительной ткани дистрофического процесса. Заключение. Патогенетически обоснованная терапия миопии должна быть направлена на увеличение функциональной активности клеток пигментного эпителия. Aim. Determination of changes in expression of lymphocyte surface receptors and concentrations of malonic dialdehyde and vitamin A in peripheral blood of patients with myopia as compared to healthy donors, and creation of a pathogenetic model. Methods. This study included 38 children with acquired moderate myopia without concurrent chronic diseases; 35 children constituted a control group. The children were 11 to 17 years old. The amount of lymphocytes expressing CD3, CD4, CD8, CD16, CD56, CD20, CD72, CD38, CD25, CD71, HLA-DR, CD95, CD54, and membrane immunoglobulins mIgM and mIgG was determined using the indirect immunofluorescence method with monoclonal antibodies. Concentrations of malonic dialdehyde (MDA) in blood and vitamin A in plasma were measured by traditional biochemical methods. Results. Patients with myopia had increased amounts of CD20-, CD54-, and CD95-positive lymphocytes. The observed immune disorders were secondary. The blood concentration of MDA was increased, and the vitamin A concentration was decreased in myopia patients compared to the control. Based on these data a pathogenetic model was proposed, in which disordered physiological regeneration of photoreceptor disks took the central place. The delayed absorption of pigment epithelial cells and impaired intracellular hydrolysis of their components result in activation of lipid peroxidation, formation of MDA, and damages of the vascular endothelium and scleral fibroblasts. The process of fibroblast remodeling is limited by influx of metabolites through the damaged endothelium, which induces synthesis of weak type V collagen with short chains. Pathogenetically substantiated therapy of myopia should be aimed at increasing the functional activity of pigment epithelial cells.

Comparison of Sensitivity and Specificity of Indirect Immunofluorescence Method with Culture and Real-Time PCR for Diagnosis of Mycoplasma hominis Infection in Genitalia of Women

Comparison of Sensitivity and Specificity of Indirect Immunofluorescence Method with Culture and Real-Time PCR for Diagnosis of Mycoplasma hominis Infection in Genitalia of Women

PHENOTYPIC COMPOSITION OF PERIPHERAL BLOOD LYMPHOCYTES AND THEIR COOPERATION IN PATIENTS WITH CHRONIC MERCURY INTOXICATION IN A POST-CONTACT PERIOD

Introduction. Based on the current understanding of the progression of professional chronic mercury intoxication, it is extremely important to study the regulatory activity of immunocompetent cells after the cessation contact with mercury to develop effective therapeutic measures. The purpose of the study was to study the population and subpopulation spectrum of peripheral blood lymphocytes and their cooperation in patients with chronic mercury intoxication in a distant postexposure period. Material and methods. Phenotyping of lymphocytes in the blood of the subjects was carried out by the method of indirect immunofluorescence using monoclonal antibodies to the molecules CD3+, CD4+, CD8+, CD9+, CD16+, CD20+, CD21+, CD23+, CD25+, CD95+. Results. In patients with chronic mercury intoxication in the long-term postexposure period, there were revealed features of the immune system functioning, indicating hyperactivation of both T- and B-components of the immune system. An increase in the total population of T-lymphocytes was established due to an increase in cells with receptors CD4+ (T-lymphocyte-helper cells) and CD16+ (killer cells), as well as an increase in the number of mature B-lymphocytes (CD20+) and pre-B-immature-lymphocytes (CD9+). Changes in the system of lymphocyte apoptosis, characterized by an increase in the number of cells expressing receptors for readiness for Fas-dependent apoptosis (CD95+), have been recorded. The established relationships between populations and subpopulations of lymphocytes indicate their importance in the implementation of the immune response, high activity and contingency between the components of the immune system in persons with chronic mercury intoxication after the termination of contact with the toxicant. Conclusion. The results obtained are the basis for long-term monitoring of the health status and improvement of the tactics of treating patients with neurointoxication with mercury in the postexposure period.

Seroprevalence of Toxoplasma gondii in Cattle and Sheep in Isfahan, Iran

Background and aims: Toxoplasma gondii is an obligate intracellular parasite which has the potential to infect all warm-blooded animals. Sheep and cattle play a main role in the economy and their infection to T. gondii, besides economic losses, can cause the infection of humans through the consumption of raw meat and other products. In this study, we aimed to survey the prevalence of T. gondii in the livestock in Isfahan, Iran. Methods: A total of 384 samples including 144 sheep and 130 bovine serum samples from Isfahan and 50 sheep and 60 bovine serum samples from Kashan slaughterhouses were tested for the presence of T. gondii antibody using the indirect immunofluorescence method. Results: In general, the antibody was found in 27.31% of sheep and 22.63% of cattle at a dilution of ≥1:200. In detail, 26.15% of bovine and 26.17% of sheep serums from Isfahan and 15% of bovine and 22% of sheep serums from Kashan were seropositive. Conclusion: The study revealed that sheep and cattle can be a significant intermediate host for the spread of the disease in this region (Isfahan province) and the health and livestock authorities in the province should be given priority in improving animal welfare and nutrition.

Zika fever immunodiagnostics: overview of test systems.

The threat of rapid spread of Zika virus beyond endemic regions has given rise to more research in field of epidemiology and clinic, as well as to the search for Zika fiver new diagnostic and preventive tools. Between 2013 and 2017 in Russia 18 cases of infection transmission by travellers were reported. Fever Zika reference monitoring center in Volgograd Research AntiPlague Institute (Volgograd, Russian Federation) provides counseling and methodological assistance on laboratory diagnosis and monitoring of Zika fever. In this regard, a literature review of commercial test systems for immunodiagnostics of this infection was performed. Currently, a number of test systems for solid-phase enzyme-linked immunoassay method (ELISA), immunochromatography and indirect immunofluorescent method (IIFT) have been developed for immunodiagnostics of Zika fever. Euroimmun Ltd. remains the only manufacturer that has access to detailed information on validation of the specificity of the produced diagnostic kits. Independent studies confirm that Euroimmun test systems have high specificity and high sensitivity, which is proved by the study of the material from various populations, including Europeans travelling to Zika virus endemic regions and people residing in these regions. A detailed overview of characteristics of Euroimmun test systems for immunodiagnostics of Zika fever allows us to conclude that there is a rationale for the use of these test systems for Russian Federation sanitary protection by identification of antibodies in patients, presumably infected with the Zika virus.

Tick-borne pathogens Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. may trigger endocarditis.

Infections caused by tick-borne pathogens such as Bartonella spp., Borrelia burgdorferi s.l., Coxiella burnetii, and Rickettsia spp. are capable of causing serious lesions of the mitral and aortic valves, leading to a need for valve replacement. The aim of the study was to determine whether such cases are sporadic or frequent. An additional goal was to establish effective diagnostic methods to detect these infections. The study involved 148 patients undergoing valve replacement. Blood samples were drawn for serological testing. Samples of the removed mitral and aortic valves were tested with polymerase chain reaction and immunohistochemical staining. Specific antibodies to Bartonella spp. were detected in 47 patients (31.7%) and in 1 of the healthy controls (1%) (p < 0.05). Antibodies to B. burgdorferi spirochetes were found in 18 of the patients (12.2%) and in 6 blood donors from the control group (5.8%) (p < 0.1). Antibodies to Rickettsia spp. were detected in 12 (8.1%) and to C. burnetii phase I and II antigens in the serum of 1 patient. All the participants in the control group were seronegative to C. burnetii and Rickettsia spp. antigens. Polymerase chain reaction (PCR) tests for detection of Bartonella spp., B. burgdorferi s.l., C. burnetii and Rickettsia spp. DNA in the valve samples were all negative. Inflammation foci with mononuclear lymphoid cells in the aortic and mitral valves were seen in sections stained with hematoxiline and eozine. In sections dyed using the indirect immunofluorescence method with hyperimmune sera, Bartonella spp. and Rickettsia spp. were found. The results obtained indicate that laboratory diagnostics for patients with heart disorders should be expanded to include tests detecting tick-borne zoonoses such as bartonelloses, Lyme borreliosis, rickettsioses and Q fever.

THE IMMUNOCOMPETENCE OF THE LYMPHOCYTIC POOL OF PERIPHERAL BLOOD IN ACUTE PURULENT LYMPHADENITIS IN CHILDREN

The substantial prevalence of different nosological forms of lymphadenitis in children is due to morpho-functional immaturity and age-related features of the structural components of the lymph node. The lymphatic system in children under 6 years of age is well developed, however, their immune competence is not perfect. Therefore, most often lymphadenitis of neodontogenic origin occurs at this age when the immune response of the organism to a significant number of bacterial and viral stimuli is insufficient for its reliable protection.&#x0D; The purpose of the work was to study the immunocompetence of the lymphocytic pool of peripheral blood in acute purulent lymphadenitis in children with overburden heredity.&#x0D; Materials and methods of research. A clinical examination and evaluation of the expression of surface markers of peripheral blood lymphocytes in 22 children with acute purulent neonotogenic submandibular lymphadenitis aged from 3 to 7 years was performed. The patients were divided into 2 groups. The first group included 17 persons with this disease, and the second one involved 5 children who have been diagnosed with a history of heredity in their families. The appearance of acute lymphadenitis in children was preceded by acute respiratory viral infection.&#x0D; The general clinical methods of examination included an assessment of the state of the somatic status, the oral cavity and the immediate inflammation cell. Incubation of peripheral blood cells was performed with monoclonal antibodies to CD3+, CD4+, CD4/25+, CD8+, CD16+, and a mechanism for incubation with FITS-conjugated goat antibodies to mouse immunoglobulins was used to detect them. The level of expression of surface markers of lymphocytes was determined as a percentage by the method of indirect immunofluorescence. Blood collection was performed at the time of hospitalization and for recovery. Prior to conducting surgical intervention, an ultrasound examination was required.&#x0D; Results of the research and their discussion. It was found out the fact that acute lymphadenitis in children of the second group occurred in the childhood of two grandparents and one grandmother, and later in their daughters, and their 4 sons. In 2 patients (a boy and a girl) it was traced only through the father or mother. In one mother, inflammation is diagnosed twice in the submandibular areas. Her sons had one-sided inflammation of the lymph nodes (right).&#x0D; The ultrasound biolocation, which was conducted with the aim of clarifying the diagnosis, determined the presence of single hypoechoic formations of ordinal form with clear contours of different sizes in all patients. At the time of hospitalization, in the group of children with hereditary imitation in the peripheral blood, it has been determined the fact that a decrease in the total number of CD3+ was in 2,2 times, CD4+ in 1,6 times, CD16+ in 2,5 times, CD4+, CD25+ in 2,1 times, immunoregulatory index in 2,5 times, the number of CD8+ in 1,6 times than in the group of healthy children.&#x0D; This clearly indicates to the presence of more pronounced immunological disorders, namely, in this group of children. At the end of treatment of patients of the second group it was followed a tendency to improve all indexes, however, the number of CD3+, CD4+ and immunoregulatory index values were lower than the control ones in 1,3, 1,4, and 1,6 times, respectively. At that time, the content of CD8+, CD16+, CD4+, CD25+ came close to healthy children.&#x0D; Thus, in acute purulent neodontogenic submandibular lymphadenitis, a decrease in the level of major lymphocyte populations in the immunoregulatory index at the T-suppressor direction of the immune response is observed, as the number of lymphocytes of this type increased by 1,3 times. More significantly, these changes were inherent for a group of children with hereditary imitation, which requires a more careful study of this phenomenon in the corporate participation of clinicians, immunologists, morphologists.

A new M23-based ELISA assay for anti-aquaporin 4 autoantibodies: diagnostic accuracy and clinical correlation

PurposeAlthough many assays have been developed to detect anti-aquaporin-4 (AQP4) antibodies, most of these assays require sophisticated techniques and are thus only available at specialized laboratories. The aim of this study was to evaluate the analytical and clinical performance of a new commercial enzyme-linked immunosorbent assay (ELISA RSR, AQP4 Ab Version 2) to detect anti-AQP4 antibodies performed on a fully automated system (SkyLAB 752).MethodsSerum samples from 64 patients with neuromyelitis optica spectrum disorders (NMOSD) (including NMO, longitudinally extensive myelitis-LETM, optical neuritis and myelitis) and 27 controls were tested for anti-AQP4 antibodies. All sera were previously tested using an indirect immunofluorescence (IIF) method on primate tissue, as the reference method. Commercial control sera were used to determine within-run, between-day and within-laboratory precision (CLSI guidelines).ResultsAt a cut-off value of 2.1 U/mL as determined by ROC curves, sensitivity and specificity for NMO were 83.3% and 100%, respectively. The ELISA assay provided 100% concordant results with the reference IIF method. The median concentration of anti-AQP4 antibodies was statistically higher in patients with NMO than in patients with LETM (p = 0.0006) or with other NMOSD and in controls (p < 0.0001). At the concentration of 12.4 and 28.1 U/mL, the within-run, between-day and within-laboratory coefficients of variation (CV) were 3.2% and 3%, 7.6% and 7.4%, and 8.2% and 8.0%, respectively.ConclusionsThis new ELISA method performed on a fully automated system, showed high sensitivity and absolute specificity, good CV in precision tests, and provided observer-independent quantitative results.

Expression and immunofluorescent localization of the Edwardsiella tarda EvpC protein in infected Pelteobagrus fulvidraco

To determine the expression and localization of immunofluorescent secretory proteins encoded by Edwardsiella tarda (E. tarda) EvpC gene in tissues of experimentally infected yellow catfish Pelteobagrus fulvidraco (P. fulvidraco), an indirect immunofluorescent staining method was performed for detecting EvpC protein. A rabbit anti-EvpC polyclonal antibody, raised against a recombinant 6× His-EvpC fusion protein expressed in Escherichia coli, was prepared, purified, and used as the primary antibody. Sixty fish (50 ± 2.2 g) were intraperitoneally administered with E. tarda (infected group). Tissues were collected and prepared for indirect immunofluorescent staining at 4, 8, 12, 24, 48, 72, and 96 h post-inoculation. E. tarda EvpC encoding protein antigens could be detected in the liver, spleen, and kidney at 4 h after challenge, in the intestine at 12 h after challenge, in brain tissue 24 h after challenge, and in renal interstitium and liver tissue at 48 h. In addition, the intensity of positive staining of E. tarda EvpCp antigen in various tissues increased sharply from 8 to 96 h, peaking around 48–72 h, and subsequently decreasing steadily after 96 h, thus suggesting the expression levels of E. tarda EvpCp in systemic organs to be closely correlated with disease progression. The distribution pattern of E. tarda EvpCp was more prevalent in the spleen, liver, and kidney after infection, than in the intestine and gills. The present study may be useful not only for describing the characteristics of EvpCp expression and distribution in the chosen tissue of P. fulvidraco, but also for understanding pathogenesis by E. tarda and providing a basis for further functional analysis of the gene.

The role of epithelial cells in atopy pathogenesis

Aim. The study of the mechanisms of atopic disease formation and a model of immunopathogenesis of the atopic diseases.Methods. Determination of surface lymphocytes receptors in peripheral blood of atopic bronchial asthma and atopic dermatitis patients with the help of monoclonal antibodies using the indirect immunofluorescence method. Expression of genes encoding TLR2, TLR4 and TLR9 receptors of airborne epithelial cells by real-time polymerase chain reaction, as well as determination of cytokine TSLP, IL-33, IL-4 and TGFβ (eBioscience) in airway flushes in atopic asthma patients and healthy people.Results. During the exacerbation of atopic diseases in peripheral blood lymphocytes, an intensive activation process develops with impaired lymphocytes activating apoptosis aimed at the formation of plasma cells capable of developing intensive IgE synthesis. To search for signals that could explain the mechanism of rearrangement of the B-cell part of the immune system during atopy, the epithelium cells of the airways were examined in a group of patients with atopic asthma and found an increase in gene expression coding for TLR2, TLR4, TLR9 in 6, 3 and 2.5 times respectively. Along with increased expression of TLRs genes in patients with bronchial asthma, an increased content of TSLP and IL-33 cytokines secreted by epithelial cells of the airways was detected. These cytokines have an immunoregulatory action - their nearby antigen presenting functions format the Th2 type of immune response, promote the production of cytokines (IL-4, IL-9, IL-13) and cause the development of an allergic type of inflammation.Conclusion. We suppose that the main link in pathogenesis is a disruption of the interaction of TLRs with the corresponding ligands caused by spontaneous dimerization of TLRs under the malonic dialdehyde influence. The intake of slowly metabolized dimers of TLRs into epithelial cells is a signal for genome activation, which leads to the synthesis of allergic cytokines IL-33 and TSLP. Thus, the main immunopathogenesis pathway of atopic diseases is the pathological functional interaction between epithelial cells and peripheral blood B-lymphocytes.

Serological diagnosis of trichinellosis in patients in Southern and Eastern Serbia in the period from 2007 to 2018

Introduction. The Nisava District has had low prevalence and incidence rates of trichinellosis for a long period. The aims of this study were to present the frequency of occurrence of trichinellosis in Nisava District and other districts in Southern and Eastern Serbia during 2007-2018, to investigate the demographic characteristics in the region and to compare the tests used for serological diagnosis of trichinellosis. Materials and Methods. This was a retrospective epidemiological study in which we analyzed the results of serological analyses of 139 serum samples taken from individual patients suspected to be infected with Trichinella spp. Indirect immunofluorescence (IIF) and immunoenzyme (ELISA) methods were used for serological diagnosis of trichinellosis. Results and Conclusions. Of 139 examined sera, 36 (25.9%) were seropositive. Most seropositive findings were encountered in 2012 and 2014 (in both these years, 12 sera were positive, i.e. 33.3% of the total 36 positive sera), when three minor outbreaks of trichinellosis were reported. Seropositivity was equally distributed according to patient sex and according to the district where they reported with illness. Positive serological findings were statistically significantly more common in hospitalized patients than in out-patients. Comparison of the findings with both IIF and ELISA test established the performance level of the tests, with the ELISA test being less sensitive than the IIF test. Immunodiagnostic procedures for the diagnosis of trichinellosis must be improved in view of the sporadic cases and minor outbreaks of the disease in the studied districts of Eastern and Southeastern Serbia.

Tissue-Clearing Technique and Cutaneous Nerve Biopsies: Quantification of the Intraepidermal Nerve-Fiber Density Using Active Clarity Technique-Pressure Related Efficient and Stable Transfer of Macromolecules Into Organs.

Background and PurposeCutaneous nerve biopsies based on two-dimensional analysis have been regarded as a creditable assessment tool for diagnosing peripheral neuropathies. However, advancements in methodological imaging are required for the analysis of intact structures of peripheral nerve fibers. A tissue-clearing and labeling technique facilitates three-dimensional imaging of internal structures in unsectioned, whole biological tissues without excessive time or labor costs. We sought to establish whether a tissue-clearing and labeling technique could be used for the diagnostic evaluation of peripheral neuropathies.MethodsFive healthy individuals and four patients with small-fiber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. The conventional methods of indirect immunofluorescence (IF) and bright-field immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efficient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fiber density (IENFD).ResultsThe mean IENFD values obtained by IF, bright-field IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fibers/mm2, respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fibers/mm2, respectively, and 3.06, 2.87, and 47.21 fibers/mm2, respectively, in the patients with PHN.ConclusionsThis study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fibers but also yielded reliable quantitative IENFD data. Quantification of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.

Linear IgA bullous dermatosis

Aim: to present a clinical case of linear IgA bullous dermatosis.Materials and methods. A 44 years old patient complaining of skin rashes on the trunk and extremities, accompanied by severe itching was examined. We carried out a morphological investigation of biopsy samples derived from the lesion and apparently unaffected skin areas using the method of indirect immunofluorescence.Results. The patient having rashes in the form of multiple vesicles and small bubbles with a tight cover, which had been grouped into figures resembling pearl necklaces, demonstrated the presence of focal subepidermal cracks (subepidermal bubble in one location), as well as a linear deposition of IgA along the epidermal basement membrane. According to the clinical picture and following the histological and immunofluorescent investigation of skin biopsies, the patient was diagnosed with linear IgA bullous dermatosis. Lesion regression was achieved as a result of systemic therapy with prednisolone at a dose of 50 mg per day.Conclusion. The diagnosis of linear IgA bullous dermatosis should be made on the basis of skin biopsy investigation by the method of indirect immunofluorescence. Systemic glucocorticosteroids are seen as an effective approach to the treatment of such patients.

Can solid-phase assays replace immunofluorescence for ANA screening?

The paper by Pisetsky et al 1 has stimulated a timely and interesting debate on a focal point in the diagnosis of autoimmune rheumatic diseases, that is, the accuracy of...

Harmonisation of laboratory tests for rheumatic diseases: still a long way to go

Antinuclear antibodies (ANAs), antineutrophil cytoplasmic antibodies (ANCAs), rheumatoid factor (RF) and antibodies to citrullinated peptides (ACPA) are widely used laboratory markers to support the diagnosis of ANA-associated systemic rheumatic diseases,...

Association of non-tuberculous mycobacteria with Mycobacterium leprae in environment of leprosy endemic regions in India

Non-tuberculous mycobacteria (NTM) are environmental mycobacteria found ubiquitously in nature. The present study was conducted to find out the presence of various species of NTM in leprosy endemic region along with Mycobacterium (M) leprae. Water and wet soil samples from the periphery of ponds used by the community were collected from districts of Purulia of West Bengal and Champa of Chhattisgarh, India. Samples were processed and decontaminated followed by culturing on Lowenstein Jensen (LJ) media. Polymerase chain reaction (PCR) was performed using 16S rRNA gene target of mycobacteria and species was confirmed by sequencing method. Indirect immune-fluorescent staining of M. leprae from soil was performed using M. leprae-PGL-1 rabbit polyclonal antibody. The phylogenetic tree was constructed by using MEGA-X software. From 380 soil samples 86 NTM were isolated, out of which 34(40%) isolates were rapid growing mycobacteria (RGM) and 52(60%) isolates were slow growing mycobacteria (SGM). Seventy-seven NTM isolates were obtained from 250 water samples, out of which 35(45%) were RGM and 42(55%) were SGM. Amongst all the RGM, we isolated M. porcinum, M. psychrotolerans, M. alsenase, M. arabiense and M. asiaticum from Indian environmental samples. M. fortuitum was the most commonly isolated species of all RGM. Out of all SGM, M. holsaticum, M. yongonense, M. seoulense, M. szulgai, M. europaeum, M. simiae and M. chimaera were isolated for the first time from Indian environment. M. intracellulare was the commonest of all isolated SGM. Presence of M. leprae was confirmed by indirect immunofluorescent microcopy and PCR method from the same environmental samples. Phylogenetic tree was showing a close association between these NTMs and M. leprae in these samples. Several NTM species of pathogenic and nonpathogenic in nature along with M. leprae were isolated from soil and pond water samples from leprosy endemic regions and these might be playing a role in causing disease and maintaining leprosy endemicity in India.

Investigation of the diagnostic value of anti-dense fine speckled 70/lens epithelium derived growth factor p75 autoantibody for autoimmune diseases

Antinuclear antibodies (ANA) are the autoantibodies that are produced against nuclear antigens in the cell nucleus and/or cytoplasm, and are one of the important diagnostic criteria in systemic autoimmune rheumatic diseases (SARD). Until today, several methods have been developed for detecting ANA's. However, indirect immunofluorescence (IIF) technique, that is also known as one of the oldest methods, is still the most commonly used one. Typically, anti-dense fine speckled 70/Lens epithelium derived growth factor p75 (anti-DFS70/LEDGF p75) autoantibody can be detected via IIF method where in HEp-2 (human larynx carcinoma) cells are used. The dense fine speckled (DFS) pattern method can be masked and remain unnoticed by the IIF method when it exists with the other ANA. Anti-DFS70 autoantibodies seldomly appear in SARD patients compared to healthy individuals. Moreover, these antibodies may appear in different chronic inflammatory conditions like interstitial cystitis, chronic fatigue syndrome, atopic dermatitis and Vogt-Koyanagi-Harada syndrome. The aim of this study was to determine the frequency of anti-DFS70 autoantibodies by immunblot (IB) method in patients sera with and without DFS70 staining pattern by IIF and to determine if the presence of anti-DFS70 has a clinical impact when included in ANA testing algorithm. In our study, a total of 60 patients' sera in which DFS pattern was defined by IIF method and 67 patients' sera in which other patterns observed were included in the study and anti-DFS70 autoantibody was investigated by IB method in these sera. In 67 patient samples which have shown the other patterns three (4.5%) samples were determined to be anti-DFS70 positive by IB. In 55 patients who were determined to have IIF-DFS pattern (+)/IB anti-DFS70 (+), 6 (11%) were diagnosed as SARD and the other antinuclear antibodies (ANA) were found as negative by IB. In the other group with the other ANA patterns detected, none of the SARD-diagnosed 22 patients had shown anti-DFS70 by IB method. Sixteen (26.6%) samples in the group that was positive for the IIF-DFS pattern were obtained from rheumatology and physical therapy and rehabilitation clinics, 32 (47.7%) samples were from the group in which other patterns observed and were also obtained from those clinics. DFS pattern was detected significantly more frequent in the samples from other clinics in comparison to the samples from rheumatology and physical therapy and rehabilitation clinics (p= 0.018). In our study, it was concluded that DFS pattern can be defined by IIF method by only specialists, however, since homogeneous-like and mixed patterns can be confused especially in low titers, there is a need for a second well-validated immunological test that could detect anti-DFS70 auto-antibody.

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.