[Actual issues of serum aquaporin-4 autoantibodies evaluation in the diagnostics of neuromyelitis optica spectrum disorders]. / Aktual'nye voprosy diagnostiki zabolevanii spektra optikoneiromielita pri opredelenii antitel k akvaporinu-4 v syvorotke krovi.

OBJECTIVE: To analyze the usage and timeliness of aquaporin-4 antibodies (AQP4-IgG) serology test in the diagnostics of neuromyelitis optica spectrum disorders (NMOSD) in routine clinical practice. MATERIAL AND METHODS: 27 patients with NMOSD were included in the study. All patients had a positive serum test for AQP4-IgG. A retrospective study of neurological manisfestations of attacks, timing and results of serology for AQP4-IgG was performed. The results were analyzed taking into account two types of attacks identified: a) HS (with highly specific manifestations for NMOSD), which are considered as indications for conducting the AQP4-IgG test and b) NS (with non-specific manifestations for NMOSD). RESULTS AND CONCLUSION: A comparison of the time from HS attack to the AQP4-IgG test administration (T1, years), from HS attack to NMOSD diagnosis (T2, years) was undertaken as well as the number of attacks during these periods (N1, N2) were counted in three groups of patients. Group 1 - with the first HS attack before or in 2008 (n=6), group 2 - from 2009 to 2013 (n=12), group 3 - from 2014 to 2018 (n=9) accordingly. A statistically significant decrease in T1, T2, N1, N2 was found in successive time intervals of 5 years (p<0.05). In 8 of 27 (28.6%) patients the first attack of NMOSD was presented with non-specific symptoms (NS attack). In 7 patients (77.8%) of 9 misdiagnosed as multiple sclerosis (MS) an increase in attack frequency was found while on disease modifying therapies (DMTs) and increase in attack severity was found in 8 (88.9%). In all 9 cases the diagnosis was revised to NMOSD after AQP4-IgG test was performed with positive result. The time interval from disease course worsening while on DMTs until the test was 7 [4; 37] months, and the number of relapses - 2 [0; 3]. In 4 of 27 patients with suspected NMOSD, the repeated AQP4-IgG test only was positive for increased antibodies titer. The time interval between first test negative and retest administered was 20 [6.1; 47.8] months. In 3 of 4 patients (75%) one or more attacks occurred during this time period. In 4 patients the presence of AQP4-IgG in the first analysis was not followed by the diagnosis of NMOSD. In recent years, apropos AQP4-IgG test administration improved, but the problem remains with the timeliness for retest with first result negative. It is advisable to expand the indications for its use. The timeliness for serum AQP4-IgG retest in cases of unexplained deterioration in the course of proposed MS on DMTs and the lack of awareness of the test diagnostic value are still relevant.

[Membranous nephropathy in a Russian population]. / Membranoznaia nefropatiia v rossiiskoi populiatsii.

AIM: To analyze the clinical and morphological manifestations of membranous nephropathy (MN) and to evaluate the efficiency of its therapy. MATERIAL AND METHODS: MN cases in 2009 to 2016 were retrospectively detected with a subsequent analysis of patients with primary MN (PMN). The titer of IgG-autoantibodies to phospholipase A2 receptor (anti-PLA2R Ab) was determined by an indirect immunofluorescence assay. Treatment outcomes, such as the time course of changes in proteinuria, nephrotic syndrome (NS), and the development of complete and partial remissions (CR and PR), were assessed. RESULTS: MN was detected in 201 cases; the secondary etiology of the disease was established in 24.9%. The prevalence of MN among morphologically confirmed glomerulopathies was 14%; that of PMN was 10.4%. The median period to diagnosis PMN was 8 (5; 19) months. 150 patients with PMN (66.7% were men; age was 50±15 years) were distributed according to the following morphological stages: Stages I (23.9%), II (48.5%), III (26.1%), and IV (1.5%). Elevated anti-PLA2R Ab levels were found in 51.6% of cases; NS in the presence of proteinuria was detected in 85.6% of patients. An estimated glomerular filtration rate (eGFR) of <60 ml/min/1.73 m2 was seen in 25% of cases. Treatment outcomes were evaluated in 80 cases; the median follow-up period was 19 (8; 40) months. 68% of cases had CR (32%) or PR (36%) with a median follow-up of 26 (13; 44) months. Spontaneous CRs or PRs were observed in 7.5% of the patients. Multivariate analysis showed that the probability of CR or PR increased 3.2-fold in the use of cyclophosphamide and/or cyclosporine and decreased as eGFR dropped. CONCLUSION: In Russia, PMN is a common type of glomerulopathy, the specific features of which should include the low rates of spontaneous remissions and detection of anti-PLA2R Abs. For renal protection, the majority of patients with PMN require timely diagnosis and treatment; individualization of the choice of treatment and its enhanced efficiency call for further investigations.

[Diagnostic value of immunoglobulin free light chains at the debut of multiple sclerosis]. / Rol' opredeleniia svobodnykh legkikh tsepei immunoglobulinov v diagnostike debiuta rasseiannogo skleroza.

AIM: To evaluate the diagnostic value of determination of free immunoglobulin light chains (IgG) in the debut of multiple sclerosis (MS). MATERIAL AND METHODS: Data from 226 patients, including 111 patients with clinically isolated syndrome with conversion to multiple sclerosis within the first 2 years of the disease (group 1), 49 patients with clinically isolated syndrome who did not develop multiple sclerosis within the first 2 years of the disease (group 2), 20 patients with other inflammatory diseases of the central nervous system (group 3) were analyzed. The control group consisted of 46 patients with non-inflammatory diseases of the central nervous system. The clonality of immunoglobulins in the CSF, concentration of kappa and lambda free light chains and their ratio were studied. RESULTS: Concentrations of free light chains were significantly higher in the first group in comparison with group 2 and the control group, but didn't differ from group 3. In group 3, concentrations of free light chains were significantly higher compared to group 2 and controls. In oligoclonal-positive patients with clinically isolated syndrome (groups 1 and 2), concentrations of kappa and lambda free light chains were significantly higher than in oligoclonal-negative patients. The production of free light chains in patients from the first group was considerably higher than in group 2 regardless of the oligoclonal status. The concentration of kappa chains and quotient of kappa free light chains in the CSF had the best diagnostic characteristics. Their use, along with the evaluation of IgG clonality, reduced the risk of false-negative results by 50%. Regardless of other factors, elevated concentrations of kappa chains increase the likelihood of MS diagnosis by 9.718 times. CONCLUSION: The use of free light chains as a laboratory marker can increase the accuracy of MS diagnosis. These markers can help indirectly assess the risk of transformation of a clinically isolated syndrome into definite multiple sclerosis within the first 2 years of disease.

[The comparative analysis of immunologic techniques of detection of anti-phospholipid antibodies].

The laboratory diagnostic of anti-phospholipid syndrome consists in detection of anti-phospholipid antibodies using technique of enzyme-linked immunosorbent assay namely in detection of anti-cardiolipin antibodies and antibodies to ß2-glycoprotein. In spite of the fact that serological diagnostic plays a key role in diagnosing anti-phospholipid syndrome application of laboratory tests s complicated by their insufficient standardization. The new approach to detection of anti-phospholipid antibodies became application of immune blotting on the basis of polyvinylidenfluoride membrane. As compared with enzyme-linked immunosorbent assay, the advantage of the mentioned technique is in using hydrophobic solid phase for sorption of antigens. The porous structure of polyvinylidenfluoride membrane orientates hydrophilic areas of phospholipids and by that ensures their more dense distribution imitating bi-lipid layer of membranes of living organism. To specify and compare value of different techniques the comparison was implemented concerning the results of measurement of anti-phospholipid antibodies in enzyme-linked immunosorbent assay test-systems of various manufacturers and reagents kits for immune blotting. The collection was assembled including bio-materials from 47 patients with non-cardioembolic ischemic strokes, 20 patients with recurrent thrombosis of deep veins of lower extremities and 50 patients with obstetrics pathology and also 30 healthy donors. In the given serums aKlaIgG, aKlaIgM, aß2glycoprotein I were measured using enzyme-linked immunosorbent assay technique assisted by test-systems of Euroimmun and Orgentes Diagnostica and the samples with the highest titre using immune blotting technique with reagents manufactured by Medipan. On the basis of measurement of anti-phospholipid antibodies by various enzyme-linked immunosorbent assay test-systems the rate of aß2glycoprotein I amounted to 31% in case of Euroimmun reagents kits for enzyme-linked immunosorbent assay, 78% in case of Orgentec Diagnistica test-systems for enzyme-linked immunosorbent assay, aKlaIgG - 2% and 30%, aKlaIgM - 31% and 54% correspondingly. The measurement of anti-phospholipid antibodies using immune blotting technique on Medipan test-systems in bio-samples with the highest titres detected aß2glycoprotein I in all patients, aKlaIgG in 70% and aKlaIgM in 30% of patients. The convergence between three commercial reagents kits varies from 20% to 88%. The standardization of commercial test-systems still to be achieved. The new technique of immune blotting can be appliedjointly with classic techniques ofserological diagnostic of anti-phospholipid syndrome. The absence of algorithms of diagnostic and standardization of different test-systems for detection of anti-phospholipid antibodies prejudices reliability of serological diagnosis of anti-phospholipid syndrome and therefore existence of anti-phospholipid syndrome as a nosologic unit.

[The association of intrathecal production of immunoglobulin free light chains and progression of multiple sclerosis]. / Sviaz' intratekal'noi produktsii svobodnykh legkikh tsepei immunoglobulinov s progressirovaniem rasseiannogo skleroza.

AIM: To assess an impact of immunoglobulin free light chains (FLC) on short-term and long-term prognosis of clinical and radiological activity and progression of disability in multiple sclerosis (MS). MATERIAL AND METHODS: A sample of 381 patients with definite MS was divided into 2 groups. In group 1, lumbar puncture was performed at the time of clinically isolated syndrome, and patients were prospectively followed up to 2 years (short-term prognosis group, n=97). In group 2, MS was diagnosed immediately after lumbar puncture, and retrospective analysis of the disease course with the duration not less than 5 years was performed (long-term prognosis group, n=284). The Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Severity Score (MSSS) were used to assess patient's status. Concentrations of kappa and lambda FLC in the CSF (K-FLCCSF, L-FLCCSF) and serum (K-FLCSERUM, L-FLCSERUM) as well as quotients of concentrations (Q-K and Q-L) were determined. Patients were stratified into subgroups with high and low concentrations of K-FLC and L-FLC using cut-offs from our previous studies: K-FLCCSF=0.595 mcg/l and L-FLCCSF=0.127 mcg/l. RESULTS: In group 1, significant correlations were found only between EDSS score and concentrations of K-FLCCSF (r=0.377, p=0.00019) and Q-K (r=0.366, p=0.0012). FLC concentrations did not correlate with the number of relapses and new T2 lesions. The age and EDSS score at the disease onset didn't differ between patients with high and low K-FLC and L-FLC (K-FLCCSF: р=0.2658; L-FLCCSF: р=0.5502). A significant decrease of EDSS score after the disease onset was observed in all groups except for patients with high concentrations of K-FLCCSF (p=0.1844), so the EDSS score after 2 years was significantly higher in this subgroup of patients (p=0.0006). In group 2, significant correlations of K-FLC with EDSS score (r=0.181, p=0.002) and MSSS score (r=0.121, р=0.044) for long-term prognosis (median (IQR) = 8 (6-13) years) were found. No correlations of FLC concentrations with the number of relapses during the first 5 years were found. Survival analysis showed that high concentrations of K-FLCCSF were associated with the high risk of progression to EDSS 6 (HR=2.055, p=0.026) but not with EDSS 4 (HR=2.388, p=0.08). CONCLUSION: Concentrations of kappa FLC can help to define the prognosis of MS early at the disease course. Although low concentrations of FLC do not exclude a severe disease phenotype, patients with high K-FLCCSF concentrations are at greater risk for faster MS progression, probably, due to impaired reparation of neural tissue. Measurement of FLC concentrations can be used to determine a therapeutic tactics in patients with MS.

Immunogenicity of Human Interferon-Beta-Containing Pharmaceuticals.

Multiple sclerosis is a severe autoimmune disease with inflammatory component that continues to be resistant to treatment. One of the approaches retarding its progression is based on using nonspecific therapy with human interferon-beta (IFN-ß)-containing pharmaceuticals. Neutralizing antibodies (NAbs) against genetically engineered pharmaceuticals developed by the patient's immune system, which reduce their therapeutic and biological activity, pose a serious problem. Cell lines sensitive to IFN-ß activity also quantifying NAb level are applied because direct measurement of IFN-ß antiviral activity is complicated. This study was aimed at standardization and validation of a reporter cell system for measuring anti-human IFN-ß NAb titers, and evaluation data were obtained with samples from 33 patients with multiple sclerosis.

[The methods of detection of binding and neutralizing antibodies to preparations of interferon-beta].

The human recombinant ß-interferon is most frequently applied for treatment of remittent recurrent form of multiple sclerosis using pharmaceuticals. The clinical response to applied therapy is absent in some of patients that can be conditioned by development of antibodies too preparations. Depending on possibility of blocking binding of human recombinant ß-interferon with its receptor, all antibodies are divided on binding and neutralizing ones. The purpose of study is to investigate analytical and clinical diagnostic parameters of tests using for detection of different types of antibodies synthesized against human recombinant ß-interferon. The study sampling consisted of 33 patients with remittent recurrent form of multiple sclerosis receiving therapy with human recombinant ß-interferon and also of 40 donors and 15 patients with multiple sclerosis without therapy with human recombinant ß-interferon. The concentration of binding antibodies was measured by enzyme-linked immunosorbent assay. Also immune blotting assay was applied. The titer of neutralizing antibodies was determined using cell line HL-116 sensitive to human recombinant ß-interferon. The binding and neutralizing antibodies were not detected in donors and patients without human recombinant ß-interferon therapy. The prevalence of binding antibodies to human recombinant ß-interferon amounted to 57.6% when analysis of samples using immune blotting assay was used and 60.6% when commercial testing system was applied. The statistical analysis of results demonstrated high convergence and correlation of values of concentrations of binding antibodies obtained using immune blotting assay and enzyme-linked immunosorbent assay (r=0.9159, p<0.0001). The clinically significant titers of neutralizing antibodies were detected in 21.21°% of patients. All patients with clinically significant titer of neutralizing antibodies were positive in relation to binding antibodies measured by immune blotting assay and enzyme-linked immunosorbent assay. The high correlation between values of titers of neutralizing antibodies and concentration of binding antibodies measured by immune blotting assay (r=0.7909, p=0.0055). The application in clinical practice of data concerning presence of binding and neutralizing antibodies to human recombinant ß-interferon can input into optimization of therapy with expensive biologic preparations in patients with multiple sclerosis and other autoimmune diseases.

[THE COMPARISON OF TECHNIQUES OF ELECTROPHORESIS, IMMUNE TURBIDYNAMIC MEASUREMENT AND PHENOTYPING OF ALPHA-1-ANTITRYPSIN FOR DIAGNOSTIC OF ALPHA-1-ANTITRYPSIN INSUFFICIENCY].

The qualitative and quantitative deficiency of alpha-1-antitrypsin (A1AT) is an inherited factor of susceptibility to a number of conditions including chronic obstructive disease of lungs and primary emphysema and liver affection. The study was carried out to evaluate analysis of alpha-1-fraction (A1F) using zonal electrophoresis technique for detecting patients with alpha-1-antitrypsin insufficiency (A1ATI). The patients with decreased (group I) and normal (group II) A1F on proteinogram. The electrophoresis was applied using the system of capillary electrophoresis Capillaris-2 Flex Piercing (Sebia, France) and also system for electrophoresis in agarose gel SAS-1/SAS-2 (Helena Biosciences, Great Britain). The commercial kit Sentinel diagnostics (Italy) and biochemical analyzer A15 (Biosystems, Spain) were used for quantitative detecting of A1AT The study results demonstrated that decreasing of A1F on proteinogram correlated with lessening of concentration of A1ATI in blood serum and with presence of its pathological phenotype. The average values of concentration of A1AT in group I and group II made up to 1148 and 1738 mg/I correspondingly. The total rate of pathological phenotypes made up to 76% (19/25) in group I that reliably differed from indicators in group II--7.1% (2/28). Thereby, electrophoresis of proteins of blood serum can be sufficiently informative for primary selection of patients requiring examination for presence of A1ATI.

[The optimization of techniques complex of serologic diagnostics of connective tissue systemic diseases].

The connective tissue systemic diseases originate from pathologic process following with antinuclear antibodies emergence. To detect these antibodies a significant number of diagnostic tests and techniques has been applied. Besides that, there is no conventional algorithm of antinuclear antibodies diagnostic. To detect antinuclear antibodies a two-fold diagnostic algorithm was applied In the capacity of screening techniques the indirect immunofluorescence technique was applied to the cells of line Hep-2 (antinuclear factor) and detection of antibodies to extractable nuclear antigen. The second stage of diagnostic included the detection of content of more specific antinuclear antibodies using the Lineblott method and the double-helical DNA antibodies. The blood serum from 981 patients with suspected connective tissue systemic diseases, 115 patients with systemic lupus erythematous and 57 healthy individuals was analyzed. The levels of antinuclear factor, nuclear antigen antibodies and double-helical DNA antibodies were detected. The antinuclear factor was detected in 84% and 86% of cases, double-helical DNA antibodies in 55% and 39% of cases depending of reagents using in detecting these characteristics. Among healthy individuals, antinuclear factor was detected in 5% (1/20) of blood serum samples in titers less than 1:160. In the group of patients with suspected connective tissue systemic diseases, antinuclear factor was detected in 48% (474/981) of cases and extractable nuclear antigen in 20% (326/981) of cases. The Lineblott test was positive in 33% (326/981) of patients with suspected connective tissue systemic diseases. Among antinuclear factor positive patients nuclear antigen antibodies were detected in 36% (171/474) and the Lineblott test was positive in 63% (298/474) of cases. Among antinuclear factor negative patients but positive under anti-nuclear antigen identification, the Lineblott test was positive in 6% (28/507) of cases. The two-fold algorithm of nuclear antigen testing is an effective technique to be applied in the clinical diagnostic laboratory. The results of effectiveness of this algorithm demonstrated that this method can ensure 33% of cost savings of testing individuals with higher incidence of diseases.

[The analytic and diagnostic characteristics of the national test system detecting cyclic citrullinated peptide antibodies].

The clinical signifcance of serodiagnostic assay of rheumatoid arthritis increased nowadays. This is a reason to include the citrullinated antigens' antibodies into the new criteria of diagnostics ACR/EULAR 2010. The approbation of national test system detecting the cyclic citrullinated peptide antibodies was implemented. The analysis was applied to 211 blood serum samples taken of 50 blood donors, 60 patients with rheumatoid arthritis, 66 patients with other rheumatoid diseases. In addition, 35 samples were concurrently analyzed with the comparative test system (CCP2 Euroimmun, Germany). The referential meanings of standard limits were established on the basis of results of study of samples taken from healthy blood donors. When the standard limit was less than 10 arbitrary units the sensitivity made up 75% and the specificity--87.9%. In the case of higher values of citrullinated antigens' antibodies which are more than 15 arbitrary units, the sensitivity made up 68% and the specificity--93.1%. The results of comparing with the comparative test system characterized by high convergence made up 94% (33 out of 35), but the comparative test system detected citrullinated antigens' antibodies in 2 samples. The positive qualitative results of both methods analysis of autoantibodies weakly correlated with one another (r = 0.14). The results testify that the parameters of national test system correspond to the publication data concerning the second generation methods of cyclic citrullinated peptide antibodies detection though yield to the best foreign analogues.