Retrospective evaluation of "Rods and Rings" pattern detected in the anti-nuclear antibody (ANA) indirect immunofluorescence (IIF) test.

Introduction: Anti-rods and rings (anti-RR) antibodies have recently been described as a cytoplasmic pattern in IIF-based screening of autoantibodies on HEp-2 cells and ICAP has named it as AC-23. It is most frequently related to drug-induced antibody generation. This study aimed to investigate the clinical significance of AC-23 positivity and its relevance to the diagnosis and/or follow-up of the associated diseases and/or drug use. Methods: A multicenter retrospective study was conducted among 10 hospitals from six different provinces in Türkiye from January 2017 to December 2021. The laboratory data and clinical information of 600 patients with positive anti-RR antibodies out of 547.558 HEp-2 IIF ANA samples were analyzed. Results: The distribution of AC-23 positive patients by year indicated a steady increase between 2017-2021. Anti-RR prevalence in post-COVID-19 period was significantly higher than that of pre-COVID-19 period (p=0.00). Concomitant ANA positivity was detected in 56.5% of patients, the most common patterns being AC-4 and AC-5 (41.1%). The most frequent pathology among the anti-RR positive patients was an autoimmune disease (19.83%); 28.57% of which had rheumatoid arthritis and 17.65% autoimmune liver disease. Among the 600 patients, 65 (10.83%) were diagnosed as hepatitis C virus (HCV) infection. Available data for 38 of the HCV patients revealed that 71.05% of them had a history of interferon alfa+ribavirin and 28.95% of them had a history of NS3/4/5A/5B polymerase inhibitor or protease inhibitor drug use. Significant increase in the rate of anti-RR positivity was observed in the post-COVID-19 period when compared to pre-COVID-19 period (p:0.00). Discussion: This is the first multicenter study in Türkiye about the clinical association of anti-RR antibodies which may be ignored during routine HEp-2 IIF testing. Pathologies other than HCV should be taken into consideration in terms of the possible role of anti-RR in autoimmune diseases and other pathologies. The preliminary data obtained in this study suggest that anti-RR antibody development might also be associated to COVID-19, supporting the several previous data related to the potential of viruses triggering the formation of autoantibodies. Large-scale prospective studies should elucidate the clinical significance of RR pattern and determine its role in patient diagnosis and follow-up.

COVID-19 vaccine response in Neuromyelitis Optica Spectrum Disorder.

OBJECTIVES AND AIMS: Neuromyelitis Optica Spectrum Disorder (NMOSD) is a disabling autoimmune disease of the central nervous system that requires immunosuppressants to control the relapses. The latter puts them at risk for more severe COVID-19 infection. Vaccines are an effective way to control the pandemic. However, we do not know how effective they are in immunologically compromised patients. We aimed to evaluate and compare antibody levels in NMOSD patients treated with disease-modifying therapies after two doses of inactivated and mRNA COVID-19 vaccines. METHODS: Patients with NMOSD diagnosis and age-sex matched healthy controls who received two doses of either inactivated and mRNA COVID-19 vaccine were recruited in the study. Serum samples were collected at least two weeks after the second dose. RESULTS: Serum samples from 24 NMOSD patients (Mean age-36.58, Female-70.83%) and 24 healthy controls (Mean age-36.71, Female-70.83%) were evaluated. Mean antibody titer was lower in the NMOSD group (Mean; SD (2.43 ± 1.51) than in healthy controls (Mean; SD 3.23 ± 0.80). Seronegativity was only seen in the rituximab group, there were no such cases in the azathioprine group. (9 vs 0). CONCLUSIONS: The study shows that NMOSD patients treated with rituximab may still be susceptible to severe COVID-19 infection even after both inactivated and mRNA vaccines.

The Ca2+ concentration impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages in vitro.

Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca2+ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional αß T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, γδ T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca2+ concentrations. This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. A similarly increased cytokine response due to elevated Ca2+ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca2+ concentrations. Furthermore, elevated Ca2+ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca2+ concentration. In summary, our data demonstrate that the Ca2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca2+ levels can boost cytokine production by both mouse and human lymphoid cells.

Three doses of COVID-19 vaccines in multiple sclerosis patients treated with disease-modifying therapies.

OBJECTIVES AND AIMS: Disease modifying therapies used in multiple sclerosis can decrease humoral response after COVID-19 vaccines. This problem must be adequately addressed because new variants evolve, and COVID-19 still poses a risk to patients with comorbidities and immunosuppression. We aimed to evaluate the antibody response after the third dose of the COVID-19 vaccine in people with multiple sclerosis on disease-modifying therapies. METHODS: People with multiple sclerosis who received the third dose of either mRNA or inactivated vaccine after two doses of inactivated vaccine were recruited for the study. Blood samples were collected at least two weeks after the third dose. RESULTS: Blood samples of 339 (female 72.5%) people with multiple sclerosis and 52 (female 71.2%) healthy controls were evaluated. Healthy controls (mean: 4.07 ± 0.66) have higher antibody titers than people with multiple sclerosis (mean: 2.79 ± 2.95). Seronegative cases were observed only in the fingolimod and ocrelizumab treatment groups. Patients on fingolimod who received mRNA as a third dose had significantly higher antibody titer than those who had inactivated vaccines. Longer disease duration, having inactivated vaccine as a third dose, and DMT use was associated with lower antibody response. CONCLUSIONS: The study shows that even after inactivated vaccine schedule, mRNA still offers more protection in people with multiple sclerosis on disease-modifying therapies.

Effect of stimulation time on the expression of human macrophage polarization markers.

Macrophages are highly plastic cells that can polarize into functionally distinct subsets in vivo and in vitro in response to environmental signals. The development of protocols to model macrophage polarization in vitro greatly contributes to our understanding of macrophage biology. Macrophages are divided into two main groups: Pro-inflammatory M1 macrophages (classically activated) and anti-inflammatory M2 macrophages (alternatively activated), based on several key surface markers and the production of inflammatory mediators. However, the expression of these common macrophage polarization markers is greatly affected by the stimulation time used. Unfortunately, there is no consensus yet regarding the optimal stimulation times for particular macrophage polarization markers in in vitro experiments. This situation is problematic, (i) as analysing a particular marker at a suboptimal time point can lead to false-negative results, and (ii) as it clearly impedes the comparison of different studies. Using human monocyte-derived macrophages (MDMs) in vitro, we analysed how the expression of the main polarization markers for M1 (CD64, CD86, CXCL9, CXCL10, HLA-DR, IDO1, IL1ß, IL12, TNF), M2a (CD200R, CD206, CCL17, CCL22, IL-10, TGM2), and M2c (CD163, IL-10, TGFß) macrophages changes over time at mRNA and protein levels. Our data establish the most appropriate stimulation time for the analysis of the expression of human macrophage polarization markers in vitro. Providing such a reference guide will likely facilitate the investigation of macrophage polarization and its reproducibility.

Comparison of SARS-CoV-2 antibody response after two doses of mRNA and inactivated vaccines in multiple sclerosis patients treated with disease-modifying therapies.

BACKGROUND: Disease-modifying therapy could weaken the immune system and decrease the immune response to vaccines. It is essential to know which vaccine is more protective against SARS-CoV-2 in the multiple sclerosis population. OBJECTIVE: To assess immune response after messenger RNA BNT162b2 (Pfizer/BioNTech) and inactivated Sinovac vaccines in people with multiple sclerosis (pwMS) treated with a disease-modifying therapy (DMT) compared to healthy controls. METHODS: This single-center cross-sectional study included 526 MS patients treated with DMT, 44 healthy controls, and 21 untreated patients with MS between May 2021 and September 2021. Serum samples were collected at least two weeks after the second dose of the vaccine. RESULTS: Participants vaccinated with BNT162b2 had a higher antibody titer than the Sinovac group (95%CI=1.023 - 1.473; p< .001). No significant difference between antibody titer of pwMS without treatment and HC was found [95%CI= -0.882; - 0.935 p > .99]. In 65 adults without DMT use (HC+pwMSwithout treatment), no seronegative cases were observed in any vaccine group. In patients treated with DMT, BNT162b2 was associated with a 16.3% greater absolute risk of seropositivity than Sinovac. CONCLUSION: The mRNA vaccine could be a preferred choice of protection against SARS-CoV-2 in pMS treated with DMT.

A Multicenter Analysis of Subjectivity of Indirect Immunofluorescence Test in Antinuclear Antibody Screening.

Objectives: This study aims to evaluate the interpretation of the antinuclear antibody (ANA)-indirect immunofluorescence (IIF) test results based on the interpreter-related subjectivity and to examine the inter-center agreement rates with the performance of each laboratory. Patients and methods: The ANA-IIF testing was carried out in a total of 600 sera and evaluated by four laboratories. The inter-center agreement rates were detected. The same results given by the four centers were accepted as gold standard and the predictive values of each center were calculated. Results: The inter-center agreement was reported for ANA-IIF test results from 392 of 600 (65.3%) sera, while 154 of 392 results were positive. Four study centers reported 213 (35.5%), 222 (37.0%), 266 (44.3%), and 361 (60.2%) positive test results, respectively. In terms of the patterns, the highest and lowest positive predictive values were 72.3% and 42.7%, respectively, while the highest and lowest negative predictive values were 99.6% and 61.5%, respectively. The agreement for semi-quantitative evaluation at three levels of fluorescence intensity stated by four centers was detected in 100 sera at 87% 3(+), while the other two levels were 6% and 7%. The highest predictive value for the highest fluorescence intensity of 3(+) was found to be 71.9%. Conclusion: Significant differences may be observed among laboratories in terms of qualitative results, patterns, and semi-quantitative determination of the fluorescence intensity in the ANA-IIF testing, particularly at low fluorescence intensity levels and in those with speckled patterns. In case of any discrepancy between ANA-IIF test and clinical prediagnosis, the test should be repeated in another laboratory, if necessary.

Effect of Blood Cell Subtypes Lysis on Routine Biochemical Tests.

BACKGROUND: The aim of this study is to establish the contribution of blood cells subtypes on hemolysis. METHODS: Separated blood cell subtype suspensions prepared with blood from 10 volunteers were serially diluted to obtain different concentrations of cell suspensions. The cells were fully lysed and cell hemolysates were added (1:20) to aliquots of serum pool. Thus, seven serum pools with different concentrations of interferent were obtained for each blood cell subtype. Biochemical parameters and serum indices were measured by an autoanalyzer. As cell lysis markers, free hemoglobin was measured by spectrophotometry while myeloperoxidase and ᵝ-thromboglobulin were measured by enzyme immunoassay. The percent changes in analyte levels of the serum pools were evaulated by Wilcoxon Signed Rank Test and compared with clinical thresholds defined for each test. RESULTS: The clinical thresholds were exceeded in lactate dehydrogenase, potassium, aspartate aminotransferase, creatine kinase, magnesium, total protein, total cholesterol, inorganic phosphate, glucose for red blood cells (RBC); lactate dehydrogenase, aspartate aminotransferase, total protein, inorganic phosphate and glucose for platelets (PLT). Free hemoglobin was significantly correlated with RBC (r=0.999; p=0.001), while myeloperoxidase and b thromboglobulin showed no significant correlation to white blood cells (WBC) and PLT, respectively. CONCLUSIONS: The effect of RBC hemolysis in serum on the routine biochemical tests are clearly established, yet, additional studies are required in order to verify this kind of effects of PLT and WBC hemolysis.

Screening of celiac disease in children with Henoch-Schoenlein purpura.

Prevalence of celiac disease (CD) is 2.42 % in healthy Turkish children. The frequency of IgA-associated disorders is increased in CD. Henoch-Schoenlein purpura (HSP) is an IgA-associated vasculitis. Association of HSP with CD has not been evaluated. We aimed to evaluate whether CD prevalence is increased in children with HSP. Children with HSP were evaluated for demographic, anthropometric, clinical, and laboratory data including urinalysis, complete blood count, albumin, creatinine, and IgA levels. In addition, tTG-IgA, EMA-IgA, anti-DGP-IgA, and IgG antibody levels were measured. Seropositive patients were evaluated by endoscopic small bowel biopsy. The rate of CD seropositivity and confirmed CD in HSP patients was compared to the rate in healthy Turkish children. There were 42 children (25 male) with HSP. No patient had classical CD symptoms, but two patients had growth failure. None of them had IgA deficiency, anemia or hypoalbuminemia. Celiac serology was positive in five (12 %) children. Endoscopic evaluation was performed in four patients, and two (5 %) of them were confirmed to have CD. Prevalence of both CD seropositivity and histologically confirmed CD in children with HSP was significantly higher compared to healthy Turkish children (p < 0.001 and p = 0.019, respectively). CD seropositivity rate in children with HSP (12 %) is significantly higher than the rate in healthy children. Although the number of children with HSP is small in this preliminary study, this result suggests that celiac screening may be considered in children with HSP.

[Implementation of "5S" methodology in laboratory safety and its effect on employee satisfaction]. / Laboratuvar güvenliginde "5S" yönteminin uygulamasi ve çalisan memnuniyeti üzerine etkisi.

Health institutions use the accreditation process to achieve improvement across the organization and management of the health care system. An ISO 15189 quality and efficiency standard is the recommended standard for medical laboratories qualification. The "safety and accommodation conditions" of this standard covers the requirement to improve working conditions and maintain the necessary safety precautions. The most inevitable precaution for ensuring a safe environment is the creation of a clean and orderly environment to maintain a potentially safe surroundings. In this context, the 5S application which is a superior improvement tool that has been used by the industry, includes some advantages such as encouraging employees to participate in and to help increase the productivity. The main target of this study was to implement 5S methods in a clinical laboratory of a university hospital for evaluating its effect on employees' satisfaction, and correction of non-compliance in terms of the working environment. To start with, first, 5S education was given to management and employees. Secondly, a 5S team was formed and then the main steps of 5S (Seiri: Sort, Seiton: Set in order, Seiso: Shine, Seiketsu: Standardize, and Shitsuke: Systematize) were implemented for a duration of 3 months. A five-point likert scale questionnaire was used in order to determine and assess the impact of 5S on employees' satisfaction considering the areas such as facilitating the job, the job satisfaction, setting up a safe environment, and the effect of participation in management. Questionnaire form was given to 114 employees who actively worked during the 5S implementation period, and the data obtained from 63 (52.3%) participants (16 male, 47 female) were evaluated. The reliability of the questionnaire's Cronbach's alpha value was determined as 0.858 (p< 0.001). After the implementation of 5S it was observed and determined that facilitating the job and setting up a safe environment created a statistically significant effect on employees, and some sufficient satisfaction was observed. In addition, the non-conformity score, which was identified in the laboratory during the previous years, was significantly reduced at a rate of 69.7% after the implementation of 5S. 5S practices have successfully contributed to the establishment and to the sustainability of laboratory safety systems in the first public ISO 15189 accredited public clinical laboratory in Turkey. It is concluded that 5S methods can be used as an effective improvement tool in order to maintain a safe environment, to facilitate the job, and to encourage employees to participate in the management process.

[Evaluation of a syphilis testing algorithm using a treponemal test for screening]. / Taramada Treponemal Test Kullanan Bir Sifiliz Tani Algoritmasinin Degerlendirilmesi.

Traditional testing algorithm for syphilis is initial screening with nontreponemal tests, then retesting positive samples for confirmation using a specific treponemal test. Commercial treponemal tests those are more sensitive than nontreponemal tests and suitable for automation are now commercially available to screen syphilis. Although the treponemal tests are offered as the first choice for syphilis screening by some of the guidelines, to date there is no consensus on recommendations in guidelines for followup testing. As some of the guidelines recommend to perform a nontreponemal test for persons with a positive treponemal screening test, the others recommend to confirm the result by another treponemal test. In this study, a testing algorithm using treponemal chemiluminescence microparticle enzyme immunoassay (CMIA; Architect Syphilis TP; Abbott Japan Co, Japan) for primary screening were retrospectively evaluated by reviewing laboratory data. A total of 12.195 serum samples obtained from 10.878 patients (7104 of them were female) who were screened by means of syphilis, between January 2007-February 2010 period have been included to the study. According to this algorithm, no further test was performed for CMIA negative samples. Samples positive by CMIA were retested by RPR (Rapid Plasma Reagin; Omega Diagnostics, UK) test. The test results of both CMIA and RPR positive samples were reported, while positive CMIA results were confirmed by TPHA (Treponema pallidum hemagglutination; Omega Diagnostics, UK) if any discrepancy in the results were identified. Screen test revealed positive results in 1.1% (120/10.878) of the patients and 2% (206/12.195) of the samples. In this study, quantitative values (sample/cut-off absorbance ratio; s/co) of positive CMIA samples were also compared with TPHA and RPR test results. It was observed that while 19.1% of CMIA positive samples with s/co ratios less than 12 were confirmed by TPHA, the confirmation rate was 100% with s/co ratios above 12. Additionally, RPR positive samples with a titer of ≥ 1/32 yielded CMIA s/co ratios above 21. As low s/co levels detected with CMIA may lead to false-positive results for syphilis, it was concluded that CMIA positive results should be confirmed by another treponemal test before RPR testing. A new syphilis testing algorithm in accordance with the results of this review and recommendations in new guidelines were established in the light of this study.

The significance of oligoclonal bands in multiple sclerosis: relevance of demographic and clinical features, and immunogenetic backgrounds.

There has been no data on oligoclonal IgG bands (OCBs) for Turkish MS population, who is believed to be placed between Western and Eastern world regarding the clinical and immunological features. In the present study, we examined the correlation between the frequency of OCB and clinical and demographical features of MS in Turkish MS population. Our objective was to determine whether, population with OCB-positive and OCB-negative MS constitutes distinct subpopulations in terms of clinical, demographic and genetic base. A total of 210 clinically definite MS patients were included in the study. Patients were assessed clinically at baseline, 1 month later, every 3 months, and at year 5. No CSF OCB could be detected in 30 (14.3%) cases. 141 of the 156 female patients (90.4%) and 39 of the 54 male patients (72.2%) were positive for OCB. The female to male ratio was higher in the OCB-positive than in the OCB-negative group (p=0.007). Clinical course and disability were similar in the two patient groups. But mean relapse severity was higher in patients without OCBs in CSF. EDSS values got worse in OCB-negative group in year 5 (p=0.008). EDSS was also significantly higher in OCB-negative group in year 5 (p=0.003). There was no statistically significant difference regarding the usage of disease modifying therapy between the two groups. HLA DR15 antigen frequency was statistically higher in the OCB-positive than in the OCB-negative patients (p=0.007) and control group (p=0.0002). In conclusion, our results suggested that, MS patients with CSF OCBs have a female predominance, better clinical course with less disability and better prognosis, are associated with HLA-DR15.

Effect of subminimal inhibitory concentrations of three fluoroquinolones on adherence of uropathogenic strains of Escherichia coli.

The effect of subinhibitory concentrations (1/2-1/32 x MIC) of ciprofloxacin, ofloxacin and levofloxacin on the adherence of three strains of Escherichia coli (a mannose-resistant haemagglutinating clinical isolate, a non-haemagglutinating clinical isolate and the mannose-resistant haemagglutinating ATCC 25922 strain) were studied. Ciprofloxacin had the lowest MIC values but only the 1/2 MIC concentration inhibited adherence of mannose-resistant haemagglutinating strains after exposure to subMIC values. Significant inhibition of adherence was observed with 1/4 x MIC ofloxacin for both haemagglutinating isolate (27096) and the ATCC strain. Levofloxacin might be more effective and safer than ciprofloxacin and ofloxacin as a long acting fluoroquinolone at subMIC values in patients with UTI.