Iwona Kotuła

Neutrophil extracellular traps (Nets) impact upon autoimmune disorders

Friend or foe? This is often asked question when it comes to neutrophil extracellular traps studies. There is no simple answer to that. At the time of their discovery they were considered to be protectors of our well-being. Excellent pathogen fighting skills were described as purely beneficial. But it was not long before those guardians of immunity reveal their dark side. What seemed to be profitable could also take its toll. They are perfectly constructed, made from nucleic deoxyribonucleic acid ornamented with cytoplasmic and granular proteins, to fight invaders. But this unique build is prone to become considered by our body as a threat. Since there is a thin line which when crossed turns a savior into enemy, it was postulated that Nets can play a significant role in autoimmune disorders pathogenesis and disease exacerbation. Recent years have brought a new insight into autoimmune disorders trying to connect the old knowledge and suspicions with modern discoveries.

A Comparison of Mindray BC-6800, Sysmex XN-2000, and Beckman Coulter LH750 Automated Hematology Analyzers: A Pediatric Study.

Modern automated laboratory hematology analyzers allow the measurement of over 30 different hematological parameters useful in the diagnostic and clinical interpretation of patient symptoms. They use different methods to measure the same parameters. Thus, a comparison of complete blood count made by Mindray BC‐6800, Sysmex XN‐2000 and Beckman Coulter LH750 was performed.

Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis.

Abstract Background: The eosin-5′-maleimide (EMA) binding test is a flow cytometric test widely used to detect hereditary spherocytosis (HS). EMA binds to plasma membrane proteins of red blood cells (RBCs), mainly to band 3 protein. The mean fluorescence of EMA-stained RBCs in HS patients is lower when compared with control RBCs due to the decreased amount of target proteins. EMA dye in aqueous solution is sensitive to light and high temperature. Its fluorescence can decrease when exposed to light or ambient temperatures higher than 4°C. The aim of the study was to evaluate the stability of fluorescence readings of EMA-labeled RBCs over a period of 24 h. Methods: The EMA test was performed in peripheral blood from 35 patients with microcytic anemia (five with HS, and 30 without HS). Peripheral blood samples were stained immediately after blood collection and analyzed using a flow cytometer at three time points: 0, after 1 and 24 h of storage at 4°C in the darkness. The results are presented as the percentage of normal control RBCs fluorescence. Flow cytometric studies were performed with Cytomics FC500 (Beckman Coulter, USA). Results: In HS patients the mean result of the test reached 66.72%±9.26% of normal controls, and in non-HS patients the EMA result was 99.48%±5.03% of normal control cells. The results of patients with HS were 66.72%±9.26%, 66.90%±10.24% and 67.86%±11.31% at 0 h, and after 1 and 24 h of storage, respectively. The results obtained from non-HS patients at time 0, after 1 and 24 h of storage reached 99.48%±5.03%, 99.49%±5.34% and 99.78%±6.13%, respectively. There was no difference between the results from each time point in samples from patients with or without HS. Conclusions: Results of the EMA binding test do not depend on storage time of stained samples when stored at 4°C up to 24 h after staining.

Influence of Sublingual Immunotherapy on the Expression of Mac-1 Integrin in Neutrophils from Asthmatic Children

Asthma can be effectively treated with sublingual immunotherapy. The influence of -sublingual immunotherapy on the function of granulocytes in asthmatic patients is largely unknown. Mac-1 integrin is a transmembrane protein containing α (CD11b) and β (CD18) chains. High expression of the complex is found on the surface of neutrophils, NK cells, and macrophages. CD11b/CD18 may bind to CD23, ICAM-1, ICAM-2, and ICAM-4. It plays a crucial role in diapedesis of neutrophils. The aim of the present study was to assess Mac-1 expression on neutrophils from asthmatic children before and after sublingual immunotherapy. Twenty five children aged of 8.1 ± 3.1 suffering from atopic asthma and allergic rhinitis, shortlisted for specific immunotherapy, served as the study group. Fifteen healthy individuals, aged 9.8 ± 3.4, served as a control group. The assessment of CD11b and CD18 expression on cells from peripheral blood was performed with a flow cytometer. The tests were performed before and after 12 months of sublingual immunotherapy. In the asthmatic children, 98.08 (90.79-99.12)% of Mac-1 positive neutrophils were detected. The group was divided into two subgroups: of more than 98% and less than 95% of neutrophils with CD11b/CD18 expression in the sample. After immunotherapy, the percentage of Mac-1 positive granulocytes increased to 99.60 (99.29-99.68)%, p = 0.01. In the control group, 90.56 (87.08-88.86)% granulocytes were Mac-1 positive, p = 0.002. We conclude that sublingual immunotherapy strongly influences the function of the immunological system, including Mac-1 expression on neutrophils.

Recurrent respiratory tract infections in children - analysis of immunological examinations.

Background Paediatric respiratory tract infections are among the most common reasons for preschool and school absences and visits to physicians. The disease mainly involves the upper respiratory tract and is associated with fever, cough, sore throat, and running nose. Children with recurrent respiratory infections (RRI), which are defined as more than six serious diseases a year, are a difficult diagnostic challenge. The aim of this study was to assess immunological deviations in laboratory tests performed in children with RRI. Material and methods In the retrospective study 25 children suffering from recurrent respiratory tract infection, aged 4.1 ±2.3 years, 13 boys and 12 girls, were involved. For all children chemiluminescence of granulocytes and immunophenotyping of lymphocytes from peripheral blood were examined. An immunophenotype of peripheral blood lymphocytes involved evaluation of T cell, B cells, and NK cells, examined with flow cytometry. Results Eleven of the studied children had decreased chemiluminescent response to stimulants, normal response was found for nine children, and five children had an increased result of the test. Five of the 25 children had decreased B cells number, and five had decreased number of T cells including decrease of CD4, as well as CD8 positive cells. Children with decreased chemiluminescence had more frequent neutropaenia than children with normal or increased chemiluminescent response, p < 0.05 (exact Fisher test). Conclusions Recurrent respiratory tract infection could be associated with improper neutrophils response to pathogens, and immunological examination should be performed to find the reason for the increased number of infections in a year.

Lymphocytes sensitivity to Fas stimulation in healthy and asthmatic children

The T cell hypothesis of asthma is based on the concept that the disease is driven and maintained by the persistence of a specialized subset of chronically activated T memory cells sensitized against an array of allergenic, occupational or viral antigens. Overreaction of CD4+ T cells in the peripheral blood and airway tissues is an invariant feature of asthma; therefore a potent mechanism for augmenting the number of activated T cells in this disease would be the resistance to the normally programmed pathway for cell death. The aim of the study was to evaluate the presence of apoptotic markers on peripheral blood lymphocytes from healthy and asthmatic children before and after stimulation with antiCD95 antibodies. The blood was collected from 21 children with atopic asthma suffering from allergic rhinitis because of house dust mite and/or grass pollen allergens and 8 healthy children matched for their age and sex. Blood was mixed with purified monoclonal antibody antiCD95 (Beckman Coulter), incubated for 24 hours and than stained with Annexin V andPI (Becton Dickinson). Prepared suspensions were analyzed with Cytomics FC 500 (Beckman Coulter) flow cytometer. Annexin V(+)/PI(-) cells were characterized as early apoptotic, Annexin V(+)/PI(+) as late apoptotic and Annexin V(-)/PI(+) as dead. In unstimulated sample from asthmatic children 21.09+/-11.20% cells were characterized as Annexin V positive/PI negative. After stimulation with antiCD95 Annexin V positive/PI negative cells constituted 18.72+/-9.42% of cells, p=0.1. In unstimulated sample from healthy children 11.69+/-6.70% cells were characterized as Annexin V positive/PI negative. In the sample stimulated with antiCD95 16.54+/-2.98% of cells were Annexin V positive/PI negative, p=0.02. There were no differences between results of late apoptotic and necrotic lymphocytes from healthy and asthmatic children. Performed research indicates that lymphocytes from asthmatic children are resistant to Fas mediated apoptosis.

Pseudohyperkalemia in capillary whole-blood samples – an occasional error or a significant problem in a pediatric hospital?

Capillary whole-blood samples are widely collected for acid-base balance evaluation in pediatric individuals. Material can be collected from fingertips, earlobes or heels. Besides typical acid-base parameters such as pH and pCO2, the sample is also referred for electrolytes (potassium and sodium) concentration analysis. Potassium is one of the most frequently tested analytes, since it plays a critical role in human homeostasis. Pre-analytical errors can result in falsely increased potassium concentrations in collected samples. Pseudohyperkalemia is defined as a difference between serum and plasma potassium concentration of more than 0.4 mmol/L [1]. Excessive leakage of potassium from cells during blood collection due to tissue squeezing can falsely elevate its concentration. Pseudohyperkalemia may also result from hemolysis, leukocytosis or blood clotting, since intracellular potassium concentration is ca. 30 times higher than extracellular concentration [2]. The phenomenon is widely known; however, it seems to be often misinterpreted [3]. The main causes of spurious hyperkalemia are presented in Table 1. We aimed to assess the frequency of hyperkalemia in capillary blood samples and the reaction of clinicians to high K+ results in a pediatric hospital. We performed a retrospective analysis of potassium concentration in plasma of capillary blood samples collected to capillary tubes filled with lithium heparin. Samples from 3280 pediatric patients, with no symptoms of hyperkalemia, median age 2 years and 7 months, from 14 hospital wards were included into this study. The analysis of capillary blood was performed directly after the samples were delivered to the laboratory, which approximately took ca. 15 min. Additional analysis of serum sample was performed for verification of potassium serum concentration. All samples collected to tubes containing clot activator were centrifuged within 30 min after the material was brought to the laboratory. The average time between blood sampling and specimen arrival to the laboratory was not longer than 15 min. Analysis of capillary samples was performed with a Radiometer ABL 90 Flex equipped with an indirect ion selective electrode for electrolytes measurement. Serum potassium concentration was measured with a Vitros 5600 Orthoclinical, using dry slide technology. Hyperkalemia was defined as an increase in serum potassium concentration over following reference ranges: – Newborns 0–1 month, 3.6 – 6.0 mmol/L – Infants 1–12 month, 3.6 – 5.8 mmol/L – Children 1–18 years, 3.5 – 5.1 mmol/L and of 0.5 mmol/L lower in plasma.

Delayed Measurement of Eosin-5'-Maleimide Binding May Affect the Test Results of Highly Hemolyzed Samples In Vivo and In Vitro-A Case Study.

Diagnosis of hereditary spherocytosis (HS) is based on clinical evaluation and eosin-5′-maleimide (EMA) test. A decrease in EMA fluorescence compared with healthy individuals is typical for HS and serves as a basis for HS diagnosis. Sensitivity and specificity of the test is high and false-positive results rarely occur. Studies have shown that anticoagulated blood sample when stored at 4°C for 7 days do not affect the test results. This case study is about an autoimmune hemolytic anemia patient who showed a primary positive result for EMA test (decrease in EMA fluorescence—47% compared with 100% for samples of healthy individual), when the test was performed in the sample stored for 48 hours after venipuncture and before staining. An irrelevant decrease (92.5% compared with 100% for samples of healthy individual) was found when freshly collected sample was analyzed. On the basis of the results obtained, it is recommended that EMA staining should be performed on the same day of blood collection for patients with significant hemolysis.

Tuberculosis infection in children with proteinuria/nephrotic syndrome

Children with nephrotic syndrome (NS) are at greater risk of infections than the general population, due to immunodeficiency in the course of the disease and the treatment. In this study we present 4 children (2 girls, 2 boys), mean age 7.6 ±5.1 years, with NS/proteinuria and latent tuberculosis in 3 children and lymph node tuberculosis in 1 child. The reasons for testing these children for tuberculosis (TB) were the evaluation of the epidemiological status before treatment with corticosteroids (GCS), leukopenia and the relapse of NS, and non-nephrotic proteinuria. The diagnosis of TB infection was based on positive IGRA (Interferon-Gamma Release Assay). Chest X-ray was normal in all the children. Chest CT scan revealed an enlargement of lymph nodes in 1 child. The children were treated with isoniazid (3 children) and isoniazid, rifampicin and pyrazinamide (1 child). Three children with idiopathic nephrotic syndrome were treated with prednisone. The child with non-nephrotic proteinuria was treated with enalapril. Proteinuria disappeared in all children during anti-TB treatment.