Prognostic and predictive role of soluble programmed death ligand-1 in head and neck cancer.

OBJECTIVES: The aim of the study was to investigate clinical significance of soluble PD-L1 (sPD-L1) serum level in head and neck cancer and to evaluate its role as a possible prognostic and predictive biomarker. METHODS: A prospective analysis of sPD-L1 levels in 60 patients diagnosed and treated due to malignant and non-malignant lesions in the region of head and neck was performed in peripheral blood by an ELISA test. RESULTS: The range of sPD-L1 in the study group was 0.16-1.63ng/mL, mean 0.64±0.32. There were no differences in the mean sPD-L1 regarding patients' age, sex, and the localization of the lesion. Statistically significant difference was revealed in the average sPD-L1 level (p= 0.006) depending on the histopathological advancement of the lesions, 0.704 ± 0.349 and 0.512 ± 0.177 respectively in the malignant and benign group. The separate analysis of laryngeal lesions confirmed statistical difference in sPD-L1 (p= 0.002) for the malignant lesions (0.741 ± 0.353) compared with the benign (0.489 ± 0.175). The sPD-L1 level of 0.765 ng/mL or higher, revealed 35% sensitivity and 95.5% specificity for the diagnosis of head and neck malignant lesions (AUC=0.664, 95% CI 0.529‒0.8, p-value=0.039). The 1-year DFS was 83.3% in the group of patients with low sPD-L1 levels (< 0.765ng/mL) and 53.8% in patients with high sPD-L1 (≥0.765ng/mL). The 2-year OS were 68% and 69.2% respectively in both groups. The log-rank test confirmed statistically significant prognostic value of sPD-L1 level for 1-year DFS (p-value=0.035). CONCLUSIONS: sPD-L1 is a promising prognostic and early recurrence predictive biomarker for head and neck cancers, most significantly for laryngeal lesions.

Serum expression of Vascular Endothelial-Cadherin, CD44, Human High mobility group B1, Kallikrein 6 proteins in different stages of laryngeal intraepithelial lesions and early glottis cancer.

Background: The study was designed to evaluate the potential validity and utility of selected molecular markers in serum samples from patients with specific stages of laryngeal intraepithelial lesions that could serve as diagnostic tools in differentiation of benign and dysplastic lesions from invasive pathologies. Methods: Prospective study included 80 consecutive patients with vocal fold lesions treated at the single otorhinolaryngology centre. All participants had surgical resection of the lesion. Blood samples were collected from each patient before the surgery. Final diagnosis was confirmed on histopathological examination and included 39 (48.75%) non-dysplastic lesions, eight (10%) low-grade dysplasia, six (7.5%) high-grade dysplasia and 27 (33.75%) invasive cancers. The ELISA procedures were performed according to the manufacturer's instruction. Individual serum concentration of selected proteins was reported in ng/ml: Vascular Endothelial-Cadherin Complex (VE-cad), CD44, Human High mobility group protein B1(HMGB1), Kallikrein 6. Results: The highest mean levels of HMGB1, KLK6 and VE-cad were detected in sera of patients with low-grade dysplasia (81.14, 24.33, 14.17 respectively). Soluble CD44 was the most elevated in patients with non-dysplastic lesions (2.49). The HMGB1, KLK6 and VE-cad serum levels were increasing from non-dysplastic to low-grade dysplasia and followed by the decrease for high-grade dysplasia and invasive cancer, however the differences were not significant (p-values 0.897, 0.354, 0.1 respectively). Patients' serum had the highest CD44 concentration in non-dysplastic and low-grade dysplasia with the following decrease through high-grade dysplasia and invasive cancer. GERD symptomatic patients had higher levels of KLK6 and CD44 than other patients (p-value 0.06 and 0.084 respectively). There were no significant differences of biomarkers levels related to patients' gender (p-value from 0.243 to 1) or smoking status (p-value from 0.22 to 0.706). Conclusions: VE-cad, HMGB1, CD44 and KLK6 did not prove to be reliable biomarkers implicating malignant potential within vocal fold hypertrophic intraepithelial lesions.

Serum neutrophil gelatinase-associated lipocalin for predicting anemia of inflammation in children with urinary tract infection.

Anemia of inflammation (IA), the second most common cause of childhood anemia, results from macrophage iron sequestration and impaired erythropoiesis. Neutrophil gelatinase-associated lipocalin (NGAL) plays an important role in innate microbial immunity through its influence on intracellular iron homeostasis and inhibition of erythropoiesis. The predictive value of NGAL in IA was assessed in 74 children (age 6.30 ±3.64 months) with the first episode of urinary tract infection (UTI). Anemia of inflammation was found in 50% of children, including those with non-febrile UTI, and delayed onset of anemia was observed in 20% of children. There were no differences in NGAL levels between the anemic and non-anemic children, and no correlations between NGAL and hemoglobin (HGB) levels and red blood cell (RBC) count. In multivariate logistic regression analysis elevated C-reactive protein (CRP) was only independently associated with the presence of anemia in children with UTI [OR (95% CI): 1.128 (1.005-1.265), p = 0.040]. In stepwise multiple analysis age independently correlated with RBC (ß = 0.051, p = 0.001), while CRP independently correlated with HGB (ß = -0.037, p = 0.027) and RBC (ß = -0.022, p = 0.014). ROC analysis demonstrated better diagnostic profiles for CRP, procalcitonin (PCT) and fever duration for predicting the risk of IA than NGAL (AUC: 0.690, 0.669, 0.678 vs. 0.638, respectively). Despite the increase in HGB levels after 4-5 weeks from the onset of UTI, HGB values were still significantly lower in the anemic than in non-anemic children. NGAL was not useful for predicting IA in UTI, since its diagnostic value was inferior to conventional inflammatory markers.

Serum and tissue expression of neuropilin 1 in precancerous and malignant vocal fold lesions.

OBJECTIVES: The study was designed to evaluate the tissue expression of NRP-1 and serum level of sNRP-1 in the same patients with intraepithelial laryngeal lesions or early staged laryngeal cancer to identify the clinical significance of these biomarkers in the diagnosis of laryngeal lesions. MATERIAL AND METHODS: A prospective analysis of tissue was performed on specimens and blood samples from 49 patients, who were admitted for surgical resection due to suspicious vocal fold lesions and were diagnosed as non-dysplasia, low-grade dysplasia, high-grade dysplasia and invasive cancers. RESULTS: ELISA was conducted on 48 blood samples. The minimum level of sNRP-1 was 0.15 ng/ml and maximum- 37.71 ng/ml. The Kruskal-Wallis one-way analysis of variance revealed no differences in sNRP-1 levels between different histopathological stages of vocal fold lesions (p = 0.234). IHC was conducted in 49 tissue samples. The evaluated mean scores of NRP-1 tissue expression were compared to histopathological stage of the lesion. The Kruskal-Wallis one-way analysis of variance revealed no differences in NRP-1 tissue expression between different histopathological stages of vocal fold lesions (p = 0.536). The correlation of tissue NRP-1 expression and serum levels of NRP-1 within analyzed group was insignificant. The Spearman's rank correlation coefficient was 0.076 (p = 0.606). CONCLUSIONS: The NRP-1 tissue expression and serum levels are unlikely to be a prognostic factor for identification of laryngeal dysplasia or early stage laryngeal cancer. Further studies investigating biomolecules involved in laryngeal carcinogenesis are necessary.

Inhibition of IDO leads to IL-6-dependent systemic inflammation in mice when combined with photodynamic therapy.

It was previously reported that the activation of antitumor immune response by photodynamic therapy (PDT) is crucial for its therapeutic outcome. Excessive PDT-mediated inflammation is accompanied by immunosuppressive mechanisms that protect tissues from destruction. Thus, the final effect of PDT strongly depends on the balance between the activation of an adoptive arm of immune response and a range of activated immunosuppressive mechanisms. Here, with flow cytometry and functional tests, we evaluate the immunosuppressive activity of tumor-associated myeloid cells after PDT. We investigate the antitumor potential of PDT combined with indoleamine 2,3-dioxygenase 1 (IDO) inhibitor in the murine 4T1 and E0771 orthotopic breast cancer models. We found that the expression of IDO, elevated after PDT, affects the polarization of T regulatory cells and influences the innate immune response. Our results indicate that, depending on a therapeutic scheme, overcoming IDO-induced immunosuppressive mechanisms after PDT can be beneficial or can lead to a systemic toxic reaction. The inhibition of IDO, shortly after PDT, activates IL-6-dependent toxic reactions that can be diminished by the use of anti-IL-6 antibodies. Our results emphasize that deeper investigation of the physiological role of IDO, an attractive target for immunotherapies of cancer, is of great importance.

Diagnostic accuracy of urine neutrophil gelatinase-associated lipocalin and urine kidney injury molecule-1 as predictors of acute pyelonephritis in young children with febrile urinary tract infection.

INTRODUCTION: We assessed whether two urinary biomarkers of acute kidney injury, neutrophil gelatinase associated lipocalin (uNGAL) and kidney injury molecule-1 (uKIM-1), can be useful for predicting acute pyelonephritis (APN) in children aged 1-24 months with the first febrile urinary tract infection (UTI). MATERIAL AND METHODS: A prospective study included 54 children divided into two groups (24 with APN, 30 with lower UTI), according to the dimercaptosuccinic acid (DMSA) renal scintigraphy results. Laboratory tests: uNGAL, uKIM-1, procalcitonin (PCT), C-reactive protein (CRP), white blood count (WBC) were performed. RESULTS: We did not find significant differences in normalized and non-normalized values of uNGAL and uKIM-1 in children with APN and lower UTI. Positive correlations were determined between uNGAL and pyuria (r = 0.28, p < 0.05) and between uNGAL/uCr and uKIM-1/uCr (r = 0.53, p < 0.001) in the all UTI groups. Univariate logistic regression analysis demonstrated that only PCT (p < 0.0001) and CRP (p < 0.05) were important diagnostic factors of APN. Receiver operating curve (ROC) analysis showed good diagnostic profiles of PCT with the best cut-off value of 1.66 ng/ml and of CRP with the best cut-off value of 4.3 mg/dl for predicting APN (area under the curve [AUC]: 0.894 and 0.719, sensitivity: 75% and 96%, specificity: 93% and 43%, respectively). CONCLUSIONS: uNGAL and uKIM-1 are not effective diagnostic markers for APN in young children with febrile UTI and cannot be used in clinical practice to differentiate APN from lower UTI.

Is copeptin a reliable biomarker of primary monosymptomatic nocturnal enuresis?

INTRODUCTION: The position of copeptin (C-terminal fragment of antidiuretin propeptide) as a marker of primary monosymptomatic nocturnal enuresis (PMNE) is under debate, and there are no data on the relation between copeptin and clinical and biochemical parameters in these patients. Aim of the study was to assess the level of serum copeptin in children with PMNE and to look for a relation between copeptin and selected clinical and biochemical parameters in these children. MATERIAL AND METHODS: Twenty-five children recruited for the trial fulfilled the following criteria: clinical diagnosis of PMNE, age 5-15 years, normal creatinine level, normal ultrasonographic image of kidneys and urinary tract. The following parameters were evaluated: serum copeptin, creatinine, sodium, potassium, hematocrit and urine specific gravity. Twenty healthy children were included in the control group. RESULTS: Children from study and control groups did not differ in serum copeptin, sex, age creatinine, sodium, hematocrit and specific gravity. Serum potassium level remained normal in subjects but was significantly higher in the study group. In children with PMNE we found no relation between serum copeptin level and sex, kidney function, sodium, and urinary specific gravity. We found a negative correlation between copeptin and bladder capacity and trends towards positive relations between copeptin and age, as well as hemoglobin. In the subgroup of children with normal bladder capacity a trend towards a positive correlation between copeptin and potassium was found. CONCLUSIONS: Copeptin may be a marker of hydration status in children with PMNE. The relation between potassium and copeptin levels and the clinical significance of the relation require further studies.

Serum neutrophil gelatinase-associated lipocalin for predicting acute pyelonephritis in infants with urinary tract infection.

INTRODUCTION: Fever and elevated inflammatory markers have been used for diagnosis of acute pyelonephritis (APN) in infants with urinary tract infection (UTI).The aim of the study was to compare the usefulness of serum neutrophil gelatinase-associated lipocalin (sNGAL) with inflammatory markers for predicting APN in infants with UTI. MATERIAL AND METHODS: The prospective study included 46 infants with a first episode of UTI, divided into two groups (APN 23, lower UTI 23), according to the DMSA scan results. The following laboratory tests were performed: sNGAL, PCT, CRP, WBC, and ESR. RESULTS: Significantly elevated levels of sNGAL, PCT, CRP, and ESR were observed in infants with APN compared to those with lower UTI. Higher sNGAL, CRP, and ESR values, presence of fever, and longer duration of fever before antibiotic treatment were associated with APN [odds ratio (OR) 1.02, 1.27, 1.03, 13.46, 2.12, respectively]. Receiver operating characteristic (ROC) analysis showed better diagnostic profiles for sNGAL, PCT, and CRP than for ESR for predicting APN [area under the curve (AUC) 0.808, 0.819, 0.841, and 0.750, respectively]. The appropriate cut-off values of sNGAL, PCT and CRP were 100.8 ng/ml, 0.15 ng/ml, 5.3 mg/dl (all sensitivity and specificity 82.6%), and that of ESR was 40 mm/h (sensitivity 78.3%, specificity 60.9%). CONCLUSIONS: sNGAL shows similar usefulness as PCT and CRP for predicting APN in infants with UTI, the diagnostic value of ESR is smaller, and WBC is not useful at all. The presence of fever and longer duration of fever are important predictors of APN in infants with UTI.

Prognostic value of serum and urine kidney injury molecule-1 in infants with urinary tract infection.

INTRODUCTION: Kidney injury molecule-1 (KIM-1) is an important diagnostic and prognostic marker in acute kidney injury and chronic kidney disease of various aetiologies. The aim of the study was to evaluate the usefulness of serum KIM-1 (sKIM-1) and urine KIM-1 (uKIM-1) for predicting febrile and non-febrile urinary tract infection (UTI) in infants. MATERIAL AND METHODS: A prospective study included 101 children divided into three groups: febrile UTI 49 children, non-febrile UTI 22 children, and healthy controls 30 children. The following laboratory tests were performed: sKIM-1, uKIM-1, white blood count (WBC), C-reactive protein (CRP), and procalcitonin (PCT). RESULTS: Median levels of sKIM-1 were significantly higher in the febrile and non-febrile UTI group compared to the healthy controls (both p < 0.05). Mean levels of uKIM-1 were significantly lower in the febrile UTI group compared to the non-febrile UTI group and healthy controls (p < 0.001 and p < 0.0001, respectively). Univariate logistic regression analysis has demonstrated a positive association of sKIM-1 with febrile and non-febrile UTI (both p < 0.05), and negative association uKIM-1 with febrile UTI (p < 0.0001). Receiver operating curve (ROC) analysis showed good diagnostic profiles of uKIM-1 with a best cut-off value of 2.4 ng/ml and sKIM-1 with a best cut-off value of 3.88 ng/ml for predicting febrile UTI (area under the curve [AUC] 0.82 and 0.67, sensitivity 73% and 63%, specificity 86% and 80%, respectively). CONCLUSIONS: sKIM-1 can be useful for predicting febrile UTI. We do not recommended use of uKIM-1 as a marker of febrile UTI because of its negative association with febrile UTI. Both markers are not useful for predicting non-febrile UTI.

Flow cytometric osmotic fragility test: Increased assay sensitivity for clinical application in pediatric hematology.

BACKGROUND: Osmotic fragility test (OFT) is widely considered as a sensitive indicator of red blood cells' sensitivity to the hypotonic solution. It is often used as a screening test for the diagnosis of hereditary spherocytosis (HS). Nowadays, the osmotic fragility test based on flow cytometric analysis (FCM OF) is widely used in laboratory practice. The purpose of this study was to optimize the assay sensitivity and to validate its clinical application in the diagnostic screening of childhood anemias. METHODS: The study was conducted on 175 children suffering from various types of anemia (including 30 children with proven hereditary spherocytosis, HS) and 16 healthy subjects. All children were aged between 3 months and 17 years, including 94 boys and 97 girls. FCM OF was performed on every subject according to two different analysis time patterns (hemolysis was analyzed for 214 or 300 s) using Cytomics FC500 flow cytometer. RESULTS: Significant higher sensitivity was demonstrated by the tests carried out according to the longer analysis time pattern (90.0 vs. 83.33%). The level of specificity of both the analysis patterns was similar. When an extended analysis time was used, the percentage of red cell survival levels in HS patients were significantly lowered compared to the same cases analyzed with shorter incubation times and all other non-HS anemic cases (9.31 ± 4.69 vs. 35.59 ± 15.30%, P < 0.05). During the shorter analysis time, the values obtained were 13.76 ± 7.92% for HS and 48.18 ± 19.04% for non-HS, P < 0.0001. The 300-s test is very useful in distinguishing thalassemia patients from patients with other types of anemias (94.74% sensitivity and 90.12% specificity) and provided the values of remaining red blood cells as 70.46 ± 12.29% for thalassemia and 27.16 ± 13.01% for nonthalassemia subjects, P < 0.0001. CONCLUSION: Flow cytometric osmotic fragility test with a longer (300-s) analysis time demonstrated an increased sensitivity in detecting HS in anemic children. © 2017 International Clinical Cytometry Society.

Neutrophil Gelatinase-Associated Lipocalin: A Biomarker for Early Diagnosis of Urinary Tract Infections in Infants.

Early diagnosis of urinary tract infection (UTI) is challenging in infants due to unspecific symptoms, difficulty in urine collection and possible contamination. The aim of this study was to assesses the usefulness of serum and urine neutrophil gelatinase-associated lipocalin (sNGAL and uNGAL, respectively) in the diagnosis of febrile and non-febrile UTI in infants. This prospective observational study enrolled 66 infants with the first episode of UTI and 18 healthy controls. At the time of enrollment, sNGAL, uNGAL, urinalysis, urine culture, white blood cell count (WBC), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), procalcitonin (PCT), and serum creatinine (sCr) were assessed. We found that, on average, both sNGAL and uNGAL levels were significantly higher in febrile UTI, compared to non-febrile UTI and controls. In turn, the mean sNGAL level, but not uNGAL, was significantly higher in the non-febrile UTI group compared to controls. sNGAL positively correlated with WBC, CRP, ESR and PCT, and uNGAL with CRP and leukocyturia. The receiver operating curves (ROC) demonstrate that the optimum cut-off of 76.2 ng/ml for sNGAL (sensitivity 92.9%, specificity 94.4%, and the area under the curve (AUC) of 0.98) and of 42.2 ng/ml for uNGAL (sensitivity 73.8%, specificity 72.2%, and AUC of 0.76) for diagnosing febrile UTI and 39.0 ng/ml for sNGAL (sensitivity 83.3%, specificity 55.6%, and AUC of 0.70) for diagnosing non-febrile UTI. In conclusion, serum NGAL is an excellent marker for the early diagnosis of febrile UTI, with sensitivity and specificity higher than those of urine NGAL. Diagnostic sensitivity of serum NGAL is smaller in non-febrile infants suffering from UTI, and urine NGAL is not useful for this purpose at all.

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.

Usefulness of Reticulocyte Parameters for Diagnosis of Hereditary Spherocytosis in Children.

Innovations in laboratory equipment have enabled a widening of the spectrum of hematological parameters obtained from single measurements of peripheral blood samples, including reticulocyte parameters. The usefulness of reticulocytes indices to confirm the diagnosis of pediatric anemia was analyzed in this study. The study group consisted of 163 children, aged 1 month-17 years, with anemia. Complete blood count extended with an analysis of reticulocyte parameters were measured using a Beckman Coulter LH 750. The mean sphered corpuscular volume (MSCV) in the group of children with hereditary spherocytosis (HS) was 66.71 ± 8.45 fL, whereas in other anemic patients MSCV was 87.76 ± 11.22 fL, p < 0.0001. In HS children the average mean corpuscular volume of red blood cells was higher than the MSCV value, while an inverse correlation was observed in the group of children with other anemias, p < 0.0001. A significant difference was found between the ratio of absolute reticulocyte count and IRF fraction (Ret#/IRF)-0.6 ± 0.28 in the HS group and 0.23 ± 0.16 in the non-HS group, respectively. Our results suggest that analysis of reticulocyte parameters is useful in the diagnosis of anemia and should be included in the routine CBC analysis in anemic children.

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.

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

BACKGROUND: 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. MATERIALS AND METHODS: A comparison of results obtained by automated analysis of 807 anticoagulated blood samples from children and 125 manual microscopic differentiations were performed. This comparative study included white blood cell count, red blood cell count, and erythrocyte indices, as well as platelet count. RESULTS: The present study showed a poor level of agreement between white blood cell enumeration and differentiation of the three automated hematology analyzers under comparison. A very good agreement was found when comparing manual blood smear and automated granulocytes, monocytes, and lymphocytes differentiation. Red blood cell evaluation showed better agreement than white blood cells between the studied analyzers. CONCLUSION: To conclude, studied instruments did not ensure satisfactory interchangeability and did not facilitate a substitution of one analyzer by another.

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.

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.

Mean corpuscular volume of control red blood cells determines the interpretation of eosin-5'-maleimide (EMA) test result in infants aged less than 6 months.

Eosin-5'-maleimide (EMA) binding test is a flow cytometric test used to detect hereditary spherocytosis (HS). To perform the test sample from patients, 5-6 reference samples of red blood are needed. Our aim was to investigate how the mean corpuscular volume (MCV) of red blood cells influences on the value of fluorescence of bounded EMA dye and how the choice of reference samples affects the test result. EMA test was performed in peripheral blood from 404 individuals, including 31 children suffering from HS. Mean fluorescence channel of EMA-RBCs was measured with Cytomics FC500 flow cytometer. Mean corpuscular volume of RBCs was assessed with LH750 Beckman Coulter. Statistical analysis was performed using Graph Pad Prism. The correlation Spearman coefficient between mean channel of fluorescence of EMA-RBCs and MCV was r = 0.39, p < 0.0001. Interpretation of EMA test depends on MCV of the reference samples. If reference blood samples have lower MCV than the patients MCV, EMA test result might be negative. Due to different MCV values of RBCs in infancy and ca. Three months later, EMA test in neonates might be interpreted falsely negative. Samples from children younger than 3 months old had EMA test result 86.1 ± 11.7 %, whereas same samples that analyzed 4.1 ± 2.1 later had results of 75.4 ± 4.5 %, p < 0.05. Mean fluorescence of EMA-bound RBC depends on RBC's volume. MCV of reference samples affects EMA test results; thus, we recommend selection of reference samples with MCV in range of ±2 fL compared to MCV of patient RBC's.