Publications by authors named "Olga Ciepiela"

40 Publications

Extremely severe anemia-Worsening a patient's condition in the COVID-19 pandemic era.

Int J Lab Hematol 2021 Feb 15. Epub 2021 Feb 15.

Department of Laboratory Medicine, Medical University of Warsaw, Warsaw, Poland.

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http://dx.doi.org/10.1111/ijlh.13493DOI Listing
February 2021

Dynamic Changes in the Ability to Release Neutrophil ExtraCellular Traps in the Course of Childhood Acute Leukemias.

Int J Mol Sci 2021 Jan 15;22(2). Epub 2021 Jan 15.

Department of Laboratory Diagnostics and Clinical Immunology of the Developmental Age, Medical University of Warsaw, 63A Żwirki i Wigury St., 02-091 Warsaw, Poland.

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.
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http://dx.doi.org/10.3390/ijms22020821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829911PMC
January 2021

Zinc Supplementation Modulates NETs Release and Neutrophils' Degranulation.

Nutrients 2020 Dec 26;13(1). Epub 2020 Dec 26.

Department of Laboratory Medicine, Medical University of Warsaw, Banacha 1a Street, 02-097 Warsaw, Poland.

Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils' functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils' functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.
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http://dx.doi.org/10.3390/nu13010051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823768PMC
December 2020

A comparison of Sysmex-XN 2000 and Yumizen H2500 automated hematology analyzers.

Pract Lab Med 2020 Nov 29;22:e00186. Epub 2020 Oct 29.

Department of Laboratory Medicine, Medical University of Warsaw, Poland.

A complete blood count is a highly automated laboratory test. The use of highly advanced measurement methods increases the accuracy and sensitivity of the determination of individual hematological parameters, especially in the case of white blood cells differentiation. Therefore, it is necessary to make comparative analyses, which involve performance of the analyzers used in daily work. The aim of the study was to indicate whether the results obtained using two compared analyzers show significant differences.

Materials And Methods: In this study a comparative analysis of 241 peripheral blood samples from adult patients was performed. The complete blood count results were obtained using two automated hematology analyzers: Sysmex XN-2000 and Horiba Yumizen H2500. The Passing-Bablok regression method and Bland- Altman analysis were also used to evaluate the results received for both analyzers.

Results: Statistically significant differences were found for four hematological parameters: eosinophil count, immature granulocytes, mean corpuscular hemoglobin concentration (MCHC) and platelet distribution width (PDW). The P value for MCHC was 0.01. Sysmex XN-2000 and Horiba Yumizen H2500 also showed disagreement in plate platelet distribution width (PDW) (P ​= ​0.04). For other parameters both analyzers showed good agreement.

Conclusion: Based on the results of the study, it was shown that there are significant differences in the measurements of hematological parameters between compared analyzers.
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http://dx.doi.org/10.1016/j.plabm.2020.e00186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649520PMC
November 2020

Laboratory work safety rules and guidelines during COVID-19 pandemic in Polish clinical laboratories - do our laboratories work according to a recent IFCC Taskforce Recommendations?

Clin Chem Lab Med 2020 09;58(10):e205-e208

Department of Laboratory Medicine, Medical University of Warsaw, Warsaw, Poland.

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http://dx.doi.org/10.1515/cclm-2020-0764DOI Listing
September 2020

Variations in Magnesium Concentration Are Associated with Increased Mortality: Study in an Unselected Population of Hospitalized Patients.

Nutrients 2020 Jun 19;12(6). Epub 2020 Jun 19.

Department of Laboratory Medicine, Medical University of Warsaw, 02-097 Warsaw, Poland.

Dysmagnesemia is a serious disturbance of microelement homeostasis. The aim of this study was to analyze the distribution of serum magnesium concentrations in hospitalized patients according to gender, age, and result of hospitalization. The study was conducted from February 2018 to January 2019 at the Central Clinical Hospital in Warsaw. Laboratory test results from 20,438 patients were included in this retrospective analysis. When a lower reference value 0.65 mmol/L was applied, hypermagnesemia occurred in 196 patients (1%), hypomagnesemia in 1505 patients (7%), and normomagnesemia in 18,711 patients (92%). At a lower reference value of 0.75 mmol/L, hypomagnesemia was found in 25% and normomagnesemia in 74% of patients. At a lower reference value of 0.85 mmol/L, hypomagnesemia was found in 60% and normomagnesemia in 39% of patients. Either hypo- or hyper-magnesemia was associated with increased risk of in-hospital mortality. This risk is the highest in patients with hypermagnesemia (40.1% of deaths), but also increases inversely with magnesium concentration below 0.85 mmol/L. Serum magnesium concentration was not gender-dependent, and there was a slight positive correlation with age ( < 0.0001, r = 0.07). Large fluctuations in serum magnesium level were associated with increased mortality ( = 0.0017). The results indicate that dysmagnesemia is associated with severe diseases and generally severe conditions. To avoid misdiagnosis, an increase of a lower cut-off for serum magnesium concentration to at least 0.75 mmol/L is suggested.
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http://dx.doi.org/10.3390/nu12061836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353204PMC
June 2020

Increased Temperature Facilitates Adeno-Associated Virus Vector Transduction of Colorectal Cancer Cell Lines in a Manner Dependent on Heat Shock Protein Signature.

Biomed Res Int 2020 8;2020:9107140. Epub 2020 Feb 8.

Department of Applied Pharmacy, Faculty of Pharmacy with Laboratory Medicine, Medical University of Warsaw, 1 Banacha Street, 02-097 Warsaw, Poland.

Colorectal cancer (CRC) is one of the most common cancers in human population. A great achievement in the treatment of CRC was the introduction of targeted biological drugs and solutions of chemotherapy, combined with hyperthermia. Cytoreductive surgery and HIPEC (hyperthermic intraperitoneal chemotherapy) extends the patients' survival with CRC. Recently, gene therapy approaches are also postulated. The studies indicate the possibility of enhancing the gene transfer to cells by recombinant adeno-associated vectors (rAAV) at hyperthermia. The rAAV vectors arouse a lot of attention in the field of cancer treatment due to many advantages. In this study, the effect of elevated temperature on the transduction efficiency of rAAV vectors on CRC cells with different origin and gene profile was examined. The effect of heat shock on the penetration of rAAV vectors into CRC cells in relation with the expression of HSP and AAV receptor genes was tested. It was found that the examined cells under hyperthermia (43°C, 1 h) are transduced at a higher level than in normal conditions (37°C). The results also indicate that studied RKO, HT-29, and LS411N cell lines express HSP genes at different levels under both 37°C and 43°C. Moreover, the results showed that the expression of AAV receptors increases in response to elevated temperature. The study suggests that increased rAAV transfer to CRC can be achieved under elevated temperature conditions. The obtained results provide information relevant to the design of new solutions in CRC therapy based on the combination of hyperthermia, chemotherapy, and gene therapy.
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http://dx.doi.org/10.1155/2020/9107140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031720PMC
November 2020

Increased temperature-related adeno-associated virus vectors transduction of ovarian cancer cells - essential signatures of AAV receptor and heat shock proteins.

Exp Ther Med 2019 Dec 21;18(6):4718-4732. Epub 2019 Oct 21.

Department of Applied Pharmacy, Faculty of Pharmacy with Laboratory Medicine, Medical University of Warsaw, Warsaw 02-097, Poland.

Recombinant adeno-associated viruses (rAAVs) are becoming more commonly used in clinical trials involving gene therapy. Additionally AAV-based drugs have already been registered. Gene therapy aims to increase transduction efficiency, increase selectivity and reduce side effects. One approach to achieve this is the use of physical factors, such as temperature or more specifically, hyperthermia, which is already utilized in oncology. The aim of the present study was to investigate the effect of hyperthermic conditions (40°C and 43°C) on the rAAV transduction efficiency of ovarian cancer cells (Caov-3 and NIH:OVCAR-3) and non-cancerous cells (AAV-293). The present study was designed to identify functional associations between the level of gene transfer and the expression of representative genes for rAAV transmission (AAVR (AAV receptor), heparan sulfate proteoglycan (HSPG) 1 and HSPG2) and heat shock proteins (HSPs). The expressions of selected genes were measured via reverse transcription-quantitative PCR and cell adhesion/invasion chamber tests were also performed. The results revealed that ovarian cancer cell lines were more efficiently transduced with rAAV vectors at an elevated temperature. Additionally, the expression patterns of AAVR, HSPG1 and HSPG2 genes were different between the tested lines. The expression of certain receptors in ascites-derived NIH:OVCAR-3 ovarian cancer cells was higher compared with tumor-derived Caov-3 cells at 37, 40 and 43°C, which indicates a higher transduction efficiency in the formerly mentioned cells. Ascites-derived ovarian cancer cells were characterized by high expressions of HSP40, HSP90 and HSP70 families. Lower levels of HSP expression were demonstrated in less-effectively transduced Caov-3 cells. Furthermore, expressions of the examined genes changed with increasing temperature. The results indicated that temperature-dependent transduction is associated with the expression of the rAAV receptor and HSP genes. The results of the current study may aid the design of effective protocols for ovarian cancer gene therapy.
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http://dx.doi.org/10.3892/etm.2019.8112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861878PMC
December 2019

Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps.

Cell Mol Life Sci 2020 Aug 24;77(15):3059-3075. Epub 2019 Oct 24.

Department of Laboratory Medicine and Clinical Immunology of Developmental Age, Medical University of Warsaw, Zwirki i Wigury 63a Street, 02-091, Warsaw, Poland.

Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.
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http://dx.doi.org/10.1007/s00018-019-03331-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366602PMC
August 2020

Pre-procedural abnormal function of von Willebrand Factor is predictive of bleeding after surgical but not transcatheter aortic valve replacement.

J Thromb Thrombolysis 2019 Nov;48(4):610-618

1st Department of Cardiology, Medical University of Warsaw, 1a Banacha, 02-097, Warsaw, Poland.

Both transcatheter aortic valve implantation (TAVI) and surgical aortic valve replacement (SAVR) have been proven to effectively correct von Willebrand Factor (vWF) pathologies, however there is limited data simultaneously comparing outcomes of both approaches. We prospectively enrolled patients with severe aortic stenosis referred for TAVI (n = 52) or SAVR (n = 48). In each case, vWF antigen (vWF:Ag), vWF activity (vWF:Ac) and activity-to-antigen (vWF:Ac/Ag) ratio were assessed at baseline, 24 h and 72 h after procedure. VWF abnormalities were defined as reduced vWF:Ac/Ag ratio (< 0.8). Bleeding events in both arms were classified according to Valve Academic Research Consortium (VARC-2) definitions. Overall, there was no difference between patients referred for TAVI and SAVR in vWF:Ac (1.62 ± 0.52 vs 1.71 ± 0.64; p = 0.593), vWF:Ag (1.99 ± 0.81 vs 2.04 ± 0.81; p = 0.942) or vWF:Ac/Ag ratio (0.84 ± 0.16 vs 0.85 ± 0.12; p = 0.950). Pathological vWF:Ac/Ag ratio was found in 20 (38%) TAVI and 15 (31%) SAVR patients (p = 0.407). Normalization of vWF:Ac/Ag ratio at day 3 after procedure was achieved in 19 (95%) TAVI and 13 (87%) SAVR patients (p = 0.439). Similar prevalence of major or life-threatening bleedings (MLTB) after TAVI and SAVR in entire groups was observed (19% vs. 23%, p = 0.652). VWF abnormalities were associated with higher incidence of MLTB in SAVR (53% vs 9%, p < 0.001), but not TAVI (15% vs. 22%, p = 0.132). Accordingly, in receiver-operating characteristic curve analysis vWF:Ac/Ag ratio < 0.8 showed significant sensitivity and specificity for predicting MLTB in SAVR group (AUC 0.735, 95% CI 0.538-0.931, p = 0.019). We proved that abnormal function of vWF is corrected successfully after both TAVI and SAVR, but vWF abnormalities are predictive of MLTB only in surgical patients.
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http://dx.doi.org/10.1007/s11239-019-01917-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800844PMC
November 2019

Clot Signature Curve Analysis as a Useful Tool of Thrombin Time Measurement in Case of Failed Optical Reading.

Hamostaseologie 2019 11 28;39(4):322-325. Epub 2019 Mar 28.

Department of Laboratory Diagnostics, Medical University of Warsaw, Warsaw, Poland.

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http://dx.doi.org/10.1055/s-0039-1685166DOI Listing
November 2019

Hypersegmentation of Granulocytes and Monocytes in a Patient with Primary Myelofibrosis Treated with Hydroxycarbamide

Turk J Haematol 2019 May 23;36(2):120-121. Epub 2019 Jan 23.

Central Laboratory at Public Central Teaching Hospital in Warsaw, Warsaw, Poland

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http://dx.doi.org/10.4274/tjh.galenos.2019.2018.0395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516095PMC
May 2019

Seroprevalence of IgG antibodies against measles in a selected Polish population - do we need to be re-vaccinated?

Cent Eur J Immunol 2019 20;44(4):380-383. Epub 2020 Jan 20.

Central Laboratory, Central Teaching Hospital of University Clinical Center, Warsaw, Poland.

Introduction: Measles is highly contagious, but preventable viral disease. Its outbreaks appear all over the world, and decreasing herd immunity excludes its elimination. High levels of IgG antibodies against a virus efficiently protects against infection.

Aim Of This Study: To assess the seroprevalence of anti-measles IgG in the serum of patients at different age levels tested for measles IgG at our laboratory.

Material And Methods: The study was conducted from March to June of 2019. Retrospective analysis included results for measles-specific IgG from 364 tested patients. The age of enrolled subjects ranged from four months to 101 years, with a median age of 46, and a mean age of 43 ±18. Quantification of anti-measles IgG was performed using indirect chemiluminescence immunoassays on the DiaSorin Liaison® automated analyzer.

Results: Our results showed a seropositivity ratio of 78.02%. The lowest number of seropositive subjects was in the group of infants (0-1 years old), with a ratio of 53.85%, and the group of adults of 19-38 years old at 55.68%. The group of the oldest patients (70-101 years old) had the highest ratio of seropositive subjects (100%), while adults of 60-69 years old had a seropositivity ratio of 97.22%.

Conclusions: These data suggest that the group of young adults who were vaccinated with one or two doses of MMR vaccine in childhood are the most susceptible for infection, and when working in contact with other people, should be re-vaccinated for protection against measles.
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http://dx.doi.org/10.5114/ceji.2019.92789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050052PMC
January 2020

Eosin-5'-maleimide binding test-Do we use appropriate reference values to detect hereditary spherocytosis in neonates?

Int J Lab Hematol 2019 06 10;41(3):e57-e60. Epub 2018 Dec 10.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland.

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http://dx.doi.org/10.1111/ijlh.12958DOI Listing
June 2019

Old and new insights into the diagnosis of hereditary spherocytosis.

Authors:
Olga Ciepiela

Ann Transl Med 2018 Sep;6(17):339

Department of Laboratory Diagnostics, Medical University of Warsaw, Warsaw, Poland.

Hereditary spherocytosis (HS) belongs to the group of congenital hemolytic anemias resulting from plasma membrane protein deficiency. When diagnosed too late, HS bares the risk of long-term complications including gall stones and severe anemia. Here, there are discussed advances in HS screening and diagnostics, with a particular focus on methodologies, most of which are available in clinical laboratories worldwide.
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http://dx.doi.org/10.21037/atm.2018.07.35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174190PMC
September 2018

Influence of Different Bacteria Strains Isolated from Septic Children on Release and Degradation of Extracellular Traps by Neutrophils from Healthy Adults.

Adv Exp Med Biol 2018;1108:1-12

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Warsaw Medical University, Warsaw, Poland.

Neutrophils are the first line of immune defense against pathogens. They use three major antimicrobial mechanisms: phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). NETs are structures which consist of nuclear DNA conjugated with antibacterial proteins. They are formed to entrap and kill pathogens. The aim of the study was to evaluate the influence of Escherichia coli (E. coli), Streptococcus pneumoniae (S. pneumoniae), Stenotrophomonas maltophilia (S. maltophilia), and Pseudomonas aeruginosa (P. aeruginosa), isolated from the peripheral blood of children with sepsis, on the release and degradation of NETs by neutrophils isolated from blood healthy adult subjects. Neutrophils were stimulated with the bacterial strains outlined above. The quantitative and qualitative analyses of NETs release were performed by fluorometric measurement and immunofluorescence, respectively. The ability of bacteria to degrade NETs was studied qualitatively. Oxidative burst was assessed by flow cytometry. Histone H3 citrullination was evaluated by Western blot. We found that NETs were formed only when neutrophils were incubated with S. pneumoniae. E. coli, P. aeruginosa, and S. maltophilia did not induce the release of the NETs. P. aeruginosa, S. pneumoniae, and E. coli induced the production of reactive oxygen species (ROS) by neutrophils. Two studied bacterial strains (S. pneumoniae and E. coli) were able to degrade NETs. However, none of the strains induced the citrullination of histone H3. We conclude that the ability of bacteria to induce and degrade NETs depends on the specific bacterial strain.
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http://dx.doi.org/10.1007/5584_2018_245DOI Listing
July 2019

The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps.

Immunol Cell Biol 2018 04 15;96(4):413-425. Epub 2018 Feb 15.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland.

Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.
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http://dx.doi.org/10.1111/imcb.12015DOI Listing
April 2018

Celiac antibodies in children with type 1 diabetes - A diagnostic validation study.

Autoimmunity 2018 03 19;51(2):81-88. Epub 2018 Jan 19.

a Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age , Medical University of Warsaw , Warsaw , Poland.

Introduction: Autoimmune diseases, such as celiac disease (CD) and diabetes mellitus type 1, tend to co-occur within the same patient. The prevalence of CD in diabetic children is higher than in the general population, and is estimated to be 0.6-16.4%. The diagnosis of CD is based on histopathological examination and serological testing, however, these methods are still imperfect and new diagnostic algorithms should be considered.

Aim: The aim of the study was to assess the diagnostic value of serological tests detecting antibodies against deamidated gliadin peptide, endomysium, tissue transglutaminase, neo-epitope tissue transglutaminase and to identify HLA-related genetic predisposition to CD in patients with type 1 diabetes mellitus (DM1).

Methods: Autoantibodies were measured in the sera of 392 children suffering from DM1 aged 1-19 years old (mean 11.76 ± 4.14 years old). Additionally, PCR-based assessment of HLA DQ2/DQ8 genotyping was performed.

Results: A positive result of at least one serological test was obtained from 81 children (20.66%). The sensitivity and specificity were 76.47% and 91.67% for anti-DGP IgA, 70.59% and 58.33% for IgG anti-DGP, respectively. A positive predictive value was 100% for the anti-TG IgA at cutoff levels of 5 and 10 times higher than upper limit of reference values. HLA DQ2 and/or DQ8 were found in 97.6% of examined children.

Conclusions: Tests based on anti-TG IgA are more accurate for detecting CD in children with type 1 diabetes than anti-DGP IgA. A high percentage of diabetic children carry HLA alleles predisposing to CD, which indicates that genetic screening in this group of patients is not obligated.
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http://dx.doi.org/10.1080/08916934.2018.1427226DOI Listing
March 2018

Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release.

Int J Mol Sci 2017 Dec 8;18(12). Epub 2017 Dec 8.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091 Warsaw, Poland.

Neutrophils are one of the first cells to arrive at the site of infection, where they apply several strategies to kill pathogens: degranulation, respiratory burst, phagocytosis, and release of neutrophil extracellular traps (NETs). Antibiotics have an immunomodulating effect, and they can influence the properties of numerous immune cells, including neutrophils. The aim of this study was to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of NETs by neutrophils. Neutrophils were isolated from healthy donors by density-gradient centrifugation method and incubated for 1 h with the studied antibiotics at different concentrations (0.5, 10 and 50 μg/mL-azithromycin and 10 and 50 μg/mL-chloramphenicol). Next, NET release was induced by a 3 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA). Amount of extracellular DNA was quantified by fluorometry, and NETs were visualized by immunofluorescent microscopy. Degranulation, apoptosis and respiratory burst were assessed by flow cytometry. We found that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of NETs. Moreover, azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils. Chloramphenicol did not affect degranulation, apoptosis nor respiratory burst. It can be concluded that antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity.
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http://dx.doi.org/10.3390/ijms18122666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751268PMC
December 2017

Awareness of blood group and blood donation among medical students.

Transfus Apher Sci 2017 Dec 18;56(6):858-864. Epub 2017 Oct 18.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Zwirki i Wigury 63a 02-091, Poland.

Background: Transfusion of blood-derived products is often used as a life-saving treatment, and being a blood donor should be common, especially among medical professionals. Thus, an awareness of one's own blood type in medical students should be common. Our aim was to assess if students of the Medical University of Warsaw know their blood type and how many of them are registered blood donors.

Material And Methods: A survey was conducted in a group of 1121 students. The survey included questions addressing awareness of the students' blood type, the frequency of blood types in Poland, being a blood donor, and willingness to become a blood donor.

Results: Of all students, 86.8% knew their blood type and 13.2% did not. Approximately 30.2% of students in the survey declared that they are blood donors, 57.9% had only considered becoming a blood donor and 11.9% had not even considered becoming a blood donor. Of all non-donors, 48.2% had contraindications to becoming a blood donor, 11.5% were afraid of blood collection and 21.9% did not have time to register as a blood donor. Most students (81%) declared that they could become a blood donor if someone closely-related needed a transfusion, and 5.2% declared that there is nothing that could force them to become a blood donor.

Conclusion: Compared to Polish society medical students are significantly more aware of the necessity of blood donation. The majority know their blood type, but they do not know what is the most common blood type in Poland.
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http://dx.doi.org/10.1016/j.transci.2017.10.002DOI Listing
December 2017

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

Indian J Hematol Blood Transfus 2017 Jun 24;33(2):239-247. Epub 2016 May 24.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Zwirki i Wigury 63a, 02-091 Warsaw, Poland.

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,  < 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,  < 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.
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http://dx.doi.org/10.1007/s12288-016-0680-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442045PMC
June 2017

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

Cytometry B Clin Cytom 2018 01 1;94(1):189-195. Epub 2017 Feb 1.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland.

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.
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http://dx.doi.org/10.1002/cyto.b.21511DOI Listing
January 2018

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

J Clin Lab Anal 2016 Nov 17;30(6):1128-1134. Epub 2016 May 17.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland.

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.
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http://dx.doi.org/10.1002/jcla.21992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6807162PMC
November 2016

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

J Pediatr Hematol Oncol 2016 11;38(8):e303-e306

Departments of *Laboratory Diagnostics and Clinical Immunology of Developmental Age †Paediatrics, Haematology and Oncology, Medical University of Warsaw, Poland.

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.
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http://dx.doi.org/10.1097/MPH.0000000000000652DOI Listing
November 2016

Different procedures of diphenyleneiodonium chloride addition affect neutrophil extracellular trap formation.

Anal Biochem 2016 09 11;509:60-66. Epub 2016 May 11.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, 02-091, Warsaw, Poland.

A unique strategy, in which invading microorganisms are being caught in web-like structures composed mainly of DNA, involves a recently described phenomenon called NETosis. This process seems to be related to the production of reactive oxygen species (ROS). In our study, the influence of diphenyleneiodonium chloride (DPI), which diminishes ROS production, was assessed in the context of neutrophil extracellular trap (NET) release. According to protocol, two distinguished procedures were compared, the first one involving DPI elimination from sample before cell activation and the second one proceeding without the step of inhibitor washout. The kinetics of DNA release was monitored by fluorometric assay, and NET formation was observed by fluorescent microscopy. The addition of DPI to the sample led to a reduction of extracellular DNA release. The strongest inhibition was noticed after treatment with 10 μM DPI, which was removed from medium before stimulation with phorbol-12-myristate-13-acetate (PMA). Our findings confirmed that DPI is able to block NET creation. However, the addition of DPI together with PMA or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of NET formation. Finally, DPI that remained in the system induced specific morphological changes in the neutrophils' nuclei that was not observed in the DPI washed out from sample.
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http://dx.doi.org/10.1016/j.ab.2016.05.003DOI Listing
September 2016

The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release.

Cent Eur J Immunol 2016 24;41(1):1-5. Epub 2016 Mar 24.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Poland.

Neutrophil extracellular traps (NETs) are threads of nuclear DNA complexed with antimicrobial proteins released by neutrophils to extracellular matrix to bind, immobilise, and kill different pathogens. NET formation is triggered by different physiological and non-physiological stimulants. It is also suggested that antibiotics could be non-physiological compounds that influence NET release. The aim of the study was to investigate the effect of clindamycin and amoxicillin on NET release and the phagocyte function of neutrophils. Neutrophils isolated from healthy donors by density centrifugation method were incubated with amoxicillin or clindamycin for two hours, and then NET release was stimulated with phorbol 12-myristate 13-acetate (PMA). After three hours of incubation with PMA NETs were quantified as amount of extracellular DNA by fluorometry and visualised by immunofluorescent microscopy. The percent of phagocyting cells was measured by flow cytometry. We showed that amoxicillin induces NET formation (increase of extracellular DNA fluorescence, p = 0.03), while clindamycin had no influence on NET release (p > 0.05), as confirmed by quantitative measurement and fluorescent microscopy. Regarding phagocyte function, both antibiotics increased bacterial uptake (43.3% and 61.6% median increase for amoxicillin and clindamycin, respectively). We concluded that the ability of antibiotics to modulate NET release depends on the antibiotic used and is not associated with their ability to influence phagocytosis.
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http://dx.doi.org/10.5114/ceji.2016.58811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829816PMC
April 2016

Flow cytometric quantification of neutrophil extracellular traps: Limitations of the methodological approach.

Am J Hematol 2016 03 9;91(3):E9-10. Epub 2016 Feb 9.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland.

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http://dx.doi.org/10.1002/ajh.24257DOI Listing
March 2016

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

Cent Eur J Immunol 2015 3;40(2):167-73. Epub 2015 Aug 3.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Poland.

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.
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http://dx.doi.org/10.5114/ceji.2015.52830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4637391PMC
November 2015

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.

Ann Hematol 2015 Aug 25;94(8):1277-83. Epub 2015 Apr 25.

Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Marszalkowska 24, 00-576, Warsaw, Poland,

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.
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http://dx.doi.org/10.1007/s00277-015-2377-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488455PMC
August 2015