Mild-to-moderate COVID-19 does not predispose to the development of autoimmune rheumatic diseases or autoimmune-based thrombosis.

An infection with severe acute respiratory syndrome coronavirus (SARS-CoV-2) may have a significant impact on the human immune system. Interactions between the virus and defence mechanisms may promote the development of autoimmune processes which manifest as antinuclear antibody (ANA) synthesis. Since many different viruses are suspected to take part in the pathogenesis of different systemic autoimmune rheumatic diseases (SARDs), we examined whether coronavirus disease 2019 convalescents who suffer from mild to moderate disease have a higher risk of developing ANA and anti-ß2-glicoprotein I IgG antibodies (ß2 GPI). In a retrospective study, we examined 294 adults among volunteer blood donors divided into convalescents (N = 215) and healthy controls (N = 79). For ANA detection, we used line-blotting, a type of indirect immunofluorescence assay (IF), to determine antigenic specificity and ELISA for ß2 GPI. We found a lower incidence of ANA in convalescents than in healthy controls, with the majority of these antibodies directed to antigens with no known clinical significance. Additionally, no participants were positive for ß2 GPI in either group. Our results show that COVID-19 with mild to moderate symptoms in the generally healthy population does not induce the development of ANA or anti-ß2 GPI antibodies for at least 6 months following the disease.

Iron excess affects release of neutrophil extracellular traps and reactive oxygen species but does not influence other functions of neutrophils.

Neutrophils apply several antimicrobial strategies including degranulation, phagocytosis, the generation of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs) to fight pathogens. Iron is considered to be an invaluable constituent of host immune defense and plays a dual role in immunity. It is a well-known component of antimicrobial proteins and is a necessary microelement for pathogen survival. The aim of this study was to broaden the knowledge regarding the impact of iron on the function of neutrophils. Neutrophils from healthy blood donors and patients with mild iron-deficiency anemia and HL-60 cells differentiated toward granulocyte-like cells were incubated with Fe2+ , Fe3+ or holo-transferrin (holo-Tf). Moreover, we isolated murine neutrophils of HFE gene knockout (KO) mice and mice fed iron-deficient, iron-equivalent and high-iron diets. We analyzed the release of NETs, phagocytosis, degranulation of azurophilic granules, ROS release, bactericidal activity of granulocytes against Escherichia coli and neutrophil elastase (NE) activity. We show that holo-Tf inhibits the release of NETs stimulated by phorbol 12-myristate 13-acetate by inhibiting NE activity. Studies performed in mice models reveal that iron overload inhibits the release of NETs and ROS production in neutrophils isolated from HFE KO mice and mice fed a high-iron diet. No impact of a low-iron diet on neutrophil phagocytosis, ROS production or release of NETs was observed. Our study underscores the physiological significance of iron in neutrophil function, specifically in the release of NETs.

Ablation of Tmcc2 Gene Impairs Erythropoiesis in Mice.

(1) Background: Transcriptomic and proteomic studies provide a wealth of new genes potentially involved in red blood cell (RBC) maturation or implicated in the pathogenesis of anemias, necessitating validation of candidate genes in vivo; (2) Methods: We inactivated one such candidate, transmembrane and coiled-coil domain 2 (Tmcc2) in mice, and analyzed the erythropoietic phenotype by light microscopy, transmission electron microscopy (TEM), and flow cytometry of erythrocytes and erythroid precursors; (3) Results: Tmcc2-/- pups presented pallor and reduced body weight due to the profound neonatal macrocytic anemia with numerous nucleated RBCs (nRBCs) and occasional multinucleated RBCs. Tmcc2-/- nRBCs had cytoplasmic intrusions into the nucleus and double membranes. Significantly fewer erythroid cells were enucleated. Adult knockouts were normocytic, mildly polycythemic, with active extramedullary erythropoiesis in the spleen. Altered relative content of different stage CD71+TER119+ erythroid precursors in the bone marrow indicated a severe defect of erythroid maturation at the polychromatic to orthochromatic transition stage; (4) Conclusions: Tmcc2 is required for normal erythropoiesis in mice. While several phenotypic features resemble congenital dyserythropoietic anemias (CDA) types II, III, and IV, the involvement of TMCC2 in the pathogenesis of CDA in humans remains to be determined.

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

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.

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

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.

Influence of iron- and zinc-chelating agents on neutrophil extracellular trap formation.

Release of neutrophil extracellular traps (NETs) is one of the neutrophils' mechanisms involved in the response to infection. NETs are released from the cell in response to a biological or synthetic stimulus to entrap, immobilize and kill pathogens. Metal ions and metal binding proteins were identified in the structure of NETs, but their role in NET release remains unclear. The aim of this study was to assess how lack of iron and zinc generated by ion sequestration using chelators affects NET release. Neutrophils were isolated from whole blood or buffy coats of healthy blood donors by density gradient centrifugation and incubated with zinc chelators: 20 µM N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), 40 µM diethylenetriaminepentaacetic acid (DTPA) or iron chelators: 400 µM deferoxamine mesylate salt (DFO) and 50 µM iminodiacetic acid (IDA). Next, 100 nM phorbol 12-myristate 13-acetate (PMA) was added to stimulate release of NETs. The amount of released DNA was measured by fluorometry and NETs were visualized by immunofluorescence microscopy. This study demonstrates that iron and zinc chelators are able to modulate NET release. Here we show that preincubation of neutrophils with TPEN and IDA inhibits NET release in cells stimulated with PMA. On the other hand, DFO stimulates NET release. Incubation of cells with DTPA does not affect release of NETs.

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

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.

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

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.

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

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.

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

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.

Awareness of blood group and blood donation among medical students.

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.

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

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.

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

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.

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.

Enzyme-linked Immunoassay Index for Anti-NC16a IgG and IgE Auto-antibodies Correlates with Severity and Activity of Bullous Pemphigoid.

Bullous pemphigoid (BP) is characterized by IgG and IgE autoantibodies to the NC16a domain of BP180. This study evaluated the correlation between body surface area (BSA), total serum IgE and enzyme-linked immunoassay (ELISA) for IgG and IgE anti-NC16a in 77 patients with BP in the active stage, and the degree of conversion to negative of the studied parameters in clinical remission. Statistically positive correlations were observed between BSA and examined parameters (correlation index 0.2548, 0.2491, 0.311, respectively). Originally, patients with BP with positive ELISAs for both IgG and IgE autoantibodies presented twice as extensive skin lesions as those with positive IgG and negative IgE ELISAs. Conversion of ELISAs for IgG and IgE to negative in clinical remission occurred in 9.3% and 81% of patients with BP, respectively. This confirmed that ELISA for IgE anti-NC16a is a helpful parameter in the modification of current treatment and the assessment of risk of relapse in BP.

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.

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

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.

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.