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In Poland, hepatitis A virus (HAV) RNA screening was performed in plasma for fractionation usually immediately before shipment.Our goal was to study epidemiology, rate of transfusion transmitted HAV during epidemic (2017-2019), and viral characteristics of infected plasma donors.HAV RNA was tested in 1,866,590 donations from 1,210,423 donors using RT-PCR in mini pools of 96 (MP96) or TMA in MP16. Virological characteristics included RNA level (RL), antibody testing, and sequencing.Twenty-one HAV infections were identified (1.13/100,000 donations; 95% confidence interval [95% CI]: 0.74-1.72) and (1.73/100,000 donors; 95% CI: 1.35-2.65). The Blood Transfusion Centers were also informed about three donors, who were hospitalized for hepatitis A soon after their blood donation. In addition, we identified a donor, who had reactive result for HAV after receiving HAV vaccination. He tested positive twice 10 days after receiving the first and the second dose. The highest RL was 16 million IU/ml, mean 1,706,905 IU/ml, and median 220 IU/ml. The longest detectable RL lasted for 113 days. HAV-infected donors were seronegative (36%) or IgM positive (64%). We followed up on 12 HAV contaminated blood components issued for transfusion. In two out of seven identified patients viral transmission was confirmed (28.6%).Based on our results, we propose a 6 month deferral after HAV infection and 14 days post HAV vaccination. The infectivity rate was below 30%. The HAV RNA testing could be considered as an additional safeguard against HAV transmission at the time of increased incidence of HAV infections in the general population.

The risk of hepatitis B virus (HBV) transmission through blood and blood components is markedly reduced through serologic testing of each donation for the presence of HBV infection markers. Testing for hepatitis B virus surface antigen (HBsAg) is obligatory almost everywhere in the world. Several countries have also implemented testing for antibodies to hepatitis B core antigen (anti-HBc) and some perform HBV DNA tests, either in single donations or minipools of up to 96 donations. In countries with obligatory hepatitis B vaccination, the incidence of HBsAg-positive infections has been on the decline. Questions are therefore raised regarding the choice of optimal strategies for blood donor screening. In Poland, screening for hepatitis B virus surface antigen (HBsAg) to identify HBV infected donors was initiated in 1971. Pursuant to the guidelines of the Council of Europe, the analytical sensitivity of 0.13 IU/ml is required for a HBsAg diagnostic test to detect a HBsAg-positive infection. In 2005, HBV-DNA testing was added to the panel of screening tests for further reduction of HBV transmission risk through blood and blood components. The study presents the analysis of the HBV-DNA screening tests used in Poland so far. The analysis is based on the author’s doctoral dissertation and the outcome may largely contribute to the process of determining the effectiveness of the methods used up to date and to development of an optimal panel of HBV screening tests for Polish blood donors.

Transfusion of blood and blood components is one of the widely used medical procedures. The responsibility for provision of blood and blood components lies with Polish blood transfusion centers (CKiK) substantively supervised by the Institute of Hematology and Transfusion Medicine. Hospital based blood banks, hospital wards, immunohematology laboratories are supervised by CKiK. Every year approximately 600 thousand people donate blood, more than 75% of which are regular donors. The annual number of donations is maintained at a constant level of about 1.3 million. The aim of this position paper is to present the current status, achievements and advancement in collection, preparation and testing procedures and methods used to obtain the best possible quality blood components dedicated for clinical use. The aim is also to show the structure of blood transfusion service in Poland. Collaboration of all organizational units involved in transfusion medicine is crucial for providing high quality health care for patients. Polish transfusiologists and blood transfusion officers have largely contributed to transfusion science with numerous publications and significant research work. The implementation of novel methods and ongoing research positions Polish blood transfusion service among those of highly developed countries.

Cold physical plasma-derived reactive oxygen specie (ROS) inactivate cells, which might be beneficial in oncology. However, several aspects of plasma oncotherapy remain elusive. These include the identification of an ROS composition with maximum toxicity and the molecular mechanisms that govern the degree of plasma-induced cell death. Using two human leukemia cell lines and the plasma jet kINPen, we identified Jurkat cells to be most sensitive to argon plasma while THP-1 cells were most sensitive to He/O2 plasma. Screening of 20 protein transcripts involved in redox regulation identified HMOX1 as a commonly regulated element, and siRNA-mediated knockdown of the HMOX1-transcribed protein HO-1 augmented plasma-derived cell death in the two leukemia cell lines. Interestingly, knockdown of the H2O2-decomposing enzyme catalase did not elevate plasma-induced cell death. By contrast, siRNA-mediated suppression of the production of the chemokine CXCL8 (IL8) markedly enhanced plasma-derived cytotoxicity up to twofold. Vice versa , by antibody-mediated blocking of IL8 receptors, a massive increase in IL8 and HO-1 secretion was observed, together with a dramatically enhanced cell death in response to plasma treatment, especially in THP-1 cells. These results suggest that the plasma-induced leukemia cell death is dictated by the ROS chemistry and the HO-1/CXCL8 axis via paracrine or autocrine IL8-mediated pro-survival signaling.

The new coronavirus — SARS-CoV-2 infections have recently become the main epidemiological and clinical challenge worldwide (Poland included). This article is based on the presentation entitled “Hematology and transfusion medicine and COVID-19” given/conferred during the webinar in May 2020. The aim was to introduce the procedure for diagnosing the new coronavirus infection. The procedure highlights the aspects of the pre-analytical and analytical phase that are critical for the quality of the performed tests and affected by both the commissioning and test-performing personnel. On the basis of current literature, further recommendations for the improvement of molecular diagnostics of SARS-CoV-2 are also presented.

For many years, the West Nile virus (WNV) has been the focus of attention of specialists in transfusion medicine due to documented transmission of the virus through blood and blood components. In the recent years, a periodic increase in the number of infections in Europe (2018) has been reported as well as outbreaks of infections among animals and people in areas where the presence of the virus has not been observed so far. The study updates biological and epidemiological data on WNV published in 2013 (Journal of Transfusion Medicine) [1] and suggests measures to be taken to prevent the spread of WNV human infections via transfusion if WNV infections occurred in Poland. National recommendations were based on international guidelines developed by specialists in transfusion medicine and infectious diseases in cooperation with research centers that monitor epidemiology of the virus.

The B-cell CLL/lymphoma 11B gene

Anti-disialoganglioside GD2 antibody ch14.18/CHO (dinutuximab beta, DB) improved the outcome of patients with high-risk neuroblastoma (HR-NB) in the maintenance phase. We investigated chemotherapeutic compounds used in newly diagnosed patients in combination with DB. Vincristine, etoposide, carboplatin, cisplatin, and cyclophosphamide, as well as DB, were used at concentrations achieved in pediatric clinical trials. The effects on stress ligand and checkpoint expression by neuroblastoma cells and on activation receptors of NK cells were determined by using flow cytometry. NK-cell activity was measured with a CD107a/IFN-γ assay. Long-term cytotoxicity was analyzed in three spheroid models derived from GD2-positive neuroblastoma cell lines (LAN-1, CHLA 20, and CHLA 136) expressing a fluorescent near-infrared protein. Chemotherapeutics combined with DB in the presence of immune cells improved cytotoxic efficacy up to 17-fold compared to in the controls, and the effect was GD2-specific. The activating stress and inhibitory checkpoint ligands on neuroblastoma cells were upregulated by the chemotherapeutics up to 9- and 5-fold, respectively, and activation receptors on NK cells were not affected. The CD107a/IFN-γ assay revealed no additional activation of NK cells by the chemotherapeutics. The synergistic effect of DB with chemotherapeutics seems primarily attributed to the combined toxicity of antibody-dependent cellular cytotoxicity and chemotherapy, which supports further clinical evaluation in frontline induction therapy.

Nucleic acid testing (NAT) was implemented in Poland in 1999 for screening of plasma for fractionation and for all blood donors in 2002. To analyze seronegative NAT‐positive samples representing hepatitis C virus (HCV) window‐period (WP) in the years 2000 to 2016 and to determine infection outcome. We analyzed results of 17 502 739 donations screened in minipools (6‐48) or individually. Index samples underwent viral load (VL) quantification, genotyping and Ag, and anti‐HCV re‐testing using chemiluminescence (CMIA), electrochemiluminescence (ECLIA), and fourth‐generation enzyme‐linked immunosorbent assay (IV EIA) assays. HCV‐seronegative infections were identified in 126 donations (7.2/mln donations; 95% confidential intervals, 6.0‐8.6). Frequency of NAT yields was decreasing over time. Of the initial 126 seronegative index cases 106 were retested: 32.1% were reactive in IV EIA, 11.3% in ECLIA, and 1.9% in CMIA. The lowest VL correlated with absent anti‐HCV and HCV Ag, while VL was highest when the antigen was detectable and then it decreased when anti‐HCV appeared at a level detectable by sensitive third generation tests while retesting. The proportion of genotype 1 was 38.9% in samples positive only for HCV RNA and 71.4% in samples that were anti‐HCV reactive in re‐testing. In parallel, genotype 3 frequency was 50% in the former group and 21% in the latter. NAT is an effective measure to limit HCV transmission by transfusion and IV EIA seems to have higher clinical sensitivity than ECLIA. Samples representing likely successive phases of early HCV infection were characterized by different genotype distribution probably due to very early elimination of genotype 3., Highlight NAT is highly effective in prevention of transmission of HCV seronegative infections.In nearly 7% of followed up donors antibodies appeared later than 50 days after index donation.The highest clinical sensitivity was demonstrated by the IVth generation tests.Among the third generation assays, ECLIA showed higher sensitivity than CMIA.Samples representing likely successive phases of early HCV infection demonstrated different genotype distribution.

Hepatitis C virus (HCV) is characterized by high genetic variability, which is manifested both at the inter-host and intra-host levels. However, its role in the clinical course of infection is less obvious. The aim of the present study was to determine the genetic variability of HCV HVR1 (hypervariable region 1) of genotype 1b and 3 in plasma of blood donors in the early seronegative stage of infection (HCV-RNA+, anti-HCV−) and in samples from chronically infected patients using next-generation sequencing. Sequencing errors were corrected, and haplotypes inferred using the ShoRAH software. Genetic diversity parameters (intra-host number of variants, number of nucleotide substitutions and diversity per site) were assessed by DNA SP and MEGA. During the early infection, the number of variants were significantly lower in subjects infected with genotype 3 than with genotype 1b (p

The Krüppel-like transcription factor (KLF) BCL11B is characterized by a wide tissue distribution and crucial functions in key developmental and cellular processes, as well as in various pathologies including cancer and HIV infection. Although the basics of BCL11B activity and relevant interactions with other proteins have been uncovered, how this exclusively nuclear protein localizes to its compartment remained unclear. Here, we demonstrate that unlike other KLFs, BCL11B does not require the C-terminal DNA-binding domain to pass through the nuclear envelope but has an independent, previously unidentified, nuclear localization signal (NLS), which is located distantly from the zinc finger domains and fulfills the essential criteria of being an autonomous NLS. First, it can redirect a heterologous cytoplasmic protein to the nucleus. Second, its mutation causes aberrant localization of the protein of origin. Finally, we provide experimental and in silico evidences of the direct interaction with importin-α. The relative conservation of this motif allows formulating a consensus sequence (K/R)K-X13–14-KR+K++ (‘+’ indicates amino acids with similar chemical properties), which can be found in all BCL11B orthologs among vertebrates and in the closely related protein BCL11A.

Surveillance on the HIV molecular variability, risk of drug resistance transmission and evolution of novel viral variants among blood donors remains an understudied aspect of hemovigilance. This nationwide study analyses patterns of HIV diversity and transmitted resistance mutations. Study included 185 samples from the first time and repeat blood donors with HIV infection identified by molecular assay. HIV protease, reverse transcriptase and integrase were sequenced using population methods. Drug resistance mutation (DRM) patterns were analyzed based on the Stanford Interpretation Algorithm and standardized lists of transmitted mutations. Phylogeny was used to investigate subtyping, clustering and recombination patterns. HIV-1 subtype B (89.2%) followed by subtype A6 (7.6%) were predominant, while in three (1.6%) cases, novel recombinant B/A6 variants were identified. Non-B variants were more common among repeat donors (14.5%) compared to the first time ones (1.8%), p = 0.011, with higher frequency (9.9%) of A6 variant in the repeat donor group, p = 0.04. Major NRTI DRMs were observed in 3.8%, NNRTI and PI in 0.6% and INSTI 1.1% of cases. Additionally, E157Q polymorphism was observed in 9.8% and L74I in 11.5% of integrase sequences. Transmission of drug resistance among blood donors remains infrequent. Subtype patters increase in complexity with emergence of novel intersubtype A6B recombinants.

The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.

Wstep: Zakazenia HIV są diagnozowane na podstawie wykrywania anty-HIV techniką EIA (enzyme immunoassay) i potwierdzane testami WB (Western Blot). W Polsce w 2003 roku rozpoczeto badania dawcow metodami biologii molekularnej, wykrywającymi zakazenie przed pojawieniem sie przeciwcial. Celem pracy byla ocena wprowadzenia badan molekularnych na bezpieczenstwo przetoczen i sprawnośc weryfi kacji wynikow testow EIA, wykrywających anty -HIV i antygen p24. Material i metody: Badania metodami EIA (Vironostika HIV Uni-Form II Ag/Ab, Bio- Merieux; HIVAg/Ab Combo Architekt, Abbott) i NAT przeprowadzano w RCKiK. Oznaczenia wykonywano w pojedynczych donacjach metodą TMA (Procleix Ultrio, Chiron) lub w pulach po 24 donacje do 2006 r. (Cobas Ampliscreen, Roche Diagn); a od roku 2006 w pulach po 6 donacji metodą PCR (Cobas Taqscreen MPX Test). Probki powtarzalnie reaktywne w EIA weryfi kowano w Instytucie Hematologii i Transfuzjologii (IHiT) testami WB (New Lav Blot I, Bio Rad; HIV1 Blot 1.3, MP Diagnostics) oraz techniką NAT (Procleix Ultrio Assy, Chiron). Wyniki: W latach 2003–2008 przeprowadzono badania przeglądowe u 2 987 066 dawcow. U 3 (0,0001%) osob wykryto zakazenie w okienku serologicznym, a u 4682 (0,16%) powtarzalnie reaktywne wyniki anty-HIV. W badaniach weryfikacyjnych u 154/4682 (3,3%) dawcow z anty-HIV wykryto RNA HIV. U 7/4682 (0,15%) wynik testu WB w pierwszej probce byl wątpliwy; u 1/4682 (0,02%) ujemny. W nastepnym badaniu, przeprowadzonym po 3–4 tygodniach test WB byl dodatni. U pozostalych 146/4682 (3,1%) osob obecnośc przeciwcial potwierdzono testem WB w pierwszej probce. Wnioski: Wprowadzenie badan molekularnych krwiodawcow w kierunku zakazenia HIV1/2 podnioslo bezpieczenstwo przetoczen, gdyz: 1) umozliwilo wykrycie zakazenia u 3 krwiodawcow, bedących w momencie oddania krwi w okresie okienka serologicznego; 2) usprawnilo weryfi kacje reaktywnych wynikow badan przeglądowych poprzez przyspieszenie diagnozy zakazenia HIV (wątpliwy wynik WB).

Background Until now, markers of hepatitis E virus (HEV) infection have not been studied in blood donors throughout Poland, and no acute case of HEV infection has been closely documented or confirmed by HEV RNA detection. The prevalence of HEV infection markers, including HEV RNA in Polish blood donors and virus genotypes was investigated. Study design and methods In total, 12,664 individual donations from 22 Polish blood transfusion centers were tested for HEV RNA by transcription-mediated amplification. In addition, 3079 first-time donors sampled throughout Poland also were screened for antibodies to HEV. HEV RNA and immunoglobulin M-positive donations were confirmed using real-time reverse transcription-polymerase chain reaction and Western blotting, respectively. Results Ten donors were identified as RNA initial reactive (one of 1266 donors), and six (one of 2109) were identified as repeat reactive and confirmed by real-time reverse transcription-polymerase chain reaction or seroconversion. Sequence analysis identified HEV Genotype 3c in one donor and Genotype 3i in two others. On average, 43.5% of donors were immunoglobulin G-positive. Immunoglobulin G seroprevalence ranged from 22.7% to 60.8% in group ages 18 to 27 years and 48 to 57 years, respectively and differed between administrative regions from 28.9% in Podlasie to 61.3% in Wielkopolska. Thirty-nine of the donors were immunoglobulin M-positive, and seven donors were IgM positive only (0.2%). Of 37 immunoglobulin M-reactive samples tested by Western blot, 24 (64.9%) were confirmed. Conclusions The current results indicate a high level of HEV endemicity throughout Poland compared with other countries. There is an urgent need to consider the protection of recipients of blood components against transfusion-transmitted HEV infection.

Blood transfusion in Poland is the safest in history. High virological level of safety has been achieved mainly by improving not only the qualification of donors and methods used for donor screening, but also applying leukoreduction, pathogen reduction technology and grace period for serum.In this article, we discuss the improvement of the epidemic situation among blood donors for hepatitis B virus (HBV) and hepatitis C virus (HCV) and the increasing trend for HIV. Preliminary results of residual risk calculation for these pathogens are presented.Hepatitis E virus (HEV) and Babesia microti were considered as new factors potentially relevant for the safety of blood transfusion in our country. Due to evidence of West Nile virus (WNV) circulation in the ecosystem in Poland, it is also necessary to monitor the infections with this pathogen.In this article, it was emphasized that the reporting of all possible complications associated with transfusion and meticulous implementation of the look-back procedure play a key role for monitoring the risk of transmission of infectious agents by blood. It is especially important in view of the increasing epidemiological problems associated with emerging infectious agents.

BACKGROUND Since the introduction of nucleic acid testing (NAT) for routine blood donor screening, hepatitis C virus (HCV) RNA-only detection rates reported from Poland have been higher than in most other European countries. STUDY DESIGN AND METHODS To examine factors that likely contribute to these window-period donations, we conducted a case-control study among 47 recently HCV-infected blood donors (cases), who gave blood between July 2002 and June 2014, and 141 controls matched by age, sex, and donation dates. Firth-corrected, conditional logistic regression models were fitted to estimate adjusted odds ratios and 95% confidence intervals. Adjusted population-attributable fractions were calculated based on the distribution of exposure among the cases. RESULTS On multivariate analysis, recent exposures in health care environments not routinely ascertained through predonation questionnaires were strongly associated with recently acquired HCV infection. These exposures included minor medical and dental procedures in the preceding 6 months (adjusted odds ratio, 5.77; 95 % confidence interval, 2.01-18.53). However, based on the population-attributable fraction, more important were behavioral deferrable risks that went unreported at the time of donation, such as high-risk sexual behaviors in the preceding 6 months (population-attributable fraction, 34%) or lifetime histories of drug use (population-attributable fraction, 28%). CONCLUSIONS This study raises questions about the effectiveness of deferral policy in excluding high-risk individuals. In addition, it provides further evidence supporting short, temporal deferrals for small medical procedures and dental treatments in Poland.

International audience

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

Le dépistage de l’AgHBs et de l’ADN viral du VHB apparait redondant en zone de faible endémie qui recherchent les anticorps anti-HBc. Des niveaux élevés d’AgHBs associés à un ADN viral indétectable ou présent à taux faible ont été observés dans ~5% des dons en France. Les mécanismes moléculaires liés à ce profil biologique sont inconnus. Par ailleurs, est essentiel d’évaluer tout impact négatif potentiel sur la sécurité transfusionnelle avant d’envisager la suppression du dépistage de l’AgHBs. Des dons AgHBs positifs (≥100 IU/mL) mais DGV non-réactifs/réactifs non-répétables avec une charge virale (CV) indétectable ou

BACKGROUND Hepatitis C virus (HCV) antigen and antibody combination assays have been launched as a cost-effective alternative to nucleic acid testing (NAT) for reducing the antibody-negative window period (WP). Later, a HCV antigen chemiluminescence immunoassay (CLIA) became available. STUDY DESIGN AND METHODS A panel composed of 337 HCV NAT–yield samples that were characterized for viral load (VL) and genotype was used to compare the sensitivity of two combination enzyme-linked immunosorbent assays (Monolisa, Bio-Rad; and Murex, formerly Abbott) and a HCV antigen CLIA (Abbott). Analytic sensitivity was compared with HCV RNA detection using Ultrio (Grifols) by testing serial dilutions of 10 genotype (gt)1 to gt4 samples. RESULTS HCV antigen CLIA detected 92.4% of samples, whereas Monolisa and Murex detected 38.3 and 47.5%, respectively. In the HCV RNA VL range of 105 to 107 IU/mL, Monolisa and Murex detected 38% to 56% of gt1, 85% to 78% of gt2, and 21% to 37% of gt3. The overall geometric mean 50% limit of detection (range) of Ultrio on gt1 to gt4 dilution series was 3.5 (1.2-7.7) copies/mL, compared to 3.3 × 106 (4.4 × 105-2.7 × 107), 3.4 × 106 (2.2 × 105-4.2 × 107), and 2728 (415-7243) copies/mL for Monolisa, Murex, and HCV antigen CLIA, respectively. CONCLUSION Analytical sensitivity of NAT was on average 1 million- and 780-fold higher than combination assays and HCV antigen CLIA, respectively. Relative sensitivities of combination assays differed for genotypes with Murex being more sensitive for gt1 and gt3 and Monolisa more sensitive for gt2. Although being less sensitive than NAT, combination assays could be considered in resource-limited settings since they detect 38% to 47% of seronegative WP donations.

The infection with more than one hepatitis C virus (HCV) genotype especially in subjects with a high risk of multiple HCV exposures has been demonstrated. The role of HCV mixed-genotype infection in viral persistence and treatment effect is not fully understood. The prevalence of such infection varies greatly depending on the technique used for genotype determination and studied population. Next-generation sequencing (NGS) which is suitable for extensive analysis of complex viral populations is a method of choice for studying mixed infections. The aim of the present study was to determine the prevalence of mixed-genotype HCV infections in the Polish seronegative, HCV-RNA-positive blood donors (n = 76). Two-step PCR was used for amplification of 5'-UTR of HCV. Using pyrosequencing altogether, 381,063 reads were obtained. The raw reads were trimmed and subjected to similarity analysis against the entire unfiltered NCBI nt database. Results obtained from NGS were compared with the standard genotyping. One (1.3%) mixed-genotype [3a, 2989 reads (94.8%); 1b, 164 reads (5.2%)] infection was found in a sample diagnosed as genotype 3a only by routine testing. Two samples were identified with different genotypes, compared to routine testing. In conclusion, NGS is a sensitive method for HCV genotyping. The prevalence of mixed-genotype HCV infections in blood donors is low.

The BCL11B gene encodes a Krüppel-like, sequence-specific zinc finger (ZF) transcription factor that acts as either a repressor or an activator, depending on its posttranslational modifications. The importance of BCL11B in numerous biological processes in multiple organs has been well established in mouse knockout models. The phenotype of the first de novo monoallelic germ line missense mutation in the BCL11B gene (encoding N441K) strongly implies that the mutant protein acts in a dominant-negative manner by neutralizing the unaffected protein through the formation of a nonfunctional dimer. Using a Förster resonance energy transfer-assisted fluorescence-activated cell sorting (FACS-FRET) assay and affinity purification followed by mass spectrometry (AP-MS), we show that the N-terminal CCHC zinc finger motif is necessary and sufficient for the formation of the BCL11B dimer. Mutation of the CCHC ZF in BCL11B abolishes its transcription-regulatory activity. In addition, unlike wild-type BCL11B, this mutant is incapable of inducing cell cycle arrest and protecting against DNA damage-driven apoptosis. Our results confirm the BCL11B dimerization hypothesis and prove its importance for BCL11B function. By mapping the relevant regions to the CCHC domain, we describe a previously unidentified mechanism of transcription factor homodimerization.

The infection with more than one hepatitis C virus (HCV) genotype especially in subjects with a high risk of multiple HCV exposures has been demonstrated. The role of HCV mixed-genotype infection in viral persistence and treatment effect is not fully understood. The prevalence of such infection varies greatly depending on the technique used for genotype determination and studied population. Next-generation sequencing (NGS) which is suitable for extensive analysis of complex viral populations is a method of choice for studying mixed infections. The aim of the present study was to determine the prevalence of mixed-genotype HCV infections in the Polish seronegative, HCV-RNA-positive blood donors (n = 76). Two-step PCR was used for amplification of 5′-UTR of HCV. Using pyrosequencing altogether, 381,063 reads were obtained. The raw reads were trimmed and subjected to similarity analysis against the entire unfiltered NCBI nt database. Results obtained from NGS were compared with the standard genotyping. One (1.3%) mixed-genotype [3a, 2989 reads (94.8%); 1b, 164 reads (5.2%)] infection was found in a sample diagnosed as genotype 3a only by routine testing. Two samples were identified with different genotypes, compared to routine testing. In conclusion, NGS is a sensitive method for HCV genotyping. The prevalence of mixed-genotype HCV infections in blood donors is low.

BACKGROUND The relative contribution of serologic screening and nucleic acid testing (NAT) to prevent hepatitis C virus (HCV) transmission has not been rigorously addressed. STUDY DESIGN AND METHODS Twenty-one blood organizations in seven geographical regions performing individual-donation (ID)-NAT in parallel with anti-HCV screening provided data from 10,897,105 donations to establish HCV infection rates in first-time, lapsed, and repeat donations. Screening efficacy was modeled for: anti-HCV alone, HCV antigen/antibody (combo), minipool (MP)-NAT in pools of 8 and 16 with anti-HCV, ID-NAT and anti-HCV, and ID-NAT alone. Probabilities of infectivity for red blood cell transfusions were estimated as 100% from window period (WP) and concordant HCV RNA/antibody-positive (concordantly positive [CP]) donations and 0.028% from anti-HCV-positive and RNA-negative probable resolved (PR) donations. RESULTS There were 5146 confirmed infections (30 WP, 3827 CP, and 1289 PR). Infection rates and transmission risks varied substantially across regions and by donation status. Residual risk with ID-NAT and serology screening was estimated at one in 250,000 in Egypt and at one in 10,000,000 in other regions combined; risk would increase to one in 7300 and one in 312,000, respectively, if NAT had not been performed. ID-NAT with or without anti-HCV testing showed higher efficacy than either MP-NAT or combo assays, particularly in lapsed or repeat donors in whom 99.2, 98.5, and 93.2% of infectious donations were estimated to be interdicted by these respective testing strategies. CONCLUSIONS The incremental efficacy of anti-HCV testing when ID- NAT screening is performed was minimal, particularly for screening lapsed and repeat donations.

Background: Mirasol® pathogen reduction technology (PRT) uses UV light and riboflavin to chemically inactivate pathogens and white blood cells in blood components. In the EU, Mirasol PRT is CE-marked for both plasma and platelet treatment. In Poland, the decision to introduce PRT treatment of the national supply of fresh frozen plasma has spurred interest in evaluating the cost-effectiveness of this strategy. Methods: A decision-analytic model evaluated the incremental costs and benefits of introducing PRT to the existing blood safety protocols in Poland. Results: Addition of PRT treatment of plasma to current screening in Poland is estimated to cost 2.595 million PLN per quality-adjusted life year (QALY) (610,000 EUR/QALY); treating both plasma and platelet components in addition to current safety interventions had a lower cost of 1.480 million PLN/QALY (348,000 EUR/QALY). Conclusions: The results suggest that in Poland the cost per QALY of PRT is high albeit lower than found in previous economic analyses of PRT and nucleic acid testing in North America. Treating both platelets and plasma components is more cost-effective than treating plasma alone. Wide confidence intervals indicate high uncertainty; to improve the precision of the health economic evaluation of PRT, additional hemovigilance data are needed.

Until now, markers of hepatitis E virus (HEV) infection have not been studied in blood donors throughout Poland, and no acute case of HEV infection has been closely documented or confirmed by HEV RNA detection. The prevalence of HEV infection markers, including HEV RNA in Polish blood donors and virus genotypes was investigated.In total, 12,664 individual donations from 22 Polish blood transfusion centers were tested for HEV RNA by transcription-mediated amplification. In addition, 3079 first-time donors sampled throughout Poland also were screened for antibodies to HEV. HEV RNA and immunoglobulin M-positive donations were confirmed using real-time reverse transcription-polymerase chain reaction and Western blotting, respectively.Ten donors were identified as RNA initial reactive (one of 1266 donors), and six (one of 2109) were identified as repeat reactive and confirmed by real-time reverse transcription-polymerase chain reaction or seroconversion. Sequence analysis identified HEV Genotype 3c in one donor and Genotype 3i in two others. On average, 43.5% of donors were immunoglobulin G-positive. Immunoglobulin G seroprevalence ranged from 22.7% to 60.8% in group ages 18 to 27 years and 48 to 57 years, respectively and differed between administrative regions from 28.9% in Podlasie to 61.3% in Wielkopolska. Thirty-nine of the donors were immunoglobulin M-positive, and seven donors were IgM positive only (0.2%). Of 37 immunoglobulin M-reactive samples tested by Western blot, 24 (64.9%) were confirmed.The current results indicate a high level of HEV endemicity throughout Poland compared with other countries. There is an urgent need to consider the protection of recipients of blood components against transfusion-transmitted HEV infection.

Blood transfusion in Poland is the safest in history. High virological level of safety has been achieved mainly by improving not only the qualification of donors and methods used for donor screening, but also applying leukoreduction, pathogen reduction technology and grace period for serum.In this article, we discuss the improvement of the epidemic situation among blood donors for hepatitis B virus (HBV) and hepatitis C virus (HCV) and the increasing trend for HIV. Preliminary results of residual risk calculation for these pathogens are presented.Hepatitis E virus (HEV) and

// Katarzyna Izykowska 1 , Grzegorz K. Przybylski 1 , Claudia Gand 2 , Floriane C. Braun 2 , Piotr Grabarczyk 2 , Andreas W. Kuss 3 , Karolina Olek-Hrab 4 , Armando N. Bastidas Torres 5 , Maarten H. Vermeer 5 , Willem H. Zoutman 5 , Cornelis P. Tensen 5 , Christian A. Schmidt 2 1 Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland 2 Clinic for Internal Medicine C, University Medicine Greifswald, Greifswald, Germany 3 Department of Functional Genomics, University Medicine Greifswald, Greifswald, Germany 4 Department of Dermatology, Karol Marcinkowski University of Medical Sciences, Poznan, Poland 5 Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands Correspondence to: Grzegorz K. Przybylski, email: grzegorz.przybylski@igcz.poznan.pl Keywords: Sezary syndrome, NGS, whole genome, RNASeq, rearrangement Received: July 26, 2016 Accepted: March 27, 2017 Published: April 24, 2017 ABSTRACT Sezary syndrome (SS) is an aggressive, leukemic cutaneous T-cell lymphoma variant. Molecular pathogenesis of SS is still unclear despite many studies on genetic alterations, gene expression and epigenetic regulations. Through whole genome and transcriptome next generation sequencing nine Sezary syndrome patients were analyzed in terms of copy number variations and rearrangements affecting gene expression. Recurrent copy number variations were detected within 8q ( MYC, TOX ), 17p ( TP53, NCOR1 ), 10q ( PTEN, FAS ), 2p ( DNMT3A ), 11q ( USP28 ), 9p ( CAAP1 ), but no recurrent rearrangements were identified. However, expression of five genes involved in rearrangements ( TMEM244, EHD1, MTMR2, RNF123 and TOX) was altered in all patients. Fifteen rearrangements detected in Sezary syndrome patients and SeAx resulted in an expression of new fusion transcripts, nine of them were in frame ( EHD1-CAPN12, TMEM66-BAIAP2, MBD4-PTPRC, PTPRC-CPN2, MYB-MBNL1, TFG-GPR128, MAP4K3-FIGLA, DCP1A-CCL27, MBNL1-KIAA2018 ) and five resulted in ectopic expression of fragments of genes not expressed in normal T-cells ( BAIAP2, CPN2, GPR128, CAPN12, FIGLA). Our results not only underscored the genomic complexity of the Sezary cancer cell genome but also showed an unpreceded large variety of novel gene rearrangements resulting in fusions transcripts and ectopically expressed genes.

The 6q23-27 region, recurrently deleted in Sézary syndrome (SS), was characterized at the molecular level in 13 SS patients and SS cell line SeAx. Using fine-tiling comparative genomic hybridization, deletions within the 6q23-27 region were detected in half of the samples (six patients and SeAx). All samples with deletions were further analyzed by ligation-mediated PCR. In addition, in one patient sample and in SeAx, paired-end next-generation sequencing was performed on the HiSeq2000 Illumina platform. Using those techniques, 23 rearrangements associated with the deletions were identified. The majority of rearrangements showed enormous complexity and diversity, including eight inversions, three transpositions, and four translocations (with chromosomes 3, 17, 10, and 12). Fifteen genes were disrupted by those rearrangements, the MYB proto-oncogene three times and the interleukin-22 receptor subunit alpha-2 gene (IL22RA2) twice. All three patients with MYB alterations showed low MYB expression, whereas seven of the remaining patients showed overexpression. Most patients overexpressing MYB also presented increased expression of MYC, HSPA8, and BCL2. Five gene fusions were identified, of which two, CCDC28A-IL22RA2 and AIG1-GOSR1, both in SeAx, were in the same orientation and were expressed at the messenger RNA level.

Background The second triplex transcription-mediated amplification (TMA) assay version (Ultrio Plus, Novartis Diagnostics) uses an additional reagent enhancing the disruption of hepatitis B virus (HBV) particles and release of DNA for the target capture probe. This study compares the performance of this new assay version with the previous one (Ultrio). Study Design and Methods For analytical sensitivity assessment the World Health Organization HBV, hepatitis C virus (HCV), and human immunodeficiency virus (HIV) international standards and various genotype dilution panels were used. Individual donations (IDs) from 9980 first-time donors were screened simultaneously by serology and both TMA assay versions. Results The 50 and 95% limits of detection (LODs) for HBV using Ultrio Plus were 0.8 (0.6-1.0) and 4.6 (3.2-7.2) IU/mL, respectively, 2.4 (1.4-4.8)-fold more sensitive than Ultrio. The TMA assay versions had comparable LODs for HIV-1 and HCV. The improvement factors on analytical sensitivity panels of HBV Genotypes A to G ranged from 1.3 to 7.3 and 50% LODs (95% confidence interval) reduced from 12.5 (10-15) to 3.8 (3.2-4.4) copies/mL. One Ultrio Plus HBV Genotype D yield sample missed by the Ultrio assay in the donor screening study was detected with ninefold higher sensitivity. The specificities of ID nucleic acid test (ID-NAT) and serologic testing in a similar repeat test algorithm were 100 and 99.41%, respectively. Conclusion More efficient target capture chemistry in the new TMA assay version significantly improved sensitivity and diminished variability in detecting HBV strains of various genotypes. We recommend a triplicate ID-NAT repeat test strategy to eliminate discriminatory tests on false-non–repeat-reactive (anti-HBc–nonreactive) donations.

Current residual infection risk by transfusion for hepatitis B and C viruses (HBV, HCV) and for human immunodeficiency virus (HIV) is the lowest in the history. It has been achieved mainly by introduction of serological markers screening (HBsAg, anti-HCV and anti-HIV) and nucleic acid testing (NAT). These procedures allow identifying donors infected in chronic and very early stages of the infection. The incidence and prevalence of HBV and HCV infection among blood donors in Poland remain stable, however in the case of HIV, in recent years, an increasing trend is seen. The residual risk of infection is associated primarily with diagnostic window. For donations tested individually, this period is estimated to be 11.6, 1.5, 3.3 days for HBV, HCV and HIV, respectively. In the case of screening in plasma pools of eight donations it is calculated for 18.2, 2.7 and 5.5 days. Particularly for HIV, polymorphism at the genomic level is an additional risk factor. Recent observations confirm that, infectivity during window period is very high – even single virions could transmit infection. At the final stage of chronic HBV infection (OBI) infectivity is still significant but much lower (about 1,000 copies). In recent years it has been demonstrated that increased analytical sensitivity translates into a significant improvement of the clinical sensitivity. Further reduction of the diagnostic window for individual donations testing is possible by increasing the efficiency of the extraction and amplification (e.g. HBV in Ultrio Plus and Ultrio Elite) or by increasing the volume of the test sample (e.g., HCV and HIV in tests based on TMA or PCR). It was demonstrated that the amplification of more than one region of the HIV genome contribute to the recovery/improvement of clinical sensitivity lost in the case of blood donations infected with escape mutants. One way to reduce the number of infected donors is to improve selection process involving questionnaire and interview before every donation. The process should be updated based on the analysis of current sources of infection.

The Epstein–Barr virus (EBV) is one of the most common human viruses, infecting more than 90�0of the world’s adult population. In some individuals the interplay between EBV replication, latency and immune control can be disrupted and evokes prolonged proliferation of EBV-infected lymphocytes and their malignant transformation. Since its discovery as the first human tumor virus, EBV has been implicated in the development of a wide range of human cancers. The evidence for an association with EBV is the strongest for Burkitt’s lymphoma, NK/T cell lymphoma, nasopharyngeal carcinoma, Hodgkin’s lymphoma and for malignant lymphomas in immune incompetent patients. Additionally, certain epithelial cell tumors, such as gastric carcinoma and breast carcinoma, have been defined as EBV related. However, the virus may be encountered in other types of malignancies. The oncogenic potential of EBV is related to its ability to infect and transform B lymphocytes into continuously growing lymphoblastoid cell lines. EBV encodes a series of products mimicking several growth, transcription and anti-apoptotic factors, to usurp control of the pathways that regulate diverse homeostatic cellular functions. However, the exact mechanism by which EBV promotes oncogenesis remains unclear. The focus of this review is to summarize the current knowledge of oncogenic potential of the Epstein–Barr virus and its role in the pathogenesis of EBV-associated lymphoproliferative disorders.

The effect of the B-cell chronic lymphocytic leukemia/lymphoma 11B gene ( BCL11B ) on human T-cell regulation remains unclear. To characterize the functions of BCL11B , recombinant BCL11B and BCL11B siRNA were transfected into human naive T cells to overexpress or knock down BCL11B expression, respectively. After BCL11B overexpression, the proliferation ability and the T-helper (Th) subset were increased, whereas no significant alteration in the expression pattern and clonality of the T-cell receptor Vβ subfamilies was observed. After BCL11B knockdown, a similar distribution of Vβ subfamilies was detected in the naive T cells; however, the proliferation capacity substantially decreased. Global gene expression profiling revealed that the dysregulated genes were mainly involved in T-cell activation and proliferation. BCL11B could selectively promote Th-cell differentiation because of increased CXCL10 and CXCL11 expression. BCL11B suppression may inhibit proliferation and induce apoptosis, which may relate to changes in the expression of CFLAR–CASP8–CASP10 in the mitochondrial pathways. In conclusion, BCL11B is required for T-cell survival; its overexpression could effectively increase the T-cell activation and proliferation abilities and Th-cell differentiation as well.

Despite recent therapeutic improvements, the prognosis for patients suffering from Sézary syndrome (SS), a disseminated form of cutaneous T-cell lymphomas, is still poor. We identified bi- and monoallelic deletions of the tumor necrosis factor-α-induced protein 3 gene (TNFAIP3; A20) in a high proportion of SS patients as well as biallelic A20 deletion in the SS-derived cell line SeAx. Furthermore, we demonstrate that inhibition of A20 activates the NF-κB pathway thereby increasing the proliferation of normal T lymphocytes. On the other hand, the reconstitution of A20 expression slowed down the cell cycle in SeAx cells. Recently A20 inactivation has been reported in various B-cell lymphomas. In this study, we show that A20 is also a putative tumor suppressor in the T-cell malignancy-SS.