An international review of the characteristics of viral nucleic acid-amplification testing (NAT) reveals a trend towards the use of smaller pool sizes and individual donation NAT.

BACKGROUND AND OBJECTIVES: Nucleic acid-amplification testing (NAT) is used for screening blood donations/donors for blood-borne viruses. We reviewed global viral NAT characteristics and NAT-yield confirmatory testing used by blood operators. MATERIALS AND METHODS: NAT characteristics and NAT-yield confirmatory testing used during 2019 was surveyed internationally by the International Society of Blood Transfusion Working Party Transfusion-Transmitted Infectious Diseases. Reported characteristics are presented herein. RESULTS: NAT was mainly performed under government mandate. Human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) NAT was performed on all donors and donation types, while selective testing was reported for West Nile virus, hepatitis E virus (HEV), and Zika virus. Individual donation NAT was used for HIV, HCV and HBV by ~50% of responders, while HEV was screened in mini-pools by 83% of responders performing HEV NAT. Confirmatory testing for NAT-yield samples was generally performed by NAT on a sample from the same donation or by NAT and serology on samples from the same donation and a follow-up sample. CONCLUSION: In the last decade, there has been a trend towards use of smaller pool sizes or individual donation NAT. We captured characteristics of NAT internationally in 2019 and provide insights into confirmatory testing approaches used for NAT-yields, potentially benefitting blood operators seeking to implement NAT.

International review of blood donation nucleic acid amplification testing.

BACKGROUND AND OBJECTIVES: Nucleic acid amplification testing (NAT), in blood services context, is used for the detection of viral and parasite nucleic acids to reduce transfusion-transmitted infections. This project reviewed NAT for screening blood donations globally. MATERIALS AND METHODS: A survey on NAT usage, developed by the International Society of Blood Transfusion Working Party on Transfusion-transmitted Infectious Diseases (ISBT WP-TTID), was distributed through ISBT WP-TTID members. Data were analysed using descriptive statistics. RESULTS: Forty-three responses were received from 32 countries. Increased adoption of blood donation viral screening by NAT was observed over the past decade. NAT-positive donations were detected for all viruses tested in 2019 (proportion of donations positive by NAT were 0.0099% for human immunodeficiency virus [HIV], 0.0063% for hepatitis C virus [HCV], 0.0247% for hepatitis B virus [HBV], 0.0323% for hepatitis E virus [HEV], 0.0014% for West Nile virus [WNV] and 0.00005% for Zika virus [ZIKV]). Globally, over 3100 NAT-positive donations were identified as NAT yield or solely by NAT in 2019 and over 22,000 since the introduction of NAT, with HBV accounting for over half. NAT-positivity rate was higher in first-time donors for all viruses tested except WNV. During 2019, a small number of participants performed NAT for parasites (Trypanosoma cruzi, Babesia spp., Plasmodium spp.). CONCLUSION: This survey captures current use of blood donation NAT globally. There has been increased NAT usage over the last decade. It is clear that NAT contributes to improving blood transfusion safety globally; however, there is a need to overcome economic barriers for regions/countries not performing NAT.

Nuclear import of BCL11B is mediated by a classical nuclear localization signal and not the Krüppel-like zinc fingers.

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.

Hepatitis A virus (HAV) RNA detection in Polish blood donors and likely transmissions through blood components during the 2017-2019 epidemic.

BACKGROUND: In Poland, hepatitis A virus (HAV) RNA screening was performed in plasma for fractionation usually immediately before shipment. OBJECTIVE: Our goal was to study epidemiology, rate of transfusion transmitted HAV during epidemic (2017-2019), and viral characteristics of infected plasma donors. STUDY DESIGN AND METHODS: 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. RESULTS: 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%). CONCLUSION: 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.

Generation of Inducible BCL11B Knockout in TAL1/LMO1 Transgenic Mouse T Cell Leukemia/Lymphoma Model.

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.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

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.

Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain.

Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

Seronegative hepatitis C virus infection in Polish blood donors-Virological characteristics of index donations and follow-up observations.

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.

Molecular and serological infection marker screening in blood donors indicates high endemicity of hepatitis E virus in Poland.

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.

Interferon alpha as antiviral therapy in chronic active Epstein-Barr virus disease with interstitial pneumonia - case report.

BACKGROUND: Chronic active Epstein-Barr virus (EBV) disease (CAEBV) is defined as a severe, progressive lymphoproliferative disorder associated with active EBV infection persisting longer than 6 months and developing in patients without recognised immunodeficiency. Rarely, interstitial pneumonitis (IP) occurs as a serious complication in CAEBV patients. The standard therapeutic regimen for IP in CAEBV has not yet been defined. Although interferon alpha (IFN-alpha) is known to suppress viral DNA replication by affecting its basal promoter activation process, it is rarely used in CAEBV patients. CASE PRESENTATION: A 22-year-old Caucasian woman, diagnosed with CAEBV 1.5 years earlier, was admitted to the Immunology Clinic due to a 4-week history of productive cough, fever and general weakness. Cultures of blood, urine and sputum were negative, but EBV DNA copies were found in the sputum, whole blood, isolated peripheral blood lymphocytes as well as in the blood plasma. Cytokine assessment in peripheral blood revealed the lack of IFN-alpha synthesis. Disseminated maculate infiltrative areas in both lungs were observed on a computed tomography (CT) chest scan. The patient was not qualified for the allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to the risk of immunosuppression-related complications of infectious IP. Inhaled (1.5 million units 3 times a day) and subcutaneous (6 million units 3 times a week) IFN-alpha was implemented. To the best of our knowledge, this was the first documented use of inhaled IFN-alpha in a patient with CAEBV and concomitant IP. Patient's status has improved, and she was eventually qualified to allo-HSCT with reduced conditioning. Currently, the patient feels well, no EBV was detected and further regression of pulmonary changes was documented. CONCLUSIONS: CAEBV should be considered in patients who present with interstitial lung infiltration and involvement of other organs. Although more promising results have been obtained with allo-HSCT, inhaled IFN-alpha may also be a therapeutic option in patients with CAEBV and a concomitant IP.

Next-Generation Sequencing of Hepatitis C Virus (HCV) Mixed-Genotype Infections in Anti-HCV-Negative Blood Donors.

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.

What weighs more-low compliance with self-deferral or minor medical procedures? Explaining the high rate of hepatitis C virus window-period donations in Poland.

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.

[Risk of transmission of blood-derived pathogens by transfusion in Poland]. / Ryzyko przeniesienia czynników zakaznych przez transfuzje w Polsce.

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.

Inclusion of human immunodeficiency virus Type 2 (HIV-2) in a multiplex transcription-mediated amplification assay does not affect detection of HIV-1 and hepatitis B and C virus genotypes: a multicenter performance evaluation study.

BACKGROUND: The Ultrio Elite assay (Hologic/Grifols) runs on the Panther blood screening system and is comparable to the Ultrio Plus assay apart from the addition of oligonucleotides for human immunodeficiency virus Type 2 (HIV-2) detection. In this multicenter evaluation study the analytical sensitivity and genotype detection efficiency of the two assay versions were compared. STUDY DESIGN AND METHODS: The analytical sensitivity and genotype detection efficiency were analyzed by replicate (18-303) testing of 27 hepatitis B virus (HBV), hepatitis C virus (HCV), HIV-1, and HIV-2 standard dilution panels calibrated in international units (IUs) and copies/mL. A wider range of subgenotypes was tested at 25 copies/mL. Specificity was evaluated in 30,756 donor samples. RESULTS: The 95% lower limits of detection (LODs) in Ultrio Elite assay on WHO standards were 4.6, 7.3, 23.5, and 23.3 IU/mL for HBV, HCV, HIV-1, and HIV-2, respectively, and ranged from 13 to 44, 7 to 23, 6 to 15, and 9 copies/mL on genotype panels of the respective viruses. Comparable LODs had been previously found on the same panels with the Ultrio Plus assay. The specificity was 99.95% on initial test and 100% in the repeat test algorithm. CONCLUSION: The change in the oligonucleotide design of the Ultrio Elite assay to enable HIV-2 detection has not affected the analytical sensitivity for the other viruses regardless of the genotype. Genotype reference panels are instrumental to compare the sensitivity of nucleic acid test assay versions and could serve as an alternative to seroconversion panels.

Sensitivity of hepatitis C virus core antigen and antibody combination assays in a global panel of window period samples.

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 10(5) to 10(7) 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 × 10(6) (4.4 × 10(5) -2.7 × 10(7) ), 3.4 × 10(6) (2.2 × 10(5) -4.2 × 10(7) ), 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.

Introducing Pathogen Reduction Technology in Poland: A Cost-Utility Analysis.

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.

Blood donors screening for blood born viruses in Poland.

Blood donor screening of viral markers in Poland is based on serologic testing for anti-HCV, HBsAg, anti-HIV1/2 (chemiluminescence tests) and on nucleic acid testing (NAT) for RNA HCV, RNA HIV-1 and DNA HBV performed in minipools of 6 with real-time PCR (MPX 2.0 test on cobas s201) or with TMA in individual donations (Ultrio Plus or Ultrio Elite). Donors of plasma for anti-D and anti-HBs production are tested for parvovirus B19 DNA. Before implementation tests and equipment are evaluated at the Institute of Hematology and Transfusion Medicine (IHTM). The last 20 years witnessed a decreasing trend for HBsAg in both first time and repeat donors (1%-0.3% and 0.1%-0.02% respectively). Prevalence of anti-HCV repeat reactive results was stable and oscillated around 0.8% for first time donors and 0.2% for repeat donors. Elevated prevalence of seropositive HIV infected donors was recently observed (7.5-9 cases/100,000 donors). Since respective molecular markers implementation HCV RNA was detected on average in 1/119,235 seronegative donations, HIV RNA in 1/783,821 and HBV DNA in 1/61,047. HBV NAT yields were mostly occult hepatitis B (1/80,248); window period cases were less frequent (1/255,146). The efficiency of HBV DNA detection depends on the sensitivity of the HBV DNA screening system.