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.

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.

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.

Introduction of EUBIS standards to upgrade inspections of blood establishments to improve the safety of blood transfusion: The Polish experience.

BACKGROUND AND OBJECTIVES: The competent authority (CA) responsible for external inspections of Polish blood establishments (BEs) and supervision of the quality system is the Institute of Haematology and Transfusion Medicine (IHTM). Before the implementation of the European Blood Inspection System (EuBIS) classification of non-compliance, the IHTM inspections were conducted according to national guidelines and the non-compliance-related recommendations were based on the inspectors' own experience and interpretation of the observed problems. Since 2009, IHTM inspections were already performed according to EuBIS guidelines. The study assessed the impact of the EuBIS classification on the IHTM recommendations. We assumed that the implementation of consistent assessment criteria contributed to the upgrading of the quality of BE inspections. MATERIALS AND METHODS: BE-inspection protocols; 30 from 2009 to 2010 and 61 from 2016 to 2019. Non-compliance-related recommendations were classified according to the seriousness of non-compliances (critical, major, other significant, and observation) and also to the area of BE activity (documentation, organisation of work, qualification and validation, pathway from donor qualification to blood component-issue, quality control of blood components, adverse events and reactions). RESULTS: The recommendations mostly referred to document-keeping and work organisation and were distributed as follows: 2009-2 critical (others unclassified), 2010-1-13 major, 4-25 other significant and 1-7 suggestions, 2016-2019-3-9 critical, 90-196 major, and 157-297 other significant as well as 14-22 suggestions. CONCLUSION: Polish BEs still require: integrated document management, analysis of IHTM recommendations, implementation of corrective and preventive measures and personnel training in identifying similar non-compliances in other procedures.

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.

Hemovigilance survey of pathogen-reduced blood components in the Warsaw Region in the 2009 to 2013 period.

BACKGROUND: In 2009 the Mirasol Pathogen Reduction Technology (PRT) was introduced to the routine blood component production of the Regional Blood Transfusion Center in Warsaw (RBTCW). The goal of this study was to investigate the safety of Mirasol-treated blood components. STUDY DESIGN AND METHODS: The accumulated passive hemovigilance data of Mirasol-treated blood components collected at the RBTCW are presented and compared to historical and contemporary data. Furthermore, active hemovigilance data collected from patients with different hematologic disorders transfused with Mirasol-treated or untreated blood components at the Institute of Hematology and Transfusion Medicine (IHTM) are presented and discussed. RESULTS: The adverse reaction (AR) reporting rate by hospitals to the RBTCW after the implementation of the Mirasol technology was 0.39% for Mirasol-treated platelet concentrates (M-PCs) and 0.05% for Mirasol-treated fresh-frozen plasma. When comparing contemporary rates of ARs recorded by RBTCW in the time period 2011 to 2012, no statistical difference was observed between Mirasol-treated and untreated blood components. No serious AR was attributed to Mirasol-treated components. At the IHTM a lower rate of ARs after transfusion of M-PCs was observed than with untreated PCs. Despite the fact that very large amounts of Mirasol-treated plasma have been transfused to patients with congenital or acquired thrombotic thrombocytopenic purpura, no significant increase in AR rates was observed. CONCLUSION: Treatment of blood components with the Mirasol PRT System has proven to be safe for patients and is not associated with increased rates and grades of adverse events in patients of hospitals in the Warsaw Region.

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.

Methods of freezing cord blood hematopoietic stem cells.

BACKGROUND: Cord blood (CB) is a valuable source of hematopoietic stem cells (HSCs). Extended storage of CB is possible provided that validated cryopreservation procedures are used. The study objective was to determine optimal methods of CB cryopreservation. STUDY DESIGN AND METHODS: In the study we 1) compared the effect of two-step cryopreservation and controlled-rate freezing method on the postthaw quality of CB (Study A) and 2) evaluated the postthaw quality of HSC fractions isolated from CB with various methods and frozen with controlled-rate freezing method (Study B). The same cryoprotectant mixture was used for 20 CB units (Study A) and 122 CB units (Study B). RESULTS: In Study A, 13.79 × 10(8) and 13.29 × 10(8) initial white blood cell (WBC) counts decreased to 6.38 × 10(8) and 6.02 × 10(8) after thaw for the two methods, respectively. The mononuclear cell (MNC) counts decreased from 5.90 × 10(8) to 3.71 × 10(8) and from 5.64 × 10(8) to 3.47 × 10(8) dependent on the method. MNC viability decreased from 99.0% to 97.4% for the former and from 98.5% to 97.2% for the latter method. The differences were insignificant. In Study B, postthaw WBC recovery in HSC fractions was 74.4% to 103.5%, MNC recovery 106.4% to 118.5%, CD34+ cell recovery 102.5% to 150.2%, and MNC viability 94.1% to 97.4%. CONCLUSION: Neither the cryopreservation procedure nor the freezing of isolated HSCs affected product quality, which may indicate that various freezing methods can be used for cell banking provided the they follow recommendations of good manufacturing practice and Directive 2004/33/EC.

Evaluation and experience from routine use of chemiluminescence assays for serological screening of blood and plasma donations on the Alinity s system and the Alinity i system, two new fully-automated immunoassay systems in Poland.

In Poland, independent evaluations under the auspices of the Institute of Hematology and Transfusion Medicine (IHTM) are mandated for any new device, assay, systems for screening samples from whole blood and plasma donors prior to implementation by Blood Transfusion Center (BTC). In last 5 years, two new systems were introduced to the market by Abbott GmbH, namely the Alinity s and the Alinity i. The evaluations performed for these two systems included the assessment of sensitivity, specificity and precision for each of the four mandatory serological screening markers in Poland: Hepatitis B Surface Antigen (HBsAg), Hepatitis C virus antibodies (Anti-HCV), HIV antibodies (anti-HIV) and Syphilis antibodies (anti-Treponema pallidum, anti-TP). Sensitivity was assessed by testing seroconversion panels, HBsAg international reference standard, well characterized local samples, and dilution panels. Specificity was assessed by testing routine donor samples. The results from Alinity i assays were compared to the results from Abbott ARCHITECT i2000SR and Ortho VITROS 3600 assays, while the results from Alinity s assays were compared to the results of ARCHITECT i2000SR assays. The evaluation of the Alinity s and Alinity i assays for sensitivity (100 %), specificity (99,92-100 %) and precision generated results that were as good as or better than generated by routinely used systems, were within acceptance criteria, and met all requirements for screening blood donor samples in accordance with Polish regulations. The specificity of the assays in routine use by BTCs, analyzed after approximately 150,000 donations on both systems, was comparable to the specificity observed during the evaluations at IHTM.

Head-to-head comparison of two transcription-mediated amplification assay versions for detection of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus Type 1 in blood donors.

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.

[Emerging infectious diseases in the context of blood safety]. / Nowo pojawiajace sie choroby zakazne w aspekcie bezpieczenstwa krwi.

The risk of transfusion-related infectious diseases, the markers for which are routinely tested, is extremely low. Recently, however, blood transfusion service faces the challenge from emerging infectious diseases (EIDs), mainly zoonotic origin. Pathogens are microorganisms, mostly viruses, that usually require vectors for their transmission to humans. The relation of some EIDs to transfusion has been proved, in other cases it is considered likely. The paper presents views on EIDs etiology and spread and explains the epidemiologic basic terminology. It describes the principles and methods of EIDs risk assessment as well as prioritization of EIDs with regard to transfusion risk. It outlines the principles of international cooperation and rapid response to newly emerging threats. More attention is devoted to such diseases as West Nile fever, malaria, dengue and chikungunya which are recently a real epidemiological threat. Preventive measures to reduce the threat of EIDs transmission have also been discussed as well as their impact on the safety and supply of blood and blood components.

[Parvovirus B19 DNA testing in Polish blood donors, 2004-2010]. / Badanie DNA parvowirusa B19 (B19V) u Polskich dawcoów krwi, 2004-2010.

UNLABELLED: Since 2004 Polish blood donors have been tested for parvovirus B19 (B19V) DNA. The screening testing has been performed in donors of plasma for fractionation and anti-D and anti-HBs production and donors of erythrocytes used for immunization. AIM is to present methods of the testing, quality control and results in period 2004-2010. MATERIAL AND METHODS: Testing was performed in individual donation testing (IDT) in Regional Blood Transfusion Center (RBTC) in Lublin or in pools of 24 in Institute of Haematology and Transfusion Medicine in Warsaw (IHTM). Quantitative testing with real-time PCR was preceded with nucleic acid isolation on silica based methods (Prepito Viral DNA/RNA, Chemagen and QIAamp DNA Mini Kit, QIAGEN). Amplification was performed initially with home made method and later with commercial assay (Artus Parvo B19 RG PCR Kit on Rotor Gene 6 000). In total 17 625 donations were tested: 8 539 in pools and 9 090 individually. Beside routine external quality control programmes in which both laboratories participated (Proficiency Study VQC,Amsterdam, Holand; EQA Programe, Glasgow, Scotland), panel containing negative samples, positive with very high DNA B 19V level and plasma infected with genotype 2 was prepared for RBTC in Lublin. RESULTS: B19V infection frequency was 1:980 donations, low viraemic donations were detected most frequently (1:1 037). It was identified only one donation with DNA load that could cause potential health risk for plasma product recipients (1:17 625). In one of the donors B 19V DNA was observed for 3 years and 3 months. In acute or persistent phase of infection no clinical or laboratory symptoms (morphology of peripheral blood, ALT) were observed. Due to risk of underestimation of viral load connected with viral genome polymorphism all donations with B19V positive result were not allowed to be clinically used.

[Analysis of the prevalence of HBV markers relevant to the risk of reactivation of infection in patients with hematologic diseases]. / Analiza wystepowania markerów wirusa HBV istotnych dla ryzyka reaktywacji zakazenia u osób z chorobami hematologicznymi.

The study assessed the incidence of HBV markers (HBsAg, anti-HBc, anti-HBs) important for determination of the risk of reactivation of infection, with particular interest of occult infection (presence of HBV DNA in the absence of HBsAg) in patients treated at the Institute of Hematology and Transfusion Medicine. Anti-HBc frequency was correlated with the age and sex of patients. HBsAg was detected in 16/468 examined patients, 98/468 (21%) were anti-HBc positive. HBV DNA was detected in 41/98 anti-HBc positives; in 13 simultaneously with HBsAg. 28 patients had occult HBV infection (HBV DNA+/HBsAg). Antibody to HBsAg was detected in 163/430 (38%) patients, 81 out of them on protective level (> 100 IU/l). It was shown that occult HBV infection occurs in approximately 6% of patients. In most of them the protective levels of anti-HBs are detected.

Human Intramuscular Hyperimmune Gamma Globulin (hIHGG) Anti-SARS-CoV-2-Characteristics of Intermediates and Final Product.

This study aims to characterize the intermediates, and the final product (FP) obtained during the production of human intramuscular hyperimmune gamma globulin anti-SARS-CoV-2 (hIHGG anti-SARS-CoV-2) and to determine its stability. Material and methods: hIHGG anti-SARS-CoV-2 was fractionated from 270 convalescent plasma donations with the Cohn method. Prior to fractionation, the plasma was inactivated (Theraflex MB Plasma). Samples were defined using enzyme immunoassays (EIA) for anti-S1, anti-RBD S1, and anti-N antibodies, and neutralization assays with SARS-CoV-2 (VN) and pseudoviruses (PVN, decorated with SARS-CoV-2 S protein). Results were expressed as a titer (EIA) or 50% of the neutralization titer (IC50) estimated in a four-parameter nonlinear regression model. Results: Concentration of anti-S1 antibodies in plasma was similar before and after inactivation. Following fractionation, the anti-S1, anti-RBD, and anti-N (total tests) titers in FP were concentrated approximately 15-fold from 1:4 to 1:63 (1800 BAU/mL), 7-fold from 1:111 to 1:802 and from 1:13 to 1:88, respectively. During production, the IgA (anti-S1) antibody titer was reduced to an undetectable level and the IgM (anti-RBD) titer from 1:115 to 1:24. The neutralizing antibodies (nAb) titer increased in both VN (from 1:40 to 1:160) and PVN (IC50 from 63 to 313). The concentration of specific IgG in the FP did not change significantly for 14 months. Conclusions: The hIHGG anti-SARS-CoV-2 was stable, with concentration up to approximately 15-fold nAb compared to the source plasma pool.