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Abstract

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

The description of the ABO blood group system by Landsteiner and coworkers marked a sea change in making blood transfusions feasible and safe for a broad range of indications. Nevertheless, with an increase in blood transfusions, side-effects such as transfusion-transmitted infections (TTIs) became more and more important. A major challenge in transfusion medicine was (and is) to develop screening assays with maximum analytical sensitivity and analytical specificity to reduce the diagnostic window period as much as possible. Until the late 1990s, blood screening for TTIs depended entirely on serological assays. Except for HBV, where the virus can be detected using HBs-antigen assays, tests for the detection of other TTIs relied almost exclusively on antibody detection. These tests, however, are associated with a relatively long diagnostic window period because they detect the response of the immune system to an infection.

In the mid 1990s, the residual risk of transfusion-associated HCV infection was estimated higher than 1:5000. New upcoming molecular technologies, such as the polymerase chain reaction (PCR), were examined to investigate how these methods could be implemented in blood donor screening to reduce this risk.

The German Red Cross Blood donor service, Frankfurt am Main, developed its own ‘in-house’ method and was the first to publish feasibility and efficiency of nucleic acid technology (NAT) blood donor screening resulting in the release of all blood components including packed red cells, fresh frozen plasma and platelet concentrates being free of HIV-1, HBV and HCV [1].

This review reports on the development of nucleic acid amplification tests and describes the current state of technology. Functional principles of the different nucleic amplification technologies (NAT) are depicted, and blood donor screening by NAT for different viruses is described. Additionally, the special situation of bacterial detection by NAT is discussed. Blood donor screening by NAT was started using in-house methods. Over the last decade, these systems were significantly improved and certified by the FDA or the EU. Currently, three fully automated, barcode-controlled NAT systems are available for blood donor screening (detection in individual donations or in mini-pools up to 96 samples per pool). In most developed countries, NAT screening for HCV-RNA and HIV-1-RNA is performed.

Depending on the screening strategy, blood donor testing by NAT is able to reduce the diagnostic window period for transfusion-transmitted infections (TTIs) such as HCV to a minimum of 4–6 days. This has led to a residual risk of TTIs of less than 1:1 million for HCV and HIV-1 in countries using NAT and underlines the efficiency of these methods.

However, in some cases, NAT detection may fail owing to mutations in the genome of the pathogen. Several cases of TTIs have been reported in the literature in which mutations in primer and probe binding regions were the major cause for a reduced analytical sensitivity and for screening failures. Amplification in at least two conserved genomic regions is a promising approach to overcome this risk. Generic bacterial detection can also be carried out by NAT, but there are a few drawbacks.

Serological combination assays may be economic alternatives to NAT but are associated with a longer diagnostic window period compared to NAT systems. Pathogen-inactivation methods are feasible for platelets and plasma products, but general inactivation methods for all three blood products are still eagerly awaited.

The description of the ABO blood group systems by Landsteiner et al. [2] was an enormous milestone for making transfusions of whole blood or blood components feasible and safe for a broad range of indications. However, concerning the principle of Hippocrates [3] of ‘primum nihil nocere’, this placed physicians in a difficult situation. On the one hand, they needed blood components for the successful treatment of many diseases, but on the other hand, the adverse side-effects, such as infections, were (and still are) potentially problematic often causing severe life-threatening disease. To avoid infection, the most critical point is the diagnostic window period [4], which is defined as the time period between the start of an infection and the first opportunity to recognize the infection by diagnostic testing. Shortening the diagnostic window period has been the focus of the last three decades of transfusion medicine. Therefore, many general safety procedures were implemented in blood donor screening, including critical donor selection [5], a donor self-exclusion opportunity [6], the storage of quarantined plasma and the development of new screening systems.

Mullis et al. [7,8] discovered a new molecular detection method, named polymerase chain reaction (PCR), that is able to produce multiple genome copies after 40–50 amplification cycles. A final concentration of approximately 1 billion genome replicates can easily be detected by agarose gel electrophoresis followed by staining with ethidium bromide. The disadvantage of first PCR version was the necessity to reopen sample tubes after amplification for detection by gel electrophoresis. This was time-consuming, and there was an associated risk of cross-contamination between different samples. In the beginning, PCR was only feasible for the analysis of individual samples and not for blood donor screening programmes. A subsequent advance was the development of real-time-NAT [9]. By adding oligonucleotides (approximately 20 basepairs of nucleotides) labelled with two different fluorochromes able to absorb and emit light at different wavelengths, the real-time NAT detection can be carried out in one step without the reopening of any sample tubes. The development of a new generation of enzymes facilitates one-step procedures for DNA and RNA amplification (including a reverse transcription step). In principle, the two technologies can be differentiated and will be discussed below:

  • 1
     transcription-mediated amplification (TMA)
  • 2
     real-time PCR technologies (with different primer and probe designs).

Transcription-mediated amplification (TMA)

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

TMA [10,11] is used to amplify portions of RNA and/or DNA. Reverse transcriptase creates a DNA copy (cDNA) of the target nucleic acid. The RNA polymerase initiates transcription, synthesizing RNA. Some of the newly synthesised RNA amplification products re-enter the TMA process and serve as templates for new rounds of amplification. The amplification process is mediated by a T7 promoter. More than 1000 amplification products are produced in one cycle, and potentially billions of copies are generated in < 1 h. Detection is carried out by acridinium ester (AE)–labelled probes specifically hybridized to the amplification products. Different AE variants are used to label the internal control specific (IC-specific) and viral-specific probes. The hybridization protection assay process selectively inactivates the AE label on unhybridized probes to minimize the background signal. Dual kinetic assay technology enables simultaneous detection of both IC-encoded RNA, through a flash of light, and viral-encoded RNA, through a longer lasting glow.

Real-time PCR technologies

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

Real-time PCR technologies can be classified into systems using intercalation dyes (e.g. SYBR green or ethidium bromide), systems with fluorescence resonance energy transfer probes [12–14] (FRET-probes) and others [15,16].

In principle, all systems use at least one sense primer, one antisense primer and any kind of probe, enzyme and nucleotide.

Systems based on FRET use specific probes labelled with one or two fluorochromes. During amplification, the DNA polymerase works also as an exonuclease. Therefore, probes bound at the target can be degraded, and the distance between both dyes can be enlarged. This changes the energy transfer between the reporter dye and the quencher dye. Classic real-time NAT systems are available as TaqMan® assays with TaqMan® probes (hydrolysis probes) or with hybridization probes. Two small hybridisation probes are labelled, each with one dye. With this configuration, a melting-curve analysis [17,18] (determination of the specific temperature at which probes bind to templates) can also be performed. Another real-time NAT system uses specific probes named molecular beacons [19,20]. These are small molecules with changing physical shapes depending on the temperature. At lower temperatures, the molecular beacons exist in a close state, the fluorophore and the quencher are held in close proximity to each other by the hairpin stem, and there is no fluorescence. However, at high temperatures, the helical order of the stem gives way to a random-coil configuration, separating the fluorophore from the quencher and restoring fluorescence. The temperature at which the stem melts depends upon the GC content and the length of the stem sequence. If a target is added to a solution containing a molecular beacon at temperatures below the melting temperature of its stem, the molecular beacon spontaneously binds to its target, thereby dissociating the stem and turning on the fluorescence. The manner in which the fluorescence of the probe–target hybrid varies with the temperature is indicated by the red fluorescence vs. the temperature trace. At low temperatures, the probe–target hybrid remains brightly fluorescent, but as the temperature is raised, the probe dissociates from the target and tends to return to its hairpin state, diminishing the fluorescence significantly. The temperature at which the probe–target hybrid melts apart depends upon the GC content and the length of the probe sequence. Other real-time NAT systems working with FRET technology (e.g. scorpion probes [21,22]) are slightly different but work on the same principle. The major benefits of real-time NAT systems compared to classical PCR systems are an improved linear range and a closed technology, as the sample cups do not need to be reopened after amplification.

Pooling and extraction methods

Blood donor services are responsible for releasing life-saving blood components for many kinds of different therapeutic strategies all over the world. Therefore, some countries centralize all blood donor screening tests in one or two laboratories. These test centres have to screen up to 10 000 samples per day. Analysing such a large number of samples by NAT daily is a big challenge. Therefore, pooling procedures were developed to reduce the total number of samples. Countries like Japan started in 1999 with mini-pools of up to 500 samples per pool for HBV, HCV and HIV-1 [23]. Other countries or blood services like ours very soon in 1996/1997 developed a mini-pool NAT (MP-NAT) system with up to 96 samples per pool [1,24,25]. After the pooling process, high-speed centrifugation was used to enrich viruses at the bottom of a centrifugation tube, followed by a manual extraction procedure using chaotropic salts [26]. At the beginning of blood donor screening by NAT, no commercial systems were available. Therefore, blood banks developed their own ‘in-house’ systems to improve blood safety by this new technology. Now, with more than 10 years of experience with NAT, the situation has completely changed. So far to our knowledge, three fully automated barcode-controlled NAT systems are on the market. Table 1 presents the analytical sensitivity for the three systems given by the manufacturers.

Table 1.   Analytical sensitivity of fully automated barcode-controlled nucleic acid technology (NAT) systems for blood donor screening
ParameterMPX Test on s201 platform 1Tigris Ultrio Plus 2Zelos x100 3
  1. NA = not available; all data represent the 95% level of detection (LOD). This virus concentration can be detected in 95 out 100 tests. Small values represent more sensitive assays. Numbers correspond to the manufacturer: 1 = Roche molecular systems, Pleasanton, CA, USA; 2 = Novartis Emmeryville, CA, USA; 3 = German Red Cross, Baden-Württemberg – Hessen, Frankfurt, Germany. According to the manufacturer’s instructions for use, the MPX test on the s201 platform can be used for individual donation NAT (ID-NAT), in pools of 6, 24, 48 or 96 samples, the Tigris ultrio plus is recommended for ID-NAT or mini-pools of 8, 16 or 24 samples and the Zelos x100 can be performed for ID-NAT or mini-pools up to a maximum number of 96 samples per mini-pool. * Test is currently within the CE certification process.

HAVNANA0·8 IU/ml
HBV3·7 IU/ml2·1 IU/ml0·6 IU/ml
HCV10·7 IU/ml3·1 IU/ml9·6 IU/ml
HIV-149·0 IU/ml27·6 IU/ml8·9 IU/ml
HIV-22·2 copies/mlNA1·3 copies/ml*
PB19NANA9·7 IU/ml

Blood donor screening by NAT is characterized by three critical processes: sample extraction, amplification and detection. All three parts must be optimized to achieve a system with a high analytical sensitivity and a low diagnostic window period. Historically, NAT systems used an enrichment centrifugation to spin down potential viruses in mini-pools to the bottom of sample tubes [24,27]. However, enrichment centrifugation process might fail for some TTI-relevant viruses (e.g. HCV) in lipid blood donations. False negative screening results could not excluded in all cases. Therefore, modern technologies use bead-based extraction processes (capture beads for the Tigris ultrio plus and magnetic beads for the MPX on s201 and the Zelos x100). Old-fashioned systems that use enrichment centrifugation are of higher risk, especially for samples with high lipid acid concentrations (e.g. blood donor samples during Christmas-time) that might reduce the efficiency of the centrifugation process and the analytical sensitivity of the PCR system.

Blood donor screening by NAT worldwide

More and more countries have already implemented NAT in blood donor screening or are currently in the process of implementation. Many countries already have approximately 10 years of experience with NAT. Starting with ‘in-house’ PCRs, the methods were currently CE-certified or FDA-approved. Most countries use fully automated NAT systems. The handling of fully automated NAT systems is currently as easy as using serological systems. With these automated systems, the pool size is reduced to six samples per pool or even to individual donation NAT (ID-NAT). Some countries like Germany and Austria continue to screen blood donor samples in mini-pools, with a maximum pool size of 96. Blood donor screening by NAT for at least HIV-1 and HCV has been implemented in different countries (e. g. USA, Canada, parts of Brazil, Spain, France, the UK, Denmark, Germany, the Netherlands, Belgium, Greece, Slovenia, the Czech Republic, South Africa, Ghana, Luxembourg, Switzerland, Italy, Japan, parts of China, Australia, Poland, Norway, Finnland and New Zealand). One exception in Europe is Sweden. Based on the very low incidence of HIV-1 and HCV in their donor population, they decided to stop blood donor screening by NAT in 2008.

Blood donor screening for pathogens by NAT can be divided into four groups:

  • 1
     transfusion-relevant pathogens that are generally tested for in many countries (HBV, HCV and HIV-1),
  • 2
     transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, B19V, Chikungunya virus and HIV-2),
  • 3
     hepatitis A virus (HAV) pathogens that are probably transfusion-relevant but that are currently not indicated as special risks for blood transfusions and not tested for in blood donor screening programmes (SARS CoV and Influenza viruses),
  • 4
     bacterial screening by NAT.

Transfusion-relevant pathogens that are generally tested

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

Human immunodeficiency virus 1 (HIV-1)

The first cases of immune deficiency after blood transfusion were reported in 1982. In 1983, HIV was described as the cause of acquired immune deficiency syndrome (AIDS). A detection assay was available in the mid 1980s, but at this time, many haemophilia A and haemophilia B patients were already infected from the transfusion of contaminated blood components. HIV is an RNA virus and can be divided into three groups (M group, O group and N group). The M group can be characterized into subtypes (A, B, C, D, F, G, H, J and K) and circulating recombinant forms. The virus doubling time is approximately 17 h. Therefore, the diagnostic window period is approximately 8–9 days by screening in mini-pools of up to 96 samples per pool and can be reduced to 5–6 days for ID-NAT. Blood donor screening by MP-NAT for HIV-1 was mandated in Germany in 2004. After this time-point, only one case of a transfusion-transmitted HIV-1 infection was reported (see below risk analysis of NAT systems) [28].

Hepatitis C virus infections

Hepatitis C viruses belong to the flavivirus family. The virus was first described in 1989, but it was known since the 1970s that a virus other than HAV and HBV existed; it was originally named ‘non-A-non-B’ hepatitis. The first antibody screening assays were available in 1990 (EIA first generation). The diagnostic window period was approximately 80 days for these assays. The virus doubling time is very short (approximately 10–11 h). Therefore, blood donor screening by NAT was able to reduce the diagnostic window period to 6–7 days for screening in mini-pools (with a maximum pool size of 96 samples per pool) or to 4–5 days for ID-NAT. Before the introduction of blood donor screening by NAT, the residual transfusion-transmitted infectious risk was estimated to be 1:200 [29], and it is currently calculated at 1:10·88 million [30]. After blood donor screening by NAT was mandated in Germany, only one single case was reported as a TTI [31]. The donation was performed in the very early infection period with a virus load of only 10 IU/ml, which was below the analytical sensitivity of the MP-NAT.

Hepatitis B virus infections

Blood donor screening for HBV is performed on a voluntary basis, although most of the fully automated NAT systems enable the detection of this pathogen. HBV belongs to the hepadnavirus family and is a DNA retrovirus. Compared to HIV-1 or HCV, the virus doubling time is very low, at approximately 2·56 days [32]. The virus can be integrated into the genome of hepatocytes, which are the primary target cells. Approximately 90% of infected patients suffer from acute infection. The immune system is able to eliminate the virus from plasma in 90% of cases. In approximately 10% of cases, HBsAg and/or HBV DNA can be detected for more than 6 months. These cases are defined as chronic HBV infections or occult HBV infections (OBI). The virus load can be very low (< 10 IU/ml), which represents special challenges for diagnostic assays. Patients with OBIs are at a higher risk of developing liver cirrhosis or liver cancer after 10–15 years. OBIs can be detected by anti-HBc, which is also tested for in blood donor screening in low epidemic areas such as the USA or Germany. Screening for anti-HBc is not feasible in high epidemic areas such as Asia because the percentage of anti-HBc reactive donors might cause an unacceptable loss of necessary life-saving blood components. General HBV vaccination programmes for infants were implemented in the mid 1990s. These might reduce the risk of HBV TTIs in the near future. Unfortunately, most vaccines induce HBV-neutralizing antibodies against genotype A. In this context, it is important that the majority of HBV infections in Asia are of genotype B or C. These HBV infections might not be sufficiently prevented by the current vaccination programmes. Therefore, blood donor screening by NAT with a high analytical sensitivity is recommended.

Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

West Nile virus infections

West Nile virus infections (WNV) occurred in birds and humans in the USA in 1999, spreading from the east coast to the west coast within 3 years [33]. In 2003, the FDA mandated blood donor screening by NAT [34] for all blood donations. The incidence of WNV infections increases in the summer season. Based on the low virus load, especially in the preseroconversion period, blood donor screening was implemented in mini-pools of eight in the winter season and changed to ID-NAT blood donor screening with the increasing WNV incidence in local districts. WNV infections were also reported in Europe in some regions, but general screening is currently not recommended [35–37].

Hepatitis A virus infections

Hepatitis A viruses are small, non-enveloped RNA viruses belonging to the picornaviridae family. Pathogen reduction by solvent and detergent methods are less efficient for HAV. The major infection route for HAV is the faecal–oral pathway, but HAV can also be detected in blood components [38–40]. Chronic infections are not described, but in rare cases, an acute HAV virus infection can cause a fulminant liver dysfunction. Although the incidence of HAV is low, blood safety can be improved by real-time NAT systems [41].

Hepatitis E virus infection

Hepatitis E virus is a small RNA virus that belongs to the caliciviridae family. Infections are frequent in Asia, the Near East, Africa and Middle America. The virus originates in drinking water contaminated with faeces or in infected animals (pigs). TTIs were reported in Japan and the UK [42–44]. Blood donor screening by NAT might possibly prevent these infections, but they are rare events. Cost–benefit analyses are still needed to calculate the value of this parameter. General screening of blood donations by NAT for HEV is not recommended.

Parvovirus B19 virus infections

Parvovirus B19 is a non-enveloped DNA virus that was detected in 1975. The virus grows to very high virus concentrations (up to 1014 IU/ml) with only mild symptoms, such as tiredness, in most cases. The virus binds to the P antigen at erythrocyte precursor cells and induces apoptosis. The B19 virus can cause haemolysis, which might be clinically relevant for infants and newborns. Transfusion transmissions by blood components are described in case reports [45]. The infections depend on the immune response of the recipients. Approximately 60% of adults 30 years of age will have relevant levels of neutralizing antibodies owing to a past infection. A recently published retrospective study by Kleinman et al. [46] could not confirm a TTI in recipients transfused with blood products with a low virus load.

Chikungunya virus infections

In recent years, large Chikungunya virus (CHIKV) outbreaks originating in Kenya have spread to islands of the Indian Ocean and parts of India, Southeast Asia and Europe [47]. The concern of transfusion transmission has been heightened for this mosquito-borne arbovirus because of high population infection incidence during outbreaks and the high-titre viraemia lasting approximately 6 days. CHIKV produces a fever–arthralgia syndrome, resulting in considerable morbidity and some mortality, particularly among older age groups and/or those with pre-existing conditions. Possible measures to prevent possible CHIKV transfusion transmission include the deferral of symptomatic donors, discontinuing blood collections in affected areas and CHIKV nucleic acid screening of donations. Even a relatively small outbreak in Italy [48] resulted in a considerable adverse impact on blood collections and economic consequences. Assays suitable for testing donations for CHIKV RNA are available as ‘in-house’ systems. Although there were many cases of potentially transfusion-transmitted CHIKV infections between 2005 and 2007 during the massive epidemic on Reunion Island, no cases are known to have been confirmed by phylogenetic analysis.

Human immunodeficiency virus 2 infections

The global distribution of the two causes of acquired immunodeficiency syndrome (AIDS), human immunodeficiency virus type 1 (HIV-1) and HIV-2, are remarkably different. In the Americas, Europe and Asia, there has been an epidemic spread of HIV-1 in certain risk groups, mostly through homosexual sex and injection drug use. In contrast, HIV-2 has been found predominantly in heterosexual populations in West Africa but has spread very little to other areas [49,50]. Based on reports from 2008 from the WHO, 33·4 million people (range 31·1–35·8 million) were living with AIDS, 2·7 million were newly infected in 2008 (range 2·4–3·0 million) and 2·0 million people died in 2008 as a consequence of AIDS (range 1·7–2·4 million). The residual risk from blood donation for HIV-1 is very low, especially in countries where blood donor screening by NAT is implemented. Although the infectious risk for HIV-2 outside the middle of Africa is very low, two fully automated NAT systems (Roche MPX test on the s201 platform and DRK HIV 1/2 PCR Kit on the Zelos ×100 platform) have already added HIV-2 to a multiplex screening procedure.

Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

Every year, a ‘new’ well-known pathogen gains attention in the daily news. In 2003, an epidemic of corona viruses was reported in Asia. Because of the general travel behaviour of the human population, infections spread all over the world within some days. The major transmission pathway of SARS CoV was airborne infection, and at the end the epidemic, it could be curtailed efficiently by compliance to strict quarantine procedures. However, in the beginning, no information existed as to whether asymptomatic patients in the early infection period were viraemic (in this case, blood products could be infectious). SARS CoV was a good example of the powerful opportunities provided by NAT systems. After sequencing a genome, a specific real-time NAT system can be developed within a few weeks [51,52].

A new antigen combination of influenza A viruses (H1N1) was found in 2009 in a young child in Mexico. The new virus supposedly originated in pigs. In history, an influenza epidemic with the same antigens occurred in 1918 (i.e. the ‘Spanish flu’) that caused the death of approximately 50 million people. Therefore, people all over the world were alert and developed risk strategies to prevent the global spreading of the new infection. In this special situation, the first effort was aimed at developing vaccines against this new influenza infection, but later on, the second or third activities were aimed at developing diagnostic NAT systems for blood and sputum [53,54].

Bacterial screening by NAT

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

Improvements in blood donor screening systems, e.g. the introduction of third and fourth generation antibody assays and the introduction of nucleic acid testing (NAT) [55], have reduced the risks of the transmission of clinically relevant viral infections to far below the risk of the transmission of bacterial infections. Therefore, bacterial contamination of blood products represents an ongoing challenge in transfusion medicine. Blood donor screening for bacterial contamination is difficult because of the very low bacterial concentration after the production process. Donors with relevant bacteraemia should have clinical symptoms and are eliminated from blood donation. Transient or resident skin bacteria in deep areas could be a source of the bacterial contamination of blood components. Leucocytes from the buffy coat and complement factors will also reduce residual bacteria. Based on reverse calculations, the amount of bacteria in contaminated platelet product is estimated at 10 CFU/bag.

Many countries have implemented culture methods such as BacT/ALERT to detect bacterial contamination of platelets. As a result of the long incubation time, platelets were released as ‘negative-to-date’. Based on the very low bacterial concentration in the platelet products after production, sample failures were reported in different countries with negative screening results and severe TTIs owing to bacterial contamination [56–58]. In Germany, the Paul-Ehrlich-Institute reduced the shelf-life of platelet products from 5 to 4 days in 2009 as a result of a statistical analysis where transfusion-transmitted fatalities were associated in four out of five cases with a transfusion of platelets on day 5 after production. Additionally, rapid bacterial detection systems were developed within the last 10 years and include NAT and FACS systems.

Bacterial detection by NAT

Target genes for the development of generic bacterial NAT systems are ribosomal structures such as 16s RNA or 23s RNA. Unfortunately, the PCR enzymes were extracted from thermoresistant bacteria such as Thermus aquaticus, and these enzymes might be contaminated with bacterial ribosomal genes, causing false-reactive results. Feng et al. described one of the first assays for the detection of Yersinia enterocolitica in blood with a sensitivity of 5000 CFU/ml [59]. This sensitivity is not acceptable for a blood screening test because a donor with 2·5 million bacteria in 500 ml of blood (5000 CFU × 500 ml) would have clinical symptoms that would exclude the donor from blood donation. Newly developed oligonucleotides with fluorescent molecules at their 5′ and 3′ ends enable detection in a closed system with improved sensitivity compared to PCR detection via agarose gel electrophoresis. This real-time PCR system for bacterial detection was recently described by Nadkarni et al. [60] and has an analytical sensitivity between 30 and 100 CFU/ml; in principle, however, it did not overcome the problem of non-specific signals. Mohammadi et al. [61] solved this challenge by pretreating the PCR mixture with the restriction enzyme Sau3AI prior to the addition of template DNA. These authors were able to improve the detection limit to 1 CFU equivalent/PCR. Another solution might be an additional filtration of all NAT reagents with plasmid binding columns [62]. Both methods can be combined to optimize the results. Other investigators have attempted to decontaminate PCR materials and reagents by UV irradiation, 8-methoxypsoralen treatment, DNase treatment or combinations of these methods [62–66]. However, most of these methods also reduce the analytical sensitivity. Therefore, some investigators recommend reductions in the number of PCR cycles as the most effective and reproducible way of avoiding false-positive results [60,64]. Real-time NAT is a powerful tool in the clinical diagnosis of bacterial contamination in blood products. The extraction method can be completely automated [67,68] and barcode-controlled to enable screening of a huge number of donations. DNA/RNA extraction can be performed with material from platelet concentrates and whole blood to include all blood products (erythrocytes, platelet concentrates and plasma) in the bacterial screening process. The analytical sensitivity is currently between 10 and 50 CFU/ml and thus is slightly behind the sensitivity of culture methods. The total screening time for NAT systems (extraction and amplification) takes approximately 4 h. Therefore, these methods offer opportunities for a late sample collection to overcome sample errors. In this context, rapid bacterial detection systems can be used for bacterial detection on day 4 platelets. In the case of negative results, the shelf-life can be extended to 5 days (see Fig. 1).

image

Figure 1.  Blood donor screening for bacterial contamination of platelets. Blood safety for platelets can be improved by two methods. Scheme a: Pathogen-inactivation method directly after the production of platelet products. Scheme b: Release of platelet products on day 1 to day 3 without any additional screening. Retesting of all platelets on day 4 with rapid bacterial screening assays (FACS or nucleic acid technology) and release of all platelets with negative screening results on day 4 to day 5. This procedure is in accordance with the guidelines from the Paul-Ehrlich-Institute.

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Special NAT risks and NAT failures

Blood safety was significantly improved by the introduction of NAT systems through the reduction of the diagnostic window period, especially for transfusion-transmitted virus infections. Nevertheless, the new technologies are not risk free, which will be discussed below.

Diagnostic window period of donation

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

The introduction of NAT systems into blood donor screening was able to reduce the diagnostic window period to only a few days for HCV and HIV-1. Currently, at least three fully automated NAT systems are available on the market that enable blood banks to reduce pool sizes even to ID-NAT. This topic is currently a point of discussion in the Transfusion Medicine Society. On the one hand, the highest analytical sensitivity will achieve the maximum blood safety. However, the benefit for ID-NAT compared to MP-NAT is limited for HCV and HIV-1. For HBV (doubling time of 2·56 days), the situation is different. NAT systems with a very high analytical sensitivity are still needed to detect infected blood donors in the preseroconversion time period as well as in the second diagnostic window period in a reasonable time. On the other hand, ID-NAT increases the total costs for public health systems. In this context, each country must come to its own decision. On a related note, it is of interest that Sweden stopped blood donor screening by NAT for HCV and HIV-1 in 2008. Currently, all countries that have implemented NAT systems into their blood donor screening programmes are using mini-pool sizes between individual donations and pools with up to 24 samples per pool. Only Germany and Austria, two countries with low incidences for HCV, HIV-1 and HBV, are screening blood donors by NAT in mini-pools up to a maximum pool size of 96 samples per pool. Independent of the maximum pool size, it should be kept in mind that a very small number of blood donors might be infected with virus concentrations below the analytical sensitivity of the test of record, as described for HCV by Kretzschmar et al. [31]. Therefore, NAT cannot guarantee 100% safety.

Virus mutation in primer/probe regions

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References

All transfusion-transmission-relevant viruses can be subdivided into different genotypes and subtypes. RNA viruses are of higher risk of genetic diversity because the RNA must be reverse-transcribed into DNA during the amplification process. The enzyme responsible for this lacks a proofreading function. Therefore, any mistakes during the process will not be corrected. These mutations represent a risk for NAT systems if they occur in the primer and probe binding regions. In 2007, the first transmission of HIV-1 in Germany after the introduction of mandatory MP-NAT was reported by Schmidt et al. [28]. The infective donation was probably missed by MP-NAT owing to mutations in the probe binding region and the antisense primer binding region. The manufacturers are aware of the methodological risk by real-time NAT and have developed screening systems with the parallel amplification of at least two conserved regions.

NAT alternatives

Within the last 5 years, new combination assays for the parallel detection of antigens and antibodies were developed for HCV and HIV, respectively. Barbara et al. compared the analytical sensitivities of different assays. The optimized antigen tests for HCV requires an additional 3 days for the diagnostic window period compared to NAT. The best HCV combo test was reactive 5 days after NAT. Such data will be comparable for HIV. These data clearly demonstrate that blood donor screening by NAT will reduce the diagnostic window to a minimum; on the other hand, if NAT technology cannot be implemented, a combo test could be a fairly good alternative to improve blood safety and should be in these cases of state-of-the-art methods.

Other alternatives to screening methods could include pathogen inactivation or reduction methods. Three different methods have been developed for platelet products and for plasma products with different pathomechanisms. Pathogen-inactivation methods can be divided into photochemical systems [69,70] (e.g. S59/Amotosalen, Intercept®, Cerus), photodynamic systems [71–73] (e.g. Riboflavin, Mirasol®, Gambro BCT) and systems using only UV-C light [74]. Independent of the method, pathogen-inactivation technologies can inactivate viruses or bacteria up to 6 log phases. For most of the pathogens, the capacity will be sufficient, especially in the early infection period. Only some viruses such as Parvovirus B19 could occur in asymptomatic donors in concentrations up to 1014 IU/ml. Pathogens such as Bacillus cereus could be other exceptions, as they can occur in both vegetative and spore states. Spores are extremely resistant against environmental conditions. Unfortunately, pathogen-inactivation reagents penetrate into spores less efficiently. These are two reasons to keep in mind that inactivation could be incomplete for pathogen reduction methods. The different types of blood products are another challenge for the inactivation methods; different systems are recommended for plasma, platelets and erythrocytes. However, there is already some experimental data that one system, such as the Mirasol system, can be used for both platelets and red cells. Based on these points, a combination of pathogen inactivation together with MP-NAT could be the blood screening procedure of the future. New unknown pathogens will be inactivated, and high concentrations of viruses can be detected by MP-NAT. In combination with pathogen-inactivation methods, the maximum number of samples pooled together for NAT will be the subject of future discussions.

Summary

Blood donor screening by NAT reduces the diagnostic window period to only a few days. Therefore, the residual transfusion transmission risk is very low. The implementation of the NAT system is as easy as the implementation of serological systems, as three fully automated NAT systems are already available on the market. NAT systems are also available for bacterial detection in platelets. This special situation requires a late sample collection. NAT systems can be used for new pathogens, as real-time NAT systems will be available immediately after the sequencing of new pathogens. The maximum pool size used for blood donor screening is still a point of discussion. On the one hand, blood donor screening by ID-NAT reduces the diagnostic window period to a minimum, but on the other hand, MP-NAT in combination with pathogen-inactivation methods may represent a new standard for blood donor screening and will probably be feasible for all three blood components in the near future.

References

  1. Top of page
  2. Abstract
  3. Transcription-mediated amplification (TMA)
  4. Real-time PCR technologies
  5. Transfusion-relevant pathogens that are generally tested
  6. Transfusion-relevant pathogens that are tested for only in some countries with special circumstances (WNV, HAV, HEV, B19V, Chikungunya virus and HIV-2)
  7. Pathogens that are probably transfusion relevant but that are currently not indicated as special risks for blood transfusions and are not implemented into blood donor screening programmes (SARS CoV, Influenza viruses and H1N1)
  8. Bacterial screening by NAT
  9. Diagnostic window period of donation
  10. Virus mutation in primer/probe regions
  11. Disclosure
  12. References