Testing strategies looking forward
Celso Bianco, America’s Blood Centers, Washington, DC, USA
The early days of the AIDS epidemic brought tragic consequences for blood recipients. However, the attention, reflection, resources and research triggered by the epidemic led to the development of highly effective approaches for the prevention of transmission of infectious agents by transfusion of blood-, components- and plasma-derived therapeutic proteins. Before the availability of screening assays, prevention focused exclusively on the deferral of individuals thought to be at higher risk of transmitting the infection from donating blood components or plasma for further manufacture. Undoubtedly, the most significant current contributors to recipient safety are the testing of blood donations for the most relevant diseases transmissible by transfusion in the area where the blood was collected, and the implementation of quality systems that guarantee the adoption of appropriate screening assays and their use according to good laboratory practices.
This summary follows two in-depth reviews about assays available for the screening of blood donors in this session; one, by M. Busch, addresses serological assays and the other, by M. Schmidt, looks at molecular assays. This summary is purposely focused on thoughts about choices of assays, current testing strategies and future technological advances in the context of different countries with different epidemiology of transfusion transmitted infectious diseases (TTID) and variable resources available for transfusion safety.
Multiple layers of safety
The concept was developed as approaches to the HIV epidemic evolved to allay stakeholder concerns and fears and to reassure blood recipients about the safety of blood products. As new screening tests, donor screening procedures and quality systems were developed that were cumulatively added to the list of donor eligibility and blood processing requirements creating many complex algorithms and procedures that are not always contribute to blood safety. Obsolete procedures were removed only under exceptional circumstances. Gradually, the multiple layers of safety became part of the regulatory requirements of many countries.
Besides testing and quality systems, other generally adopted ‘multiple layers of safety’ are (1) donor selection and eligibility, in which donors are questioned about potential exposure to transmissible agents; (2) deferral files listing donors who were deferred because of travel, risk behaviour or reactive screening tests results; (3) quarantine of untested and unsuitable components, kept separately to avoid inappropriate release for transfusion. It should be noted that donor history questions have lower sensitivity and poorer predictive value (e.g. history of risk behaviour, travel or hepatitis)  and deferral files have a more limited role in an age of computers and reproducible tests .
It is reassuring that, in the midst of all the complexity of qualification of donors and products for transfusion, blood safety relies primarily on currently available assays that have excellent sensitivity, specificity, positive and negative predictive value for the detection of TTID recognized as significant. However, sometimes technology evolves faster than biological knowledge, and agents with questionable relevance for transfusion recipients are detected in a proportion of blood donors otherwise negative for HIV, HBV, HCV and HTLV. Among these are Hepatitis G or GVBC and SEN V . More recently, concerns have been raised about HHV-8, a herpes virus associated with Kaposi’s sarcoma  and xenotropic murine leukemia virus-related virus (XMRV), a gamma retrovirus detected in individuals with prostate cancer, in some cohorts of patients with chronic fatigue syndrome and in some blood donors .
Pathogen reduction and screening tests
Obviously, the practice of adding screening tests for every newly recognized pathogen transmissible by transfusion is not feasible. This concern became even clearer after the recent publication of a comprehensive list of relevant emerging infectious agents . Hopefully, in the foreseeable future, safe and effective pathogen inactivation technologies (PI) will allow us to limit screening assays to the most relevant needed to ensure the safety of the blood supply . PI has been applied successfully to proteins derived from human plasma as for instance albumin, IVIg, Factor VIII, alpha-1 anti-trypsin, etc. Unfortunately, the methods of inactivation used for proteins (solvent detergent, heat, nanofiltration, etc.) are not compatible with cellular components. It should be noted that even after the introduction of effective PI for plasma proteins, regulatory agencies require and advisory bodies like the World Health Organization recommend the exclusive use of plasma that tested negative for HIV, HBV and HCV in screening tests for the manufacture of derivatives . Likewise, the availability of safe and effective PI processes will not eliminate the need for screening of cellular blood components for the most relevant transfusion transmitted diseases using serological and molecular tests. Essentially, many of the current testing strategies are here to stay, at least for the foreseeable future.
The selection of appropriate assays and the algorithms used for screening receive careful attention from those involved in the collection and processing of blood for transfusion into recipients. Among the factors considered for the selection are sensitivity, specificity, positive and negative predictive values. Also, critical are the epidemiology and the geographical distribution of infectious diseases of interest.
Decisions about implementation of screening assays for TTIDs of concern in the geographical area where blood is collected are usually made by policymakers and regulatory authorities, based on the prevalence and incidence of disease. Under certain circumstances, these decisions have been made for political reasons in the absence of sufficient information showing the expected contribution of the assay in terms of blood safety. This was the case with the implementation of screening for HIV-1 p24 antigen in the United States in the mid-90s. No individual positive for the antigen and negative for antibodies to HIV-1 was found in the screening of over 500 000 donors . Despite this result, the US Food and Drug Administration recommended test implementation to show to the public that everything possible was being done to ensure the safety of the blood supply. The test produced no significant yield and was abandoned after implementation of NAT for HIV.
TTIDs like HIV, HBV, HCV and Syphilis are worldwide concerns. Other agents like Trypanosoma cruzi, Babesia spp., HTLV-I, Q Fever, affect donors in more restricted geographical areas and donor screening, when appropriate, is applied locally frequently under a ‘selective’ screening protocol, i.e. only donors at risk are screened. Examples are T. cruzi for prospective donors born in endemic areas (Spain) or on the first time they donate after test availability (US), one time screening for HTLV-I/II in certain EU countries and screening of individuals coming from areas affected by Q Fever in The Netherlands.
Factors affecting the choice of serological and molecular (Nucleic Acid Amplification tests, NAT) assays
Different technologies address different issues. Serological assays for antibodies to HIV, HBV and HCV detect antibodies generated in response to infection by the agent. They do not depend on the presence of the agent, and ‘signal amplification’ occurs in the body of the prospective donor as a result of the immune response. Serological assays for antibodies recognize the immunological scar resulting from infection and are not necessarily associated with infectivity. Serological assays for antigens recognize antigenic products of the infectious agent; screening for HBsAg, the surface antigen of HBV is widely utilized for donor screening because the protein is produced in large amounts by infected individuals and is highly correlated with the ability of infected individuals to transmit the virus.
Nucleic acid amplification tests (NAT), or molecular assays, detect nucleic acid sequences (RNA or DNA) of the infectious agent through a process of amplification that takes place as part of the assay. NAT are extremely sensitive and specific and are able to detect very small number of copies of the sequence. The major limitation of NAT is the chance of encountering the sequence of the agent in question in the small volume of specimen usually used for testing (Poisson distribution).
In terms of assay selection, serological assays for antibodies are effective for identification of prevalent infections, i.e. infections that are present or have occurred in a given population of prospective blood donors. While there is correlation between prevalence and transfusion transmission, not all individuals positive on a serological test are able to transmit infection. Molecular assays, on the other hand, are in general more sensitive and are more effective for the detection of recent infections in a population, or incident cases. Essentially, the yield of individuals who are positive on NAT and negative on the corresponding serological assay is higher among populations in which an infection is having a high rate of spread. Serological screening for HIV is highly effective for the detection of infected individuals in populations with high prevalence of infection. In these situations, the NAT yield maybe rather small when compared with that of serological assays. However, in situations where the infection is spreading rapidly, even the less sensitive serological assay for HIV-1 p24 Ag can contribute significantly to blood safety . Similarly, NAT can also be quite effective  because it becomes positive earlier than serological tests.
While serological assays are extremely useful tools for prevention of transmission of infections like HIV, HBV and HCV by transfusion, they are not useful for arboviruses like West Nile Virus because most transmissions occur in the early stages of infection, prior to the appearance of antibodies. Viremia declines and gradually disappears as antibody titres increase. This would also be true for other arboviral infections like dengue, yellow fever or Chikungunya.
Testing strategies, to NAT or not to NAT
Ideally, a combination of serological tests and NAT brings the highest degree of safety to blood products for transfusion. Unfortunately, NAT implementation is not feasible in every environment. Despite public demand for the unattainable ‘zero risk’, investments in transfusion safety need to be balanced with available resources and other healthcare priorities. Thus, choices need to be made. Many areas of the world have attempted to introduce NAT for HIV, HCV and also for HBV and have not yet succeeded because of a series of obstacles that include costs, assay complexity (the technology is very different from ELISA), the requirement for collection of separate specimens and the issues of contamination of negative specimens with amplified products from positive specimens (resolved in most commercial assays). Homebrew assays have been developed and validated by the plasma fractionation industry in the nineties and some have been licensed by North-American and European regulatory bodies. However, the issues of high development and implementation costs and patent royalties remain as obstacles.
It should be noted that improvements in technology have brought sensitivity of newer generation serological assays for antibodies and combined assays for both antigens and for antibodies to HIV, HBV and HCV closer to that of NAT. In some instances, like the detection of parasitic infections, serological tests are more effective than NAT because the few parasites present in a unit of component may be sufficient to transmit infection but, as mentioned before, may not be present in the small specimen obtained for testing.
Multiple serological tests used for the screening of donors for the same agent
This has been another approach to increase effectiveness of serological screening. For instance, some countries in Latin America have required the use of two different assays for HIV or for antibodies to T. cruzi to increase the sensitivity of donor screening tests. The US has used in the past a second FDA licensed serological assay before notifying donors that they are infected with HTLV-I/II . Unfortunately, one of the tests is not anymore manufactured. There is hope that a new assay will be submitted for licensure by another manufacturer in the near future.
Another example is the use of an assay for antibodies to the core antigen of HBV (HBcAb) in addition to the serological assay for HbsAg. This is the practice in North America, an area with relatively low prevalence for HBV, but is not common practice in European countries or in Asia. HBcAb was initially adopted in the 80s in the US as a correlate of HIV infection and non-A, non-B hepatitis. The assay remains in use until today, now to reduce transmission of HBV. Several European countries adopted NAT for HBV instead of HBcAb, depending on availability and resources. The use of HBcAb is problematic in high prevalence areas because a large segment of the population has been exposed to HBV and is positive in the assay, with great impact in blood availability. Often, resources for the implementation of NAT are not available in these areas; thus, more frequently screening relies exclusively on the high sensitivity of serological assays for HbsAg. The more recent introduction of sensitive, multiplex NAT assays for HIV, HCV and HBV became an additional tool for the prevention of transmission of HBV and in a certain way diminished the contribution of HBcAb to blood safety. Again, feasibility of its use in resource limited environments is questionable.
Future technologies, microarray chips, nanotechnology
There has been great progress in the development of assays based on new technologies, including microarray chips and nanotechnology, with high sensitivity and specificity that allow simultaneous screening for multiple agents in a cost effective manner. As an example, nanotechnology-based assays operate without the need for enzymes and have multiplexing capabilities with high degree of sensitivity and specificity. One specific example is an assay that uses nanoparticles (NP) such as the NP-based biobarcode amplification (BCA) assay and can detect HIV-1 p24 antigen with a sensitivity 150-fold higher than that of conventional ELISA .
Hopefully, these and many other new technologies, including those that allow detection of multiple agents simultaneously at a lower cost , will become available for blood donor screening and together with PI will make transfusion of blood and blood products an extremely safe procedure in the entire world.