NAT in blood screening around the world

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LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. list and describe various tumor biomarkers;
2. recognize rates of higher risk blood donations of the total blood donations in the world;
3. recognize current testing procedures used for testing for infectious diseases in blood donations; and
4. recognize advantages and disadvantages of different testing methods in testing for infectious disease in blood donations.

Annually, millions of people worldwide receive blood transfusions or blood-derived products. Around the world, more than 92 million blood donations
are collected every year.¹
From these, a single whole-blood donation can be transfused in up to three people, while blood-derived products from a single donation may be given to hundreds of patients.^2,3
Although testing and policy decisions have combined to make blood supplies in many countries among the safest in the world, there still exists some risk of
transfusion-transmitted infection (TTI) with bloodborne diseases (e.g., HIV, hepatitis, or West Nile virus [WNV]). Laboratory screening of donated blood
and blood products for infectious diseases is a key safety measure in protecting patients and preventing the spread of serious diseases.

Compared to 30 years ago, TTI risk in countries worldwide has decreased substantially.^4,5
Active screening and hemovigilance programs are credited with having impacted the rate of TTIs. Nucleic-acid testing (NAT) has also played an important
role as the most advanced technology for directly detecting infections in blood. Today, NAT has been adopted in countries around the world, including
in the U.S., Canada, France, Australia, New Zealand, South Africa, and many countries in Europe and Asia. TTIs, however, are still a real concern in some parts of the world.
It has been estimated that, on an annual basis, unsafe blood transfusions around the world have been responsible for up to

• 16 million new hepatitis B viral (HBV) infections;
• 5 million new hepatitis C viral (HCV) infections; and
• 160,000 cases of HIV infections — in fact 5% to 10% of HIV infections worldwide are due to transfusions of contaminated blood or blood products.⁶

The World Health Organization states that “screening for TTIs to exclude blood donations at risk of transmitting infection from donors to recipients
is a critical part of the process of ensuring that transfusions are as safe as possible.” As NAT becomes a part of countries' blood-screening protocols, they will likely experience
a significant TTI reduction and help ensure that their blood supplies meet international safety standards.

The value of NAT

NAT detects the presence of viral infection by directly testing for viral nucleic acids and can be used to screen whole blood and plasma samples.
Commonly used NAT assays detect HIV-1 RNA, HCV RNA, HBV DNA, and WNV RNA.

Countries that have not yet adopted NAT may use the traditional method for screening blood donations, known as immunoassay (or serology) testing.
Immunoassays detect antibodies to viruses or viral antigens. With immunoassays, however, there is an interval between the donor's exposure to a virus until antibodies against the virus
are produced, known as the detection “window period.” It is during this period that the risk of infection in donated blood can be missed.

NAT shortens this window period, thereby offering blood centers a much higher sensitivity for detecting viral infections. For example, with serology tests,
it takes about two months after infection for anti-HCV antibodies to be detected, while NAT testing can detect HCV RNA in about five days after infection.⁷

Given the impact that NAT can have in lowering the risk of TTIs, many countries employ NAT testing as a complement to traditional serology testing,
performing both to fully optimize the safety of their blood supplies.

PCR and TMA

Commonly used NAT-screening technologies include polymerase chain reaction (PCR), and transcription-mediated amplification (TMA). PCR is a method
that replicates (or amplifies) single or small amounts of DNA segments by several orders of magnitude to detectable levels, producing thousands or millions of copies of specific DNA
sequences from the target sequences.⁸
TMA is a transcription-amplification process that uses two enzymes — reverse transcriptase and RNA polymerase — to produce hundreds
of millions of copies of the targeted RNA sequences. TMA consists of three main steps: target capture, amplification, and detection.⁹
Compared to other NAT assays, the
advantages of TMA are primarily shown in these ways: TMA allows for simultaneous testing of multiple viruses in a single test tube, thereby dramatically simplifying the assay process.
TMA is convenient, allowing laboratories to reduce the steps for blood screening and shortening processing time, thereby obtaining results faster.

With various screening platforms, fully integrated and automated current Good Manufacturing Practices environments can reduce the number of steps
in which materials are transferred, decreasing the potential for operator-induced errors and reducing the risk of contamination.

Screening platforms

Common NAT platforms — fully integrated and automated, semi-automated, and modular automated types — are used with assays to conduct
NAT screening in two ways: individual donor testing (IDT) and pooled testing. IDT is done on a sample from each unit of donated blood with no dilution of viral genetic
materials required before testing. IDT is the most sensitive method for NAT testing. The viral titers during window periods are often low, and IDT maintains a high level of sensitivity.
Pooled testing, in which samples from multiple donors are combined before testing, is preferred by many blood centers that need to process large volumes of blood.

Trends in application of NAT worldwide

Around the world, more than 53 million units of blood are screened with NAT. To increase blood safety, U.S. blood banks began using
NAT to supplement serology testing in the late 1990s, first for HIV and HCV, and later for HBV and WNV. Today, 100% of the U.S. blood supply is screened with NAT
for HIV-1, HCV, WNV, and HBV. In the U.S., TMA-based NAT technology is used to screen more than 80% of the blood supply.

NAT implementation in developing countries^1,6,10

Since the late 1990s, many other countries have adopted NAT testing on all or some portion of blood donations.
Despite this widespread adoption of NAT, approximately 40% of the 92 million donations of blood are still not tested with NAT technology.
Developing and low-resource countries represent the most recent group of countries to start to explore advanced blood screening with NAT.
Currently, countries such as Thailand, Malaysia, Indonesia, Egypt, and Mexico have been using NAT.¹¹

The risks and implications of TTIs in developing countries remain substantial.
For example, developing countries are more likely to use blood that is contaminated. This may be due to a higher disease prevalence, or
because there is inadequate serology-based screening. In addition, a significant portion of blood goes to treat younger patients, including infants and children,
victims of trauma, and mothers with blood loss due to childbirth. In comparison, in developed nations, blood typically goes toward treating older patients.

China and India are two countries with large populations where the adoption of NAT could have a significant impact on the rate of TTIs.

IndiaThe prevalence of HIV, HBV, and HCV in India is, respectively, about 0.9%, 4%, and 1%. Blood screening in India is performed by three groups:
regional governments, private hospitals and non-government organizations (NGOs). Private hospitals and NGOs have started to make strides in adopting NAT. Lions Delhi — operated by
Lions International, a global non-profit service organization — became the first NGO to adopt NAT technology in November 2010 to test between 35,000 and 50,000 donations each year.

Study data published in The Indian Journal of Medical Research underscored the value of NAT for Indian blood centers. In this multicenter study
in which 12,000 donations from eight Indian blood centers were screened in IDT, the results indicated six HBV, one HIV-1 yield, and one HIV-HCV co-infection.
The authors said, “Based on the results of this study, screening would be predicted annually to interdict 3,272 infectious donations,
including 818 HIV, 409 HCV, and 2,454 HBV infected donations.”¹²
Studies on the feasibility of NAT implementation in low-resource countries help extend the
message to blood centers that NAT can be an effective method for safeguarding the blood supply.

China:In China, the number of people affected by HIV has been estimated at between 430,000 and 1.5 million¹³
; and according to
China's Ministry of Health, more than 38 million people are infected with HCV. There are approximately 130 million carriers of HBV and 30 million chronically infected
with HBV in China.^14,15
In 2010, the Ministry of Health commenced a NAT pilot program in 15 blood centers in 12 provinces across the country.
Through the pilot study, the country is evaluating NAT technology as a method for safeguarding the country's 10 million blood donations that centers receive annually.

The future of global NAT adoption

NAT technology has revolutionized the ability of blood centers to efficiently test for and intercept potentially infectious pathogens
while continuing to ensure on-time blood availability for patients and hospitals. The global trend towards adopting this technology helps underscore its effectiveness
for increasing the safety of blood supplies. As reducing the rate of transfusion-transmitted disease gains speed in more countries, NAT can serve as a valuable addition to
existing safety efforts.

Frank Strobl, MD, PhD,

a board-certified clinical pathologist and transfusion-medicine specialist, is global head of Scientific Affairs at Novartis Diagnostics.
In December 2009, he joined Novartis Vaccines & Diagnostics, which collaborates with Gen-Probe to offer the PROCLEIX ULTRIO assays, and the PROCLEIX TIGRIS system.

Note: The study published in The Indian Journal of Medical Research mentioned in Dr. Stobl's article used his company's ULTRIO screening product.

References

1. Global Blood Safety and Availability: Facts and Figures from the 2007 Blood Safety Survey. WHO Fact Sheet #279, November 2009.
2. Piotrowicz-Theizen D, Schoeffter C. An example of traceability up to the patient. STP Pharma Pratiques. 2004;14(5):476-481.
3. Yu MW, Mason BL, Guo ZP, et al. Hepatitis-C Transmission Associated with Intravenous Immunoglobulins. Lancet. 1995;345:1173-1174.
4. Bihl F, et al. Transfusion Transmitted Infections. J Transl Med. 2007;5:25.
5. WHO Screening Donated Blood from Transfusion Transmissible Infections: Recommendations. World Health Organization. 2010.
6. Editorial. Improving Blood Safety Worldwide. Lancet. 2007:370(9603):1879-1974.
7. Busch MP, et al. A new safety strategy for estimating risks of transfusion-transmitted viral infections based on rates of detection of recently infected donors. Transfusion. 2005;45:254-264.
8. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239(1):488-491.
9. Giachetti C, Linnen JM, Kolk DP, et al. Highly sensitive multiplex assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA. J Clin Microbiol. 2002;40(7):2408-2419.
10. Transfusion Transmitted Infections: How Many More? Asian J Transfus Sci. 2010;4(2);71-72.
11. Blood Transfusion Should Be Safe and Not Transmit HIV or Hepatitis B, C infections. Patients for Patient Safety News. April 2008, No. 11.
12. Makroo RN, Choudhury N, Jagannathan L, Parihar-Malhotra M, et al. B & C viruses in Indian Blood Donors. Indian J Med Res. 2008;127(2):140-147.
    
13. Steinbrook R. The AIDS Epidemic in 2004. NEJM. 2004;351(2):115-117.
14. Custer B, Sullivan SD, Hazlet TK, Iloeje U, et al. Global epidemiology of hepatitis B virus. J Clin Gastroenterol. 2004;38(10) Suppl 3):S158-S168.
15. Liu J, Fan D. Hepatitis B in China. Lancet. 2007;369:1582-1583.