Improving blood donor screening by nucleic acid technology (NAT)

Improving blood donor screening by nucleic acid technology(NAT)M. Schmidt & E. SeifriedInstitute of Transfusion Medicine and Immunohematology, German Red Cross, Johann Wolfgang Goethe University, Frankfurt, Germany

The description of the ABO blood group system by Landsteiner and coworkersmarked a sea change in making blood transfusions feasible and safe for a broadrange of indications. Nevertheless, with an increase in blood transfusions, side-effects such as transfusion-transmitted infections (TTIs) became more and moreimportant. A major challenge in transfusion medicine was (and is) to developscreening assays with maximum analytical sensitivity and analytical specificity toreduce 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 ofother TTIs relied almost exclusively on antibody detection. These tests, however, areassociated with a relatively long diagnostic window period because they detect theresponse 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 tech-

nology. 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 coun-

tries, NAT screening for HCV-RNA and HIV-1-RNA is per-

formed.

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 muta-

tions 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. Gen-

eric bacterial detection can also be carried out by NAT, but

there are a few drawbacks.

Serological combination assays may be economic alter-

natives to NAT but are associated with a longer diagnostic

window period compared to NAT systems. Pathogen-inacti-

vation methods are feasible for platelets and plasma prod-

ucts, but general inactivation methods for all three blood

products are still eagerly awaited.

Correspondence: Michael Schmidt, MD, Institute of Transfusion Medicineand Immunohematology, Johann Wolfgang Goethe University, GermanRed Cross, Sandhofstr. 1, 60528 Frankfurt am Main, GermanyE-mail: [email protected]

ISBT Science Series (2010) 5, 219–229

STATE OF THE ART 5C-S36-02 ª 2010 The Authors.Journal compilation ª 2010 International Society of Blood Transfusion

219

The description of the ABO blood group systems by Land-

steiner 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, concern-

ing 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-threaten-

ing 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 quaran-

tined plasma and the development of new screening sys-

tems.

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 ampli-

fication cycles. A final concentration of approximately 1

billion genome replicates can easily be detected by agarose

gel electrophoresis followed by staining with ethidium bro-

mide. The disadvantage of first PCR version was the neces-

sity 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 nucleo-

tides) 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 tran-

scription 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)

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 syn-

thesised RNA amplification products re-enter the TMA pro-

cess 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 tech-

nology 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

Real-time PCR technologies can be classified into systems

using intercalation dyes (e.g. SYBR green or ethidium bro-

mide), 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 Taq-

Man� 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 spe-

cific temperature at which probes bind to templates) can

also be performed. Another real-time NAT system uses spe-

cific 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 temper-

ature 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

220 M. Schmidt & E. Seifried

� 2010 The Authors.Journal compilation � 2010 International Society of Blood Transfusion, ISBT Science Series (2010) 5, 219–229

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 chal-

lenge. 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) sys-

tem 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 chao-

tropic salts [26]. At the beginning of blood donor

screening by NAT, no commercial systems were avail-

able. 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-con-

trolled NAT systems are on the market. Table 1 presents

the analytical sensitivity for the three systems given by

the manufacturers.

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 diagnos-

tic 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 mag-

netic 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 Christ-

mas-time) that might reduce the efficiency of the centrifu-

gation 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 approxi-

mately 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

Table 1 Analytical sensitivity of fully automated barcode-controlled

nucleic acid technology (NAT) systems for blood donor screening

Parameter

MPX Test ons201 platform1

Tigris Ultrio Plus2

Zelos x1003

HAV NA NA 0Æ8 IU ⁄ ml

HBV 3Æ7 IU ⁄ ml 2Æ1 IU ⁄ ml 0Æ6 IU ⁄ ml

HCV 10Æ7 IU ⁄ ml 3Æ1 IU ⁄ ml 9Æ6 IU ⁄ ml

HIV-1 49Æ0 IU ⁄ ml 27Æ6 IU ⁄ ml 8Æ9 IU ⁄ ml

HIV-2 2Æ2 copies ⁄ ml NA 1Æ3 copies/ml*

PB19 NA NA 9Æ7 IU ⁄ ml

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 Emmery-

ville, 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.

Blood donor screening by NAT 221


maximum pool size of 96. Blood donor screening by NAT

for at least HIV-1 and HCV has been implemented in differ-

ent countries (e. g. USA, Canada, parts of Brazil, Spain,

France, the UK, Denmark, Germany, the Netherlands, Bel-

gium, Greece, Slovenia, the Czech Republic, South Africa,

Ghana, Luxembourg, Switzerland, Italy, Japan, parts of

China, Australia, Poland, Norway, Finnland and New Zeal-

and). 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 tes-

ted 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 aregenerally tested

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 diag-

nostic window period is approximately 8–9 days by screen-

ing 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-trans-

mitted HIV-1 infection was reported (see below risk analy-

sis 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 anti-

body screening assays were available in 1990 (EIA first

generation). The diagnostic window period was approxi-

mately 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 win-

dow 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 screen-

ing by NAT, the residual transfusion-transmitted infectious

risk was estimated to be 1:200 [29], and it is currently cal-

culated at 1:10Æ88 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 2Æ56 days [32]. The virus can be integrated

into the genome of hepatocytes, which are the primary tar-

get cells. Approximately 90% of infected patients suffer

from acute infection. The immune system is able to elimi-

nate the virus from plasma in 90% of cases. In approxi-

mately 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 repre-

sents 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. Screen-

ing 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. Unfor-

tunately, most vaccines induce HBV-neutralizing antibod-

ies 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 aretested for only in some countries with specialcircumstances (WNV, HAV, HEV, B19V,Chikungunya virus and HIV-2)

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 infec-

tions 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 path-

way, 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 pre-

vent 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 con-

centrations (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 apopto-

sis. 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 retrospec-

tive 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 height-

ened 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 consid-

erable 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 rela-

tively small outbreak in Italy [48] resulted in a considerable

adverse impact on blood collections and economic conse-

quences. 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 mas-

sive 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 immu-

nodeficiency 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, 33Æ4million people (range 31Æ1–35Æ8 million) were living with

AIDS, 2Æ7 million were newly infected in 2008 (range 2Æ4–

3Æ0 million) and 2Æ0 million people died in 2008 as a conse-

quence of AIDS (range 1Æ7–2Æ4 million). The residual risk

from blood donation for HIV-1 is very low, especially in

countries where blood donor screening by NAT is imple-

mented. Although the infectious risk for HIV-2 outside the



middle of Africa is very low, two fully automated NAT sys-

tems (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 transfusionrelevant but that are currently not indicatedas special risks for blood transfusions and arenot implemented into blood donor screeningprogrammes (SARS CoV, Influenza viruses andH1N1)

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 epi-

demic, 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 exam-

ple of the powerful opportunities provided by NAT systems.

After sequencing a genome, a specific real-time NAT sys-

tem 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 approx-

imately 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 sit-

uation, the first effort was aimed at developing vaccines

against this new influenza infection, but later on, the sec-

ond or third activities were aimed at developing diagnostic

NAT systems for blood and sputum [53,54].

Bacterial screening by NAT

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 trans-

mission of bacterial infections. Therefore, bacterial contam-

ination of blood products represents an ongoing challenge

in transfusion medicine. Blood donor screening for bacte-

rial contamination is difficult because of the very low bac-

terial 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 plate-

lets. As a result of the long incubation time, platelets were

released as ‘negative-to-date’. Based on the very low bacte-

rial concentration in the platelet products after production,

sample failures were reported in different countries with

negative screening results and severe TTIs owing to bacte-

rial 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 2Æ5 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 Nadkar-

ni 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. Moham-

madi 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-meth-

oxypsoralen treatment, DNase treatment or combinations

of these methods [62–66]. However, most of these meth-

ods 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 bac-

terial contamination in blood products. The extraction

method can be completely automated [67,68] and bar-

code-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, plate-

let 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 sensi-

tivity of culture methods. The total screening time for

NAT systems (extraction and amplification) takes

approximately 4 h. Therefore, these methods offer oppor-

tunities 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).

Special NAT risks and NAT failures

Blood safety was significantly improved by the introduc-

tion of NAT systems through the reduction of the diagnos-

tic 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

The introduction of NAT systems into blood donor screen-

ing 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 2Æ56 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 rea-

sonable time. On the other hand, ID-NAT increases the total

costs for public health systems. In this context, each coun-

try 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

All transfusion-transmission-relevant viruses can be subdi-

vided 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 proofread-

ing 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 Ger-

many 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

Days0 1 2 3 4 5

Release of plateletsproducts form day 1

to day 3 withoutany bacterial tests

Blo

od d

onat

ion

(don

or s

elec

tion,

sta

bdar

d sk

in d

esin

fect

ion,

pre-

dona

tion

sam

oplin

g)

Pro

duct

ion

of p

late

lets

(sel

f-st

erili

zing

effe

cts)

Rapid bacterial test on day 4

Self life forplatelets withneg. testresults for5 days

Shelf life afterpathogen inactivation for 5 days

Pat

ho

gen

in

acti

vati

on (a)

(b)

Fig. 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 bac-

terial 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.



region. The manufacturers are aware of the methodological

risk by real-time NAT and have developed screening sys-

tems with the parallel amplification of at least two con-

served regions.

NAT alternatives

Within the last 5 years, new combination assays for the

parallel detection of antigens and antibodies were devel-

oped for HCV and HIV, respectively. Barbara et al. com-

pared 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 imple-

mented, 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 differ-

ent methods have been developed for platelet products

and for plasma products with different pathomecha-

nisms. 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, patho-

gen-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 concentra-

tions up to 1014 IU ⁄ ml. Pathogens such as Bacillus cereus

could be other exceptions, as they can occur in both veg-

etative and spore states. Spores are extremely resistant

against environmental conditions. Unfortunately, patho-

gen-inactivation reagents penetrate into spores less effi-

ciently. 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 ery-

throcytes. 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 win-

dow period to only a few days. Therefore, the residual

transfusion transmission risk is very low. The implementa-

tion 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 compo-

nents in the near future.

Disclosure

No potential conflicts declared.

References

1 Roth WK, Weber M, Seifried E: Feasibility and efficacy of rou-

tine PCR screening of blood donations for hepatitis C virus,

hepatitis B virus, and HIV-1 in a blood-bank setting. Lancet

1999; 353:359–363

2 Landsteiner K: Individual differences in human blood. Science

1931; 73:403–409

3 Rosahl SK, Samii M: ‘‘Nihil nocere’’ and beyond: the issue of

leadership in healthcare. Healthc Pap 2003; 4:78–82; discus-

sion 8–90

4 Dodd R, Kurt Roth W, Ashford P, Dax EM, Vyas G: Transfusion

medicine and safety. Biologicals 2009; 37:62–70

5 Epstein JS: Alternative strategies in assuring blood safety: an

overview. Biologicals 2010; 38(1):31–35

6 Brennan MT, Hewitt PE, Moore C, Hall G, Barbara JA: Confiden-

tial unit exclusion: the North London Blood Transfusion Cen-

tre’s experience. Transfus Med 1995; 5:51–56

7 Mullis PE, Brickell PM: The use of the polymerase chain reac-

tion in prenatal diagnosis of growth hormone gene deletions.

Clin Endocrinol (Oxf) 1992; 37:89–95

8 Mullis KB: Target amplification for DNA analysis by the

polymerase chain reaction. Ann Biol Clin (Paris) 1990;

48:579–582

9 Fronhoffs S, Bruning T, Ortiz-Pallardo E, Brode P, Koch B,

Harth V, Sachinidis A, Bolt HM, Herberhold C, Vetter H, Ko Y:

Real-time PCR analysis of the N-acetyltransferase NAT1 allele



*3, *4, *10, *11, *14 and *17 polymorphism in squamous cell

cancer of head and neck. Carcinogenesis 2001; 22:1405–1412

10 Benjamin RJ: Nucleic acid testing: update and applications.

Semin Hematol 2001; 38:11–16

11 Allain JP: Genomic screening for blood-borne viruses in trans-

fusion settings. Clin Lab Haematol 2000; 22:1–10

12 Kashida H, Takatsu T, Sekiguchi K, Asanuma H: An efficient

fluorescence resonance energy transfer (FRET) between pyrene

and perylene assembled in a DNA duplex and its potential

for discriminating single-base changes. Chemistry 2010; 16(8):

2479–2486

13 Lassauniere R, Kresfelder T, Venter M: A novel multiplex real-

time RT-PCR assay with FRET hybridization probes for the

detection and quantitation of 13 respiratory viruses. J Virol

Methods 2010; 165(2):254–260

14 Olsen EV, Gibbins CS, Grayson JK: Real-time FRET PCR assay

for Salmonella enterica serotype detection in food. Mil Med

2009; 174:983–990

15 Buh Gasparic M, Tengs T, La Paz JL, Holst-Jensen A, Pla M,

Esteve T, Zel J, Gruden K: Comparison of nine different

real-time PCR chemistries for qualitative and quantitative

applications in GMO detection. Anal Bioanal Chem 2010;

396(6):2023–2029

16 Kusser W: Use of self-quenched, fluorogenic LUX primers

for gene expression profiling. Methods Mol Biol 2006;

335:115–33

17 Erali M, Wittwer CT: High resolution melting analysis for gene

scanning. Methods 2010; 50(4):250–261

18 Crews N, Wittwer CT, Montgomery J, Pryor R, Gale B: Spatial

DNA melting analysis for genotyping and variant scanning.

Anal Chem 2009; 81:2053–2058

19 Sandhya S, Chen W, Mulchandani A: Molecular beacons: a

real-time polymerase chain reaction assay for detecting Escher-

ichia coli from fresh produce and water. Anal Chim Acta 2008;

614:208–212

20 Gubala AJ, Proll DF: Molecular-beacon multiplex real-time

PCR assay for detection of Vibrio cholerae. Appl Environ

Microbiol 2006; 72:6424–6428

21 Stroup SE, Roy S, McHele J, Maro V, Ntabaguzi S, Siddique A,

Kang G, Guerrant RL, Kirkpatrick BD, Fayer R, Herbein J, Ward

H, Haque R, Houpt ER: Real-time PCR detection and speciation

of Cryptosporidium infection using Scorpion probes. J Med

Microbiol 2006; 55:1217–1222

22 Arya M, Shergill IS, Williamson M, Gommersall L, Arya N, Patel

HR: Basic principles of real-time quantitative PCR. Expert Rev

Mol Diagn 2005; 5:209–219

23 Mine H, Emura H, Miyamoto M, Tomono T, Minegishi K,

Murokawa H, Yamanaka R, Yoshikawa A, Nishioka K: High

throughput screening of 16 million serologically negative blood

donors for hepatitis B virus, hepatitis C virus and human immu-

nodeficiency virus type-1 by nucleic acid amplification testing

with specific and sensitive multiplex reagent in Japan. J Virol

Methods 2003; 112:145–151

24 Roth WK, Weber M, Buhr S, Drosten C, Weichert W, Sireis W,

Hedges D, Seifried E: Yield of HCV and HIV-1 NAT after screen-

ing of 3.6 million blood donations in central Europe. Transfu-

sion 2002; 42:862–868

25 Roth WK, Seifried E: The German experience with NAT. Trans-

fus Med 2002; 12:255–258

26 Roth WK, Buhr S, Drosten C, Seifried E: NAT and viral safety in

blood transfusion. Vox Sang 2000; 78(Suppl. 2):257–259

27 Roth WK, Weber M, Petersen D, Drosten C, Buhr S, Sireis W,

Weichert W, Hedges D, Seifried E: NAT for HBV and anti-HBc

testing increase blood safety. Transfusion 2002; 42:869–875

28 Schmidt M, Korn K, Nubling CM, Chudy M, Kress J, Horst HA,

Geusendam G, Hennig H, Sireis W, Rabenau HF, Doerr HW, Ber-

ger A, Hourfar MK, Gubbe K, Karl A, Fickenscher H, Tischer BK,

Babiel R, Seifried E, Gurtler L: First transmission of human

immunodeficiency virus Type 1 by a cellular blood product

after mandatory nucleic acid screening in Germany. Transfu-

sion 2009; 49:1836–1844

29 Kubanek B, Cardoso M, Gluck D, Koerner K: [Risk of infection

transmission by blood components]. Infusionsther Transfu-

sionsmed 1993; 20:54–59

30 Hourfar MK, Jork C, Schottstedt V, Weber-Schehl M, Brixner V,

Busch MP, Geusendam G, Gubbe K, Mahnhardt C, Mayr-Wohlf-

art U, Pichl L, Roth WK, Schmidt M, Seifried E, Wright DJ:

Experience of German Red Cross blood donor services with

nucleic acid testing: results of screening more than 30 million

blood donations for human immunodeficiency virus-1, hepati-

tis C virus, and hepatitis B virus. Transfusion 2008; 48:1558–

1566

31 Kretzschmar E, Chudy M, Nubling CM, Ross RS, Kruse F, Tro-

bisch H: First case of hepatitis C virus transmission by a red

blood cell concentrate after introduction of nucleic acid ampli-

fication technique screening in Germany: a comparative study

with various assays. Vox Sang 2007; 92:297–301

32 Biswas R, Tabor E, Hsia CC, Wright DJ, Laycock ME, Fiebig EW,

Peddada L, Smith R, Schreiber GB, Epstein JS, Nemo GJ, Busch

MP: Comparative sensitivity of HBV NATs and HBsAg assays

for detection of acute HBV infection. Transfusion 2003;

43:788–798

33 Busch MP, Wright DJ, Custer B, Tobler LH, Stramer SL, Klein-

man SH, Prince HE, Bianco C, Foster G, Petersen LR, Nemo G,

Glynn SA: West Nile virus infections projected from blood

donor screening data, United States, 2003. Emerg Infect Dis

2006; 12:395–402

34 Busch MP, Caglioti S, Robertson EF, McAuley JD, Tobler LH,

Kamel H, Linnen JM, Shyamala V, Tomasulo P, Kleinman SH:

Screening the blood supply for West Nile virus RNA by nucleic

acid amplification testing. N Engl J Med 2005; 353:460–467

35 Kantzanou MN, Moschidis ZM, Kremastinou G, Levidiotou S,

Karafoulidou A, Politis C, Marantidou O, Kavallierou L, Kape-

roni A, Veneti C, Hatzakis A: Searching for West Nile virus

(WNV) in Greece. Transfus Med 2010; 20(2):113–117

36 Pfleiderer C, Blumel J, Schmidt M, Roth WK, Houfar MK, Eckert

J, Chudy M, Menichetti E, Lechner S, Nubling CM: West Nile

virus and blood product safety in Germany. J Med Virol 2008;

80:557–563

37 Costa AN, Grossi P, Porta E, Venettoni S, Fehily D: Measures

taken to reduce the risk of West Nile virus transmission by

transplantation in Italy. Euro Surveill 2008; 13(42):19009

38 Nainan OV, Armstrong GL, Han XH, Williams I, Bell BP, Margo-

lis HS: Hepatitis a molecular epidemiology in the United States,



1996–1997: sources of infection and implications of vaccina-

tion policy. J Infect Dis 2005; 191:957–963

39 Henriques I, Monteiro F, Meireles E, Cruz A, Tavares G, Ferreira

M, Araujo F: Prevalence of Parvovirus B19 and Hepatitis A

virus in Portuguese blood donors. Transfus Apher Sci 2005;

33:305–309

40 Heitmann A, Laue T, Schottstedt V, Dotzauer A, Pichl L: Occur-

rence of hepatitis A virus genotype III in Germany requires the

adaptation of commercially available diagnostic test systems.

Transfusion 2005; 45:1097–1105

41 Lee DH, Prince AM: Automation of nucleic acid extraction for

NAT screening of individual blood units. Transfusion 2001;

41:483–487

42 Tamura A, Shimizu YK, Tanaka T, Kuroda K, Arakawa Y, Takah-

ashi K, Mishiro S, Shimizu K, Moriyama M: Persistent infection

of hepatitis E virus transmitted by blood transfusion in a patient

with T-cell lymphoma. Hepatol Res. 2007; 37:113–120

43 Boxall E, Herborn A, Kochethu G, Pratt G, Adams D, Ijaz S, Teo

CG: Transfusion-transmitted hepatitis E in a ‘nonhyperendemic’

country. Transfus Med 2006; 16:79–83

44 Matsubayashi K, Nagaoka Y, Sakata H, Sato S, Fukai K, Kato T,

Takahashi K, Mishiro S, Imai M, Takeda N, Ikeda H: Transfu-

sion-transmitted hepatitis E caused by apparently indigenous

hepatitis E virus strain in Hokkaido, Japan. Transfusion 2004;

44:934–940

45 Yee TT, Cohen BJ, Pasi KJ, Lee CA: Transmission of symptom-

atic parvovirus B19 infection by clotting factor concentrate. Br

J Haematol 1996; 93:457–459

46 Kleinman SH, Glynn SA, Lee TH, Tobler LH, Schlumpf KS, Todd

DS, Qiao H, Yu MY, Busch MP: A linked donor–recipient study

to evaluate parvovirus B19 transmission by blood component

transfusion. Blood 2009; 114:3677–3683

47 Brouard C, Bernillon P, Quatresous I, Pillonel J, Assal A, De

Valk H, Desenclos JC: Estimated risk of Chikungunya viremic

blood donation during an epidemic on Reunion Island in the

Indian Ocean, 2005 to 2007. Transfusion 2008; 48:1333–1341

48 Liumbruno GM, Calteri D, Petropulacos K, Mattivi A, Po C,

Macini P, Tomasini I, Zucchelli P, Silvestri AR, Sambri V,

Pupella S, Catalano L, Piccinini V, Calizzani G, Grazzini G: The

Chikungunya epidemic in Italy and its repercussion on the

blood system. Blood Transfus 2008; 6:199–210

49 De Cock KM, Brun-Vezinet F: Epidemiology of HIV-2 infection.

AIDS 1989; 3(Suppl. 1):S89–S95

50 De Cock KM, Brun-Vezinet F, Soro B: HIV-1 and HIV-2

infections and AIDS in West Africa. AIDS 1991; 5(Suppl.

1):S21–S28

51 Schmidt M, Brixner V, Ruster B, Hourfar MK, Drosten C, Preiser

W, Seifried E, Roth WK: NAT screening of blood donors for

severe acute respiratory syndrome coronavirus can potentially

prevent transfusion associated transmissions. Transfusion

2004; 44:470–475

52 Hourfar MK, Roth WK, Seifried E, Schmidt M: Comparison of

two real-time quantitative assays for detection of severe acute

respiratory syndrome coronavirus. J Clin Microbiol 2004;

42:2094–2100

53 Hindiyeh M, Ram D, Mandelboim M, Meningher T, Hirsh S,

Robinov J, Levy V, Orzitzer S, Azar R, Grossman Z, Mendelson

E: Rapid Detection of Influenza A Pandemic (H1N1) 2009 Virus

Neuraminidase Resistance Mutation H275Y by Real-Time

RT-PCR. J Clin Microbiol 2010; 48(5):1884–1887

54 Gimeno C, Navarro D: Real-time reverse-transcription PCR in

the diagnosis of influenza A (H1N1)v in intensive care unit

adult patients. Crit Care 2009; 13:428; author reply

55 Busch MP, Glynn SA, Stramer SL, Strong DM, Caglioti S,

Wright DJ, Pappalardo B, Kleinman SH: A new strategy for esti-

mating risks of transfusion-transmitted viral infections based

on rates of detection of recently infected donors. Transfusion

2005; 45:254–264

56 Eder AF, Kennedy JM, Dy BA, Notari EP, Weiss JW, Fang CT,

Wagner S, Dodd RY, Benjamin RJ: Bacterial screening of apher-

esis platelets and the residual risk of septic transfusion reac-

tions: the American Red Cross experience (2004–2006).

Transfusion 2007; 47:1134–1142

57 Munksgaard L, Albjerg L, Lillevang ST, Gahrn-Hansen B,

Georgsen J: Detection of bacterial contamination of platelet

components: six years’ experience with the BacT ⁄ ALERT sys-

tem. Transfusion 2004; 44:1166–1173

58 te Boekhorst PA, Beckers EA, Vos MC, Vermeij H, van Rhenen

DJ: Clinical significance of bacteriologic screening in platelet

concentrates. Transfusion 2005; 45:514–519

59 Feng P, Keasler SP, Hill WE: Direct identification of Yersinia

enterocolitica in blood by polymerase chain reaction amplifica-

tion. Transfusion 1992; 32:850–854

60 Nadkarni MA, Martin FE, Jacques NA, Hunter N: Determination

of bacterial load by real-time PCR using a broad-range (univer-

sal) probe and primers set. Microbiology 2002; 148:257–266

61 Mohammadi T, Reesink HW, Vandenbroucke-Grauls CM, Save-

lkoul PH: Optimization of real-time PCR assay for rapid and

sensitive detection of eubacterial 16S ribosomal DNA in platelet

concentrates. J Clin Microbiol 2003; 41:4796–4798

62 Mohammadi T, Reesink HW, Vandenbroucke-Grauls CM, Save-

lkoul PH: Removal of contaminating DNA from commercial

nucleic acid extraction kit reagents. J Microbiol Methods 2005;

61:285–288

63 Dreier J, Stormer M, Kleesiek K: Two novel real-time reverse

transcriptase PCR assays for rapid detection of bacterial con-

tamination in platelet concentrates. J Clin Microbiol 2004;

42:4759–4764

64 Harris KA, Hartley JC: Development of broad-range 16S rDNA

PCR for use in the routine diagnostic clinical microbiology ser-

vice. J Med Microbiol 2003; 52:685–691

65 Schmidt M, Hourfar MK, Nicol SB, Wahl A, Heck J, Weis C,

Tonn T, Spengler HP, Montag T, Seifried E, Roth WK: A

comparison of three rapid bacterial detection methods under

simulated real-life conditions. Transfusion 2006; 46:1367–

1373

66 Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski

EB, Fox AJ: Contamination and sensitivity issues with a real-

time universal 16S rRNA PCR. J Clin Microbiol 2000; 38:1747–

1752

67 Hourfar MK, Schmidt M, Seifried E, Roth WK: Evaluation of an

automated high-volume extraction method for viral nucleic

acids in comparison to a manual procedure with preceding

enrichment. Vox Sang 2005; 89:71–76



68 Stormer M, Kleesiek K, Dreier J: High-volume extraction of

nucleic acids by magnetic bead technology for ultrasensitive

detection of bacteria in blood components. Clin Chem 2007;

53:104–110

69 Osselaer JC, Messe N, Hervig T, Bueno J, Castro E, Espinosa

A, Accorsi P, Junge K, Jacquet M, Flament J, Corash L: A

prospective observational cohort safety study of 5106 plate-

let transfusions with components prepared with photochemi-

cal pathogen inactivation treatment. Transfusion 2008;

48:1061–1071

70 Janetzko K, Cazenave JP, Kluter H, Kientz D, Michel M,

Beris P, Lioure B, Hastka J, Marblie S, Mayaudon V, Lin L,

Lin JS, Conlan MG, Flament J: Therapeutic efficacy and

safety of photochemically treated apheresis platelets pro-

cessed with an optimized integrated set. Transfusion 2005;

45:1443–1452

71 Janetzko K, Hinz K, Marschner S, Goodrich R, Kluter H: Patho-

gen reduction technology (Mirasol) treated single-donor plate-

lets resuspended in a mixture of autologous plasma and PAS.

Vox Sang 2009; 97:234–239

72 Goodrich RP, Edrich RA, Li J, Seghatchian J: The Mirasol PRT

system for pathogen reduction of platelets and plasma: an over-

view of current status and future trends. Transfus Apher Sci

2006; 35:5–17

73 AuBuchon JP, Herschel L, Roger J, Taylor H, Whitley P, Li J,

Edrich R, Goodrich RP: Efficacy of apheresis platelets treated

with riboflavin and ultraviolet light for pathogen reduction.

Transfusion 2005; 45:1335–1341

74 Mohr H, Steil L, Gravemann U, Thiele T, Hammer E, Greinacher

A, Muller TH, Volker U: A novel approach to pathogen reduc-

tion in platelet concentrates using short-wave ultraviolet light.

Transfusion 2009; 49:2612–2624



Documents

Improving blood donor screening by nucleic acid technology (NAT)