to increase blood safety , we introduced minipool nat screening in our blood donor service in 1997 for hepatitis b virus ( hbv ) , hepatitis c virus ( hcv ) , and hiv-1 and in 2000 for hepatitis a virus ( hav ) and parvovirus b19 ( 4 ) .
for these purposes , 100-l aliquots of up to 96 blood samples were pooled . the complete pool of up to 9.6 ml was centrifuged at 58,000 g for 60 min at 4c .
viruses were extracted by using spin columns , and nucleic acid was eluted in a total volume of 75 l . only 60 l of extract is needed for routine nat screening . a residual volume of 15 l can then be used for additional nat testing ( 5 ) for influenza viruses .
the real - time quantitative amplification of influenza / h5 was performed according to the manufacturer s instructions ( artus influenza / h5 lc rt - pcr kit , qiagen , hamburg , germany ) by using a thermocycler ( lightcycler ; roche applied science , mannheim , germany ) .
the specificity of this reaction was demonstrated for all subtypes of influenza a ( h1h15 , n1n9 ) and all subtypes of influenza b. samples with a positive test result in the first pcr were analyzed in a second pcr with influenza ( h5n1)specific primers and probes .
therefore , the assay allows differentiation between avian influenza ( h5n1 ) and other influenza virus strains . to mimic a situation like an h5-positive donation , a purified culture supernatant of vero cells infected with influenza ( h5n1 ) ( strain a / thailand/1 ( kan-1)/2004 ) ( 6 ) was used as an external quantification standard .
the viral rna concentration was determined in an external laboratory by multiple quantitative real - time pcr determinations ( 7 ) .
different dilutions of the external influenza ( h5n1 ) subtype quantification standard ( 0.0 , 0.91 , 1.96 , 3.91 , 7.81 , 15.63 , 31.25 , 62.5 , 125 , and 250 pfu / ml ) were prepared , and 100 l of each dilution was spiked into 9.5-ml negative plasma pools . each dilution was repeatedly spiked and tested in 8 minipools .
five microliters of the extract was analyzed with the generic influenza nat as well as with the specific influenza ( h5n1 ) nat .
probit analysis of these data yielded a detection probability of > 95% in parallel tests when an average of at least 13.4 pfu / ml ( 95% confidence interval [ ci ] 8.3184 pfu / ml ) and 7.4 pfu / ml ( 95% ci 5.214.7 pfu / ml ) for influenza generic assay and for the influenza ( h5n1)specific test , respectively , were present in individual plasma samples before pooling . *
influenza ( h5n1 ) standard was extracted from 9.6 ml of 96 pooled donor samples after centrifugation .
the 95% detection limit was 13.4 pfu / ml ; the 50% detection limit was 4.8 pfu / ml .
* influenza ( h5n1 ) standard was extracted from 9.6 ml of 96 pooled donor samples after centrifugation .
the 95% detection limit was 7.4 pfu / ml ; the 50% detection limit was 2.5 pfu / ml .
a total of 117 routine minipools , representing 10,272 blood donor samples , containing an average of 88  8 samples per pool , had previously been tested for hiv-1 , hbv , hcv , hav , and parvovirus b19 .
all pools were negative for influenza virus when tested with the generic influenza pcr and the influenza ( h5n1)specific pcr .
one pool had invalid results ( failed amplification of internal control rna , representing 0.01% of all analyzed runs ) .
( 8) , nat significantly increased the safety of blood products . at the german red cross ,
look - back examinations showed only 1 transfusion had transmitted hiv-1 ( 1998 ) after the introduction of nat testing .
blood donor screening by nat was made technically and financially feasible by creating minipools of up to 96 individual samples per pool .
roth et al . demonstrated an efficient enrichment for all tested viruses in plasma samples ( 9 ) . in the absence of an infective donor , different concentrations of the new influenza genotype h5n1 were spiked into minipools of 95 samples . as shown in table 2 , the influenza ( h5n1 ) subtype was detected by the generic influenza primers as well as by the influenza ( h5n1)specific primers when our routine minipool screening procedure was used .
sensitivity was expressed as pfu / ml and can be converted into viral genome copy number according the calculation of yoshikawa et al .
therefore , the analytical sensitivity was 804 geq / ml and 444 geq / ml for a generic influenza and for the influenza ( h5n1 ) subtype , respectively . after screening 10,272 samples by minipool nat
, none of the samples were found to be infected by influenza , which corresponds with the low eiss index ( european influenza surveillance scheme index ) of < 20 during the study period ( february  april , 2006 ) ( 10 ) .
an eiss index > 80 is expected during an influenza epidemic , as was seen in 2005 .
as with other viruses , a viremic phase of infection can be assumed to precede clinical symptoms such as fever ( 13,14 ) .
( 15 ) detected influenza ( h5n1 ) virus ( 3,080 copies / ml ) in the plasma of a 5-year - old boy , which indicates a viremic phase of influenza ( h5n1 ) infection .
in addition to quarantine of infected patients , treatment with antiviral drugs , and development of avian influenza vaccines , blood donors should be tested during a pandemic to avoid transfusion - transmitted infections .
our study demonstrates that nat screening could be incorporated into blood testing without delay and that the influenza virus could be sufficiently enriched by centrifugation .
sensitivity of our influenza - screening method would have been sufficient to detect recently reported virus concentrations in plasma of infected persons ( 15 ) .
however , as with all minipool methods , infections can be transmitted to transfusion recipients on rare occasions because the viremia level in the donor is below the analytical sensitivity of the screening assay . to reduce this risk , a selective infectious dose nat strategy ( e.g. , triggering of infectious dose nat testing when at least 1 viremic donation is collected per week with the standard minipool screening algorithm ) , as performed for west nile virus ( wnv ) screening in the united states
implementation of wnv - nat in the united states in 2003 interdicted well over 1,000 donations from persons infected with wnv and is a good example of successful implementation of nat screening for emerging viruses .
the collective fight against new viruses such as severe acute respiratory syndrome virus , wnv , or influenza ( h5n1 ) presents an immense challenge for the whole community , but new molecular - biologic methods offer opportunities to overcome this challenge .
nat screening tests are now available soon after the sequencing of new viruses . in the absence of a general pathogen inactivation method for all blood products ( erythrocytes , platelets , and plasma )
 influenza viruses , including highly pathogenic avian influenza virus ( h5n1 ) , could threaten blood safety . 
 we analyzed 10,272 blood donor samples with a minipool nucleic acid amplication technique . 
 analytical sensitivity of the method was 804 geq / ml and 444 geq / ml for generic influenza primers and influenza ( h5n1 ) subtype  specific primers . 
 this study demonstrates that such screening for influenza viruses is feasible . 
The Study
Conclusions