sensitivity of hcv rna and hiv rna blood screening assays

T R A N S F U S I O N C O M P L I C A T I O N S

Sensitivity of HCV RNA and HIV RNA blood screening assays

P. Nico Lelie, Harry A.J. van Drimmelen, H. Theo M. Cuypers, Susan J. Best, Susan L. Stramer,

Catherine Hyland, Jean-Pierre Allain, Pierre Moncharmont, Christine Defer, Micha Nubling,

Andreas Glauser, Marcia da Silva Cardoso, Jean-Francois Viret, Mervi H. Lankinen, Lena Grillner,

Urs Wirthmuller, Joliette Coste, Volkmar Schottstedt, Barbara Masecar, and Elizabeth M. Dax

BACKGROUND: The FDA requirement for sensitivityof viral NAT methods used in blood screening is a 95-percent detection limit of 100 copies per mL, whereas theNAT screening system should have a sensitivity of at least5000 copies per mL per individual donation. According tothe Common Technical Specifications of the EuropeanDirective 98/79/EC for in vitro diagnostics, viral standarddilutions (calibrated against the WHO standard) should betested at least 24 times for a statistically valid assessmentof the 95-percent detection limit.STUDY DESIGN AND METHODS: Viral standard dilutionpanels (PeliCheck, VQC-CLB) were prepared for HCVRNA genotypes 1 and 3 and for HIV RNA genotypes Band E. In a multicenter study, 23 laboratories tested thepanels all together in 8 to 91 test runs per NAT method.RESULTS: The following 95-percent detection limits (and95% CIs) were found on the HCV RNA genotype 1 refer-ence panels (shown as geq/mL): Gen-Probe TMA, 85(64-118); AmpliScreen, 126 (83-225); AmpliScreen withNucliSens Extractor, 21 (13-44); Amplicor with NucliSensExtractor, 69 (50-102), and Amplicor with Qiagen extrac-tion technology, 144 (74-102). On HIV RNA genotype B dilu-tion panels, the following 95-percent detection limits werefound (shown as geq/mL): Gen-Probe TMA, 31 (20-52); Am-pliScreen, 126 (67-311); AmpliScreen with NucliSens Extrac-tor, 37 (23-69), and NucliSens QL assay, 123 (51-566). HIVRNA genotype E panels were detected with equal sensitivityas HIV RNA genotype B panels. In the Gen-Probe TMA as-say, the 50-percent detection limits on HIV RNA type B andtype E were 3.6 (2.6-5.0) and 3.9 (2.4-5.8) geq per mL, re-spectively. The HCV RNA genotype 1 and 3 standards weredetected with equal sensitivity.CONCLUSION: The differences in sensitivity betweenNAT assays can be explained by the input of isolated viralnucleic acid in the amplification reactions. The FDA re-quirements for sensitivity of NAT blood screening assayscan be met by the Gen-probe TMA, as well as by theAmpliScreen assays, particularly when combined withthe NucliSens Extractor.

In the late 1990s, manufacturers of plasma productshave introduced minipool NAT (MP-NAT) to reducethe viral burden in the starting pools for fraction-ation. Since July 1, 1999, there is an official require-

ment of the European Medicine Evaluation Agency thatthe starting pools for fractionation need to test negativefor HCV RNA by using an HCV NAT assay with a sensi-tivity of at least 100 IU per mL. To avoid dual standardsfor the safety of plasma and cellular blood components,the Paul Ehrlich Institute required from April 1999 that alllabile components in Germany be tested for HCV RNAwith a sensitivity of at least 5000 IU per mL per individualdonation. For blood banks that also perform HIV RNAscreening with MP-NAT, the Paul Ehrlich Institute re-cently proposed a detection limit of 10,000 IU per mL perindividual donation. The FDA requires a 95-percent de-tection limit of at least 100 copies per mL for both HCV

ABBREVIATIONS: geq = genome equivalent; MP-NAT =

minipool NAT; MWP = microwell plate; S/CO = sample to cut-

off value.

From the VQC Laboratory, Sanquin-CLB Diagnostic Division,

Amsterdam, the Netherlands; National Serology Reference

Laboratory, Melbourne, Australia; American Red Cross, Gai-

thersburg, Maryland; Australian Red Cross Blood Service, Bris-

bane, Australia; University of Cambridge, Cambridge, United

Kingdom; EFS, Lyon, France; EFS, Lille, France; Paul-Ehrlich

Institut, Langen, Germany; MSD Pharma Services, Wangen-

Zurich, Switzerland; DRK-Blood Transfusion Service of Baden-

Wurttemberg, Baden-Wurttemberg, Germany; Swiss Serum

and Vaccine Institute, Bern, Switzerland; Finnisch Red Cross,

Helsinki, Finland; Department of Clinical Microbiology, Karo-

linska Institut, Stockholm, Sweden; ZLB, Bern, Switzerland;

EFS, Montpellier, France; DRK-Blutspendedienst, Hagen, Ger-

many; Bayer Corporation, Clayton

Address reprint requests to: P. Nico Lelie, PhD, Viral Qual-

ity Control Laboratory, CLB-Sanquin, Jan Steenstraat 1, 1816

CT Alkmaar, the Netherlands.

Received for publication May 21, 2001; revision received

November 30, 2001, and accepted December 24, 2001.

TRANSFUSION 2002;42:527-536.

Volume 42, May 2002 TRANSFUSION 527

and HIV NAT blood screening assays and a sensitivity ofat least 5000 copies per mL per donation when MP-NATis performed. Anticipating these developments, the bloodtransfusion organizations have introduced or are in theprocess of implementing MP-NAT of blood donations forthe presence of HCV RNA, HIV RNA, and HBV DNA.1-5

With the introduction of NAT, there was an urgent needfor standardization to allow interlaboratory comparisonof NAT assays. In the early 1990s, it was demonstratedthat there was considerable variation between laborato-ries in the quality of performance of NAT assays.6,7 In1995, the WHO International Laboratories for BiologicalStandards (National Institute for Biological Standardsand Control, London, UK, and The Central Laboratory ofThe Blood Transfusion Service, Amsterdam, the Nether-lands) started annual expert meetings for standardizationof NAT assays. Since then, NIBSC, CLB, and CBER (FDA)have established International Standards for HCV RNA,HBV DNA, HIV RNA, and parvovirus B19 DNA.8-11 Theseinternational standards are available in limited supplyand are intended for use as primary reference standardsfor calibration of secondary standards. Saldanha et al.12

have calibrated a number of HCV RNA quality controlreagents against the international standard in a collabo-rative study. For example, the PeliSpy HCV RNA 3800 geqper mL run control (VQC, CLB, Amsterdam, the Nether-lands) and a CBER #1 reference sample with 1000 copiesper mL were found to contain 1000 and 250 IU per mL,respectively. Thus, for the VQC and CBER HCV RNA ge-notype 1 standards, the conversion factor was 1 IU toapproximately four copies. Preliminary calibration resultsof the VQC HIV RNA genotype B standard compared withthe WHO standard10 shows that 1 IU of HIV RNA isequivalent to approximately 1 geq. However, differentconversion factors from IU to geq were found for differ-ent NAT assays. Even when an attempt has been made tocalibrate a working standard in IU, one cannot simplypredict the NAT reactivity of dilutions of the reagent.Therefore, we have examined the reactivity of the VQCstandard dilution panels in different NAT assays used byblood screening laboratories all over the world. In thisreport, the sensitivity of widely used NAT blood screeningassays is compared for HCV RNA genotype 1 and geno-type 3 standards, as well as for HIV RNA genotype B andgenotype E standards.

MATERIALS AND METHODSHCV RNA and HIV RNA standardsThe characteristics of the HCV RNA and HIV RNA stan-dards are described in the package inserts of referencepanels (PeliCheck, VQC-CLB). The standard dilution pan-els were prepared from the following standards.

VQC HCV RNA genotype 1 standard. The VQC HCVRNA genotype 1 standard is prepared from a plasma poolof subtype 1a and 1b units. Genotype analysis showed a

pattern mainly indicative of subtype 1b. The concentra-tion in the VQC HCV RNA genotype 1 standard was de-termined by calibration against the Eurohep HCV RNAgenotype 1 standard in repeated branched DNA assays(Quantiplex 2.0, Chiron, Emeryville, CA). The concentra-tion of 8.76 2 106 geq per mL in the VQC standard com-pared with 3.79 2 106 geq per mL in the Eurohep stan-dard was confirmed by limiting dilution data of the VQCproficiency study in 1997.13

Eurohep HCV RNA genotype 3 standard. The Euro-hep genotype 3 HCV RNA standard is derived from asingle FFP unit. Recently, the Eurohep HCV RNA geno-type 3 standard dilutions were recalibrated against theVQC HCV RNA genotype 1 dilutions by a series of tests inan HCV branched DNA assay (Versant 3.0, Bayer Diag-nostics, Emeryville, CA). From these results, it could beestablished that the Eurohep HCV RNA genotype 3 stan-dard contains a concentration of 270,000 geq per mL.This result was confirmed by quantitative results on thegenotype 3 and genotype 1 standards obtained by labo-ratories testing the VQC proficiency panels in 1999.

VQC HIV RNA genotype B standard. The VQC HIVRNA genotype B standard is prepared from a field isolate,propagated on MT2 cells. The culture supernatant wasdiluted in a pool of clarified citrate plasma. After repeatedtesting of standard dilutions in HIV assays (AmplicorMonitor 1.0 [Roche Molecular Systems, Pleasanton, CA],NucliSens assay [bioMerieux, Marcy-l’Etoile, France],and Quantiplex 2.0 branched DNA assay[Chiron]), a con-centration of 2.5 2 108 geq per mL was assigned to theundiluted VQC HIV RNA genotype B standard.

VQC HIV RNA genotype E standard. The VQC HIVRNA genotype E standard has been prepared as describedpreviously here for the genotype B standard. For calibra-tion of the VQC HIV RNA genotype E standard against theVQC genotype B standard, we assessed the results of NATassays based on mixed gag primers or pol primers ondilutions of the genotype B and genotype E standards inthe VQC proficiency panel of 1997. Therefore, we wereable to establish a concentration of 7.5 2 108 geq per mLin the undiluted VQC genotype E standard.

Reference panelsThe viral standards described previously here were usedto prepare half-log dilution panels with concentrationsvarying between 25,000 geq per mL and 0.1 geq per mL.For HCV RNA, standard dilutions were included aroundconcentrations of 100 and 380 geq per mL (100 IU/mL),the NAT detection limits required by the regulatory bod-ies. The homogeneous dilutions were divided into bio-freeze vials, which were snap frozen in liquid nitrogenand stored at –707C. For preparation of the reference pan-els for this study, the viral dilutions were aliquoted involumes of either 1.1 or 4.3 mL. Before release of thepanels, each lot was tested in the standard qualitative and

LELIE ET AL.

528 TRANSFUSION Volume 42, May 2002

quantitative CLB NAT procedures. Each dilution in thepanels was divided into four vials to allow testing in mul-tiple test runs. The preparation of dilutions is traceableby gravimetric records. The instructions for specimenhandling and the data for stability of the referencesamples are described in the package insert. The viralstandard dilutions have been shown to be stable for atleast 4 years when stored frozen at –307C and for 8 hourswhen stored in liquid phase at 47C. The reference panelswere distributed internationally, and 23 NAT laboratoriesreturned their test results for statistical analysis. Theprincipal investigators in those blood centers and refer-ence laboratories are the coauthors of this report.

NAT blood screening assaysEach laboratory tested the reference panels (PeliCheck)at least four times, and some laboratories tested the pan-els in 24 individual test runs by using one of the methodslisted later here.

HIV-1/HCV TMA system. This semiautomated sys-tem from Gen-Probe (San Diego, CA) combines samplepreparation, amplification, and detection in a one-tubeassay so that a transfer of amplicons is not necessary. Theviral RNA is lyzed from 0.5 mL of plasma and is specifi-cally captured by oligomers on magnetic particles. In themultiplex assay potentially present, wild-type HIV and/orHCV RNA and an internal HIV-1-based control are bothamplified by isothermal transcription-mediated amplifi-cation (TMA). In the detection step, a multiplex probereagent can discriminate between wild-type RNA signaland internal control signal, which are recorded by a lu-minometer. Gen-Probe TMA assays on the reference pan-els were performed in Australia, the United States,France, and the United Kingdom.

HCV AmpliScreen 2.0 and HIV AmpliScreen 1.5 as-says. Viral particles are pelleted from 1-mL plasmaminipools by ultracentrifugation for 1 hour at 47C. For thenucleic acid isolation, a separate kit (AmpliScreen Multi-prep Specimen Preparation, Roche) is used. The pellet isresuspended in 600 mL lysis buffer to which approxi-mately 80 copies of internal HCV and HIV control areadded. Nucleic acid is extracted by the standard organicisolation method by using alcohol precipitation (Roche).The pellet is dissolved in 200 mL of specimen diluent(Multiprep), of which 50 mL is used in the PCR amplifi-cation reaction. For both the HCV and HIV assay, a vol-ume of 50 mL of working master mix is added to 50 mL ofnucleic acid isolate for amplification and detection. Theassays (AmpliScreen 2.0 and 1.5, Roche) were usually per-formed on the Cobas platform (HCV Cobas AmpliScreen2.0 and HIV Cobas AmpliScreen 1.5), but one laboratoryused the microwell plate (MWP) assay version (HCVMWP AmpliScreen 2.0 and HIV MWP AmpliScreen 1.5).With the Multiprep extraction protocol, RNA isolatedfrom an equivalent of 250 mL of plasma and approxi-

mately 20 copies of the internal control (assuming 100%extraction efficiency) is amplified and detected in boththe HCV AmpliScreen 2.0 and HIV AmpliScreen 1.5 as-says. AmpliScreen assays on the reference panels wereperformed in the United States, Sweden, and the Neth-erlands.

Cobas AmpliScreen in combination with NucliSensExtractor. Two-mL aliquots of plasma pools are mixedwith 9 mL of lysis buffer using the NucliSens isolation kit(bioMerieux), to which 11 mL of the AmpliScreen HCVand HIV internal control (approx., 130 copies) has beenadded. Nucleic acid is released by incubating the samplelysis mixture for 30 minutes at 377C. Nucleic acid is thenabsorbed to 50 mL added silica suspension by using end-over-end inversion for 5 to 10 minutes. In 1999, themanufacturer recommended the use of the “decant”method when 2-mL volumes are lyzed. With this method,10 mL of the plasma-lysis buffer solution is removed afterpelleting the silica particles by centrifugation for 3 min-utes at 4000 2 g. The residual 1-mL content of the lysistubes is transferred to the plastic cartridges that areplaced in one of the 10 NucliSens Extractor stations. Inthe Extractor cartridge, the silica particles are subjectedto a number of washing cycles with buffer and organicsolution by an airpump mechanism, whereas the silicaparticles are captured by a membrane. The silica is driedat 567C, and RNA is eluted in a 60 to 70 mL aliquot byusing the high-elution volume (EluHigh) NucliSens Ex-tractor protocol. Of the 60- to 70-mL eluate, 25 mL is trans-ferred to the Cobas A rings to which 25 mL of Multiprepspecimen diluent is added for both the HCV and HIVAmpliScreen assays (EluHigh NucliSens-HCV Ampli-Screen 2.0 and EluHigh NucliSens-HIV AmpliScreen 1.5).A result is valid if the internal control (approx., 45 copies/PCR assuming 100 percent extraction efficiency) is reac-tive. In the NucliSens Cobas AmpliScreen assay, anequivalent of 735 mL of plasma (average) is amplified anddetected in both the HCV AmpliScreen 2.0 and HIVAmpliScreen 1.5 assays. The NucliSens-AmpliScreen re-sults were obtained at CLB in the Netherlands.

HCV Amplicor 2.0 assays in combination with Nu-cliSens Extractor. This method was first introduced inEurope 1998 by Cuypers et al.14 The procedure was simi-lar to the NucliSens AmpliScreen method described pre-viously here. However, at that time, 6 mL of the HCVinternal control (approx., 80 copies) was added to theplasma-lysis buffer solution, and the “decant” methodwas not yet applied. The whole 11 mL of plasma-lysisbuffer solution was subjected to the NucliSens Extractorprocedure, which generated 40- to 50-mL eluate volumes(current EluLow Extractor protocol). In the EluLow Nu-cliSens HCV Amplicor 2.0 assay, HCV RNA isolated froman equivalent of on average 1100 mL of plasma is ampli-fied and detected. The NucliSens Amplicor assays onPeliCheck panels were performed in the Netherlands,

SENSITIVITY OF HCV/HIV-NAT ASSAYS


Germany, Finland (Cobas Amplicor assays), and Switzer-land (MWP assays).

HCV Amplicor 2.0 assay in combination with Qia-gen nucleic acid isolation. Some laboratories used thespin columns (Qiagen, Hilden, Germany) or the Biorobot(Qiagen) for isolation of viral RNA and processed the elu-ate in the Amplicor 2.0 assay (Qiagen-Amplicor 2.0). Vol-umes of 140 to 200 mL of plasma were used for HCV RNAextraction, and 6 mL of the Amplicor internal control (ap-prox., 85 copies) was added to the lysis buffer. The pro-tocols for sample preparation slightly varied betweenlaboratories. The volume of plasma extract that was as-sayed in the Cobas Amplicor 2.0 varied from 100 to 167mL. The Amplicor results on the dilution panels obtainedwith the different Qiagen extraction protocols were com-bined. The Qiagen extraction method in combinationwith Amplicor was used for testing HCV RNA referencepanels in Switzerland, Germany, and France.

NucliSens HIV-1 QL assay. In CLB, one reagentbatch of the NucliSens HIV-1 QL assay (bioMerieux) wasanalyzed. For the sample preparation, the EluLowNucliSens Extractor protocol was used. The NucliSensHIV-1 QL assay is almost identical to the widely usedquantitative NucliSens HIV-1 QT assay(bioMerieux). The assay is based onnucleic acid sequence-based amplifica-tion, an isothermal amplificationmethod. In the qualitative version ofthe test, instead of three calibrators,only one synthetic RNA is used as in-ternal control. The wild-type RNA andthe internal control RNA are coampli-fied and separately detected by electro-chemiluminescence.

Statistical analysisThe overall percentage of positive re-sults of each of the laboratories usingthe same method was plotted againstthe log concentration. From the dose-response curve, the 95- and 50-percentdetection limits (and 95% CIs) were cal-culated by probit analysis.15 For the cal-culation, a statistical software package(SPSS, version 7.5, Chicago, IL) wasused. To compare the sensitivity of as-says for different genotypes, the detect-ability of one genotype was expressedrelative to the detectability of the othergenotype. For each assay, the test po-tencies of one genotype relative to theother were calculated from the shift be-tween the probability curves in a paral-lel line model. Some laboratories also

submitted sample and/or cutoff (S/CO) values. For theGen-Probe TMA assay and AmpliScreen assays, the geo-metric mean S/CO ratios (and range) were plotted againstthe concentrations in the panels.

RESULTS

Detection of HCV RNA genotype 1standard dilutionsTable 1 shows the percentage positive results found onthe HCV RNA genotype 1 reference panel with five assays.From these results, the probability of detecting limitingdilutions of the VQC HCV RNA genotype 1 standard (Fig.1) and the 95- and 50-percent detection limits (Table 2)can be calculated by probit analysis. In 1998 and 1999,eight European laboratories combined the HCV Amplicormethod with the NucliSens Extractor (EluLow NucliSens-HCV Amplicor 2.0) and tested the panel all together in102 test runs. The overall 95-percent detection limit inthese laboratories was 69 geq per mL. However, when theresults of 49 test runs at CLB were assessed, the 95-percent detection was 30 geq per mL. The laboratoriescombining different protocols of the Qiagen extraction

TABLE 1. Percentage of positive results found with different HCV NATblood screening assays on VQC HCV RNA genotype 1 and Eurohep HCV

RNA genotype 3 standard dilution panelsEluLow

NucliSens-HCVAmplicor 2.0*

Qiagen-HCVAmplicor 2.0†

HCVAmpliScreen

2.0‡

EluHighNucliSens-HCV

AmpliScreen 2.0§Gen-Probe

TMA\

VQC HCV RNA genotype 1 (geq/mL)1140 100 100 100379 100 100 100 100270 100 94 100 100 100140 100 94 100 100 97100 97 94 93 100 9738 88 73 71 100 9111 73 28 29 79 603.8 21 10 26 50 181.1 13 0 0 8 110.4 5 6 3 0 20.1 0 0 0 0 2

Eurohep HCV RNA genotype 3 (geq/mL)2700 100 100810 100 100 100270 100 100 9881 97 100 9527 91 56 758.1 63 28 332.7 41 6 220.8 13 0 60.3 3 0 20.1 3 0 0

* Performed by 8 laboratories in 102 test runs for VQC HCV RNA genotype 1 and per-formed by 5 laboratories in 32 test runs for Eurohep HCV RNA genotype 3.

† Performed by 4 laboratories in 36 test runs for VQC HCV RNA genotype 1 and forEurohep HCV RNA genotype 3.

‡ Performed by 3 laboratories in 34 test runs for VQC HCV RNA genotype 1.§ Performed by 1 laboratory in 24 test runs for VQC HCV RNA genotype 1.\ Performed by 8 laboratories in 65 test runs for VQC HCV RNA genotype and in 8

laboratories in 67 test runs for Eurohep HCV RNA genotype 3.

LELIE ET AL.


technology with the Amplicor 2.0 assay (Qiagen-HCVAmplicor 2.0) found a 95-percent detection limit of144 geq per mL. More recently, the HCV RNA genotype1 panel was also tested in 30 Cobas AmpliScreen 2.0runs and four times in the AmpliScreen 2.0 MWP assay.For calculation of the detection limits, all AmpliScreenresults were combined, and a 95-percent detection limitof 126 geq per mL was found. However, when the resultsof 24 Cobas AmpliScreen 2.0 test runs in CLB were as-sessed, a 95-percent detection limit of 77 geq per mL wasachieved. CLB also tested the HCV RNA reference panelin 24 AmpliScreen 2.0 assay runs in combination with theNucliSens Extractor (EluHigh NucliSens-HCV Ampli-Screen 2.0). With the introduction of the AmpliScreenHCV and HIV assays, a higher NucliSens Extractor elutionvolume was necessary, and therefore, the EluHigh optionhas been introduced. With the EluHigh NucliSens-AmpliScreen 2.0, a 95-percent detection limit of 21 geqper mL was found. Thus, replacement of the MultiprepAmpliScreen sample preparation method by the EluHighNucliSens isolation method increases the sensitivity ap-proximately threefold, which can be explained by the in-crease of 250 to 735 mL in the equivalent of the plasma

volume used for amplification. As com-pared with the EluLow protocol (Elu-Low NucliSens-Extractor Amplicor 2.0),the sample input in the PCR reducedfrom an equivalent of 1100 to 735 mL ofthe original 2 mL of plasma used forextraction. Nevertheless, the replace-ment of the HCV Amplicor 2.0 assay bythe HCV AmpliScreen 2.0 assay (andthe concurrent transition from the Elu-Low to EluHigh Extractor protocol) didnot have a significant effect on the de-tection limit, which indicates that theHCV AmpliScreen 2.0 assay is slightlymore sensitive than the HCV Amplicor2.0 assay. Finally, when 65 HCV Gen-Probe TMA test runs on the HCV RNAgenotype 1 standard dilutions wereanalyzed, an overall 95-percent detec-tion limit of 85 geq per mL was found.

For three assays, we also studiedthe S/CO response values that weresubmitted by some laboratories. Figure2 shows that in CLB the saturationpoint of the Cobas HCV AmpliScreen2.0 assay was reached at a concentra-tion of 270 geq per mL and at a lowerconcentration of 100 geq per mL whenthe EluHigh Nuclisens-HCV Ampli-Screen 2.0 assay was used. The meanS/CO values in Gen-Probe TMA assayruns of six laboratories reached a pla-

teau at a concentration of 270 geq per mL (S/CO valuesabove 270 geq/mL level off and are not shown).

Detection of HCV RNA genotype 3standard dilutionsTable 1 shows the percentage of positive results on therecalibrated HCV RNA concentrations in the PeliCheckgenotype 3 panel. We performed probit analysis on thegenotype 3 dilution series in a parallel line model withthe genotype 1 response curves of the same assay takenas a reference. The 95- and 50-percent detection limits onthe Eurohep HCV RNA genotype 3 standard dilutions areshown in Table 2. Assuming parallel response curves inthe probit analysis, one can calculate the test potency ofgenotype 3 dilutions relative to genotype 1 dilutions. Thetest potencies of each assay for genotype 3 relative togenotype 1 are shown in Table 3. When the 95-percentCIs overlap a potency of 1.0, there is not a significantdifference in detection efficiency of the genotype 1 andgenotype 3 standards. The results in Table 3 show thatthe differences in detection efficiency of genotype 3 andgenotype 1 are not significant.

Fig. 1. Probability of detection of lower concentrations of the HCV RNA genotype 1

(A) and HIV RNA genotype B (B) reference panels by three NAT blood screening

assays, as calculated by probit analysis on the percentage of positive results on the

viral standard dilutions



Detection of HIV RNA genotype B standard dilutionsTable 4 shows the proportion of positive results found onthe HIV RNA genotype B standard dilution panel. Figure1 shows the probabilities of detecting the lower viral stan-dard concentrations in the panel, as found by probitanalysis for three assays. The 95- and 50-percent detec-tion limits of assays on the VQC HIV RNA genotype Bstandard dilutions are shown in Table 5. Twenty-eighttest runs have been performed on the reference panelwith the HIV AmpliScreen 1.5 assay, of which 24 runswere on the Cobas analyzer and four on the MWP ver-sion. When all HIV AmpliScreen test results were com-bined, a 95-percent detection limit of 126 geq per mL wasfound. However, when only the results of the Cobas Am-pliScreen 1.5 tests in CLB were assessed, a 95-percentdetection limit of 92 geq per mL was achieved. In a firststudy at CLB, the AmpliScreen 1.5 assay was combinedwith the EluLow NucliSens Extractor protocol (EluLowNucliSens-HIV AmpliScreen 1.5), and after testing theHIV RNA panel eight times, a 95-percent detection limitof 14 geq per mL was found. Later, the EluHigh option onthe NucliSens Extractor was introduced to allow simulta-neous detection of HCV RNA and HIV RNA. Testing thepanel 24 times in the EluHigh NucliSens-HIV Ampli-

TABLE 3. Potency comparison of assays forHCV RNA

Assays

Number of test runson genotype 3

standard/Numberof test runs on

genotype 1 standard

Potency ofgenotype 3

to genotype 1(95% CI)

Qiagen-HCVAmplicor 2.0 36/36 1.08 (0.41-2.78)

EluLow NucliSens-HCV Amplicor 2.0 32/102 1.73 (0.50-2.78)

Gen-Probe TMA 67/65 0.80 (0.26-1.11)

Fig. 2. Geometric mean S/CO ratios and range of the re-

sponse values as observed in one laboratory testing the HCV

RNA genotype 1 reference panel 24 times in the AmpliScreen

assay with regular Multiprep sample preparation (A) and

with NucliSens extraction technology (B). The results are

also shown for six laboratories that reported response values

of 56 test runs in the Gen-Probe TMA assay (C).

TABLE 2. The 95 and 50-percent detection limits (and 95% CIs) calculated by probit analysis on the reactivityrates found with different NAT assays on VQC HCV-RNA genotype 1 and Eurohep HCV RNA genotype 3

standard dilutions

Assays LaboratoriesTestruns

Plasmainput (µL)

Amplificationinput (µL)

95-percentdetection limit

(95% CI)


(95% CI)

HCV RNA genotype 1EluLow NucliSens HCV Amplicor 2.0 8 102 2000 ∼1100 69 (50-102) 6.4 (5.0-8.1)EluLow NucliSens HCV Amplicor 2.0 1* 49 2000 ∼1100 30 (18-72) 4.9 (3.3-7.8)Qiagen-HCV Amplicor 2.0 4 36 140-200 100-167 144 (74-402) 18 (10-32)HCV AmpliScreen 2.0 (Cobas and MWP) 3 34 1000 250 126 (83-225) 14 (11-19)HCV Cobas AmpliScreen 2.0 1* 24 1000 250 77 (43-203) 9.9 (6.3-15)EluHigh NucliSens-HCV AmpliScreen 2.0 1* 24 2000 ∼735 21 (13-44) 4.3 (3.1-5.9)Gen-Probe TMA 8 65 500 500 85 (64-118) 7.9 (6.3-9.9)

HCV RNA genotype 3Qiagen-HCV Amplicor 2.0 4 36 140-200 100-167 132 (59-449) 16.2 (8-35)EluLow NucliSens-HCV Amplicor 2.0 5 32 2000 ∼1100 41 (26-70) 3.7 (2.5-5.5)Gen-Probe TMA 8 67 500 500 107 (78-152) 9.9 (7.9-12.5)

* CLB.

LELIE ET AL.


Screen 1.5 assay, the 95-percent detection limit was 37geq per mL. The introduction of the EluHigh option madethe assay slightly less sensitive by shifting the 50-percentdetection limit from 2.0 to 4.6 geq per mL. The EluLowNucliSens-HIV-1 QL assay was also evaluated at CLB.This assay was less sensitive than the EluLow NucliSens-HIV AmpliScreen 1.5 assay (Table 5). The HIV RNA geno-type B panel was tested 91 times in six laboratories byusing the Gen-probe TMA assay. The overall 95- and 50-percent detection limits of this assay were 31 and 3.6 geqper mL. The Gen-Probe TMA assay was as sensitive as theEluHigh NucliSens-HIV AmpliScreen 1.5 assay, eventhough the TMA assay uses a lower input of extractedRNA from plasma in the amplification reaction.

Figure 3 compares the geometric mean and range ofS/CO values found with three NAT assays on the HIV RNAstandard dilutions. The AmpliScreen data with Multiprepspecimen preparation (Cobas HIV AmpliScreen 1.5) andwith NucliSens extraction (EluHigh NucliSens-HIV Am-pliScreen 1.5) were obtained in one laboratory (CLB). TheCobas-HIV AmpliScreen 1.5 assay reached its saturationpoint at a concentration of 250 geq per mL, whereas the

EluHigh NucliSens-HIV AmpliScreen1.5 responses saturated at a level of 75geq per mL. The Gen-Probe TMA re-sponses were reported by six laborato-ries and reached maximum values at alevel of 250 geq per mL (S/CO valuesabove 750 geq/mL level off and are notshown).

Detection of HIV RNA genotype Estandard dilutionsIn Table 4, the percentage of positiveson the HIV RNA genotype E panel isshown. The 95- and 50-percent detec-tion limits on the VQC genotype E stan-dard dilutions were comparable to theones found on the VQC genotype B di-lutions (Table 5). Assuming parallel re-sponse lines of one assay on the geno-type B and genotype E dilution panels,one can express the detectability of ge-notype E relative to genotype B by pro-bit analysis. When the genotype E ver-sus B test potencies are compared(Table 6), it can be concluded that allassays detected the genotype E stan-dard dilutions with equal efficiency asthe genotype B standard dilutions.

DISCUSSIONIn the late 1990s, the NAT method forblood screening went through a rapid

development phase. In a short period, manufacturers ofin vitro diagnostics (IVDs) have developed semiauto-mated assays that can be applied to MP-NAT screening ofblood donations. Meanwhile, regulatory bodies devel-oped guidelines for validation of NAT assays and have setstandards for sensitivity.16-18 In Europe, a new IVD direc-tive became into force in June 2000, and the performanceevaluation criteria of IVDs have been documented in theCommon Technical Specifications.18 According to theCommon Technical Specifications, the 95-percent detec-tion limit of NAT assays should be determined by testinga standard dilution series at least 24 times. For this pur-pose, the standards have to be calibrated against theWHO International Standards.8-12 The WHO standardsfor NAT are ampoules with lyophilized material, and theviral nucleic acid content is expressed in IUs. There is nota universal relationship between IUs and the number ofviral target sequences. Calibration of VQC standardsagainst the WHO standard has demonstrated that thereare large differences in conversion factors from IU to geqbetween different analytes. One IU corresponds to ap-proximately 4 geq for HCV RNA,12 1 geq for HIV RNA,10

TABLE 4. Percentage of positive results found with different HIV NATblood screening assays on VQC HIV RNA genotype B and genotype E

standard dilution panels

NucliSensHIV-1 QL*

EluLow NucliSens-HIV Ampliscreen

1.5*

HIVAmpliScreen

1.5†

EluHighNucliSens-HIV

AmpliScreen 1.5‡Gen-Probe

TMA§

VQC HIV RNA genotype B25,000 100 100

7,500 100 1002,500 100 100

750 100 100 100 100250 100 100 100 100

75 100 100 89 100 9825 63 100 86 96 100

7.5 38 100 32 58 762.5 0 38 25 25 270.8 13 13 18 13 50.3 0 3 0 40.08 0 0 2

VQC HIV RNA genotype E25,000 100

7,500 1002,500 100

750 100 100 100250 100 100 100

75 88 100 100 9925 50 100 96 90

7.5 40 88 67 722.5 50 25 46 370.8 0 38 4 90.3 0 8 10.08 0 1

* Performed by 1 laboratory in 8 test runs for HIV RNA genotype B and by 1 laboratoryin 8 test runs for HIV RNA genotype E.

† Performed by 2 laboratories in 28 test runs for HIV RNA genotype B.‡ Performed by 1 laboratory in 24 test runs for HIV RNA genotype B and by 1 labora-

tory in 24 test runs for HIV RNA genotype E.§ Performed by 6 laboratories in 91 test runs for HIV RNA genotype B and by 6 labora-

tories in 89 test runs for HIV RNA genotype E.



and 7 geq for HBV DNA.9 Thus far, the FDA has not ex-pressed their standards for sensitivity of NAT assays in IUper mL. The FDA requirement for sensitivity of NATmethods used in blood screening is a 95-percent detec-tion limit of 100 copies per mL, whereas the NAT screen-ing system should achieve a sensitivity of at least 5000copies per mL per individual donation.

One has to bear in mind that the 95-percent detec-tion limit is influenced by the proficiency of the techni-cians. One “false”-negative result at a critical concentra-tion can have a profound effect on the outcome of the95-percent detection limit. For example, a 95-percent de-tection limit of 126 geq per mL was found for all labora-tories with both the HCV and HIV AmpliScreen method.However, CLB showed that it is feasible to achieve a 95-percent detection limit with the Cobas AmpliScreen 1.5or 2.0 that lies below the sensitivity requirement of 100copies per mL set by the FDA.

The differences in sensitivity between assays can bet-ter be estimated by comparing the 50-percent detectionendpoints. Doing this, it appears that half-log differencesin sensitivity between the NAT assays can be found. Thedifferences in sensitivity could not be attributed to a cer-tain amplification method. The amplification method ofthe Gen-probe TMA seems to be as powerful as the Am-pliScreen PCR method. More likely, the differences insensitivity between assays are caused by differences inthe volume of plasma from which RNA was extracted andused as input in the amplification reactions. For example,replacement of the Multiprep AmpliScreen samplepreparation method by the NucliSens Extractor increasedthe 50-percent AmpliScreen detection limit from 9.9 to4.3 geq per mL for HCV RNA and from 6.6 to 4.6 geq permL for HIV RNA. These results show that the EluHighprotocol on the NucliSens Extractor is approximately

twofold more efficient in isolating viral RNA than the Am-pliScreen specimen preparation method.

We performed probit analysis in a parallel line modelto compare the detection limits of two divergent geno-types of HCV and HIV, respectively. The detection effi-ciencies of assays for HIV RNA genotypes B and E werecomparable because the potencies of the assays for de-tection of genotype E relative to genotype B were close toone. The same holds for the detection efficiencies of as-says for HCV RNA genotypes 1 and 3. One has to bear inmind that the potency values are directly related to theoriginal quantification of the viral standards of differentgenotypes. A certain degree of imprecision is inevitablewith the currently available methods for quantification ofnucleic acid concentrations in viral plasma-derived stan-dards. The quantification and calibration of VQC stan-dards of different genotypes in CLB are mainly based onmultiple historical Quantiplex branched DNA assays,which have been validated for accurate quantification ofviral genotypes.19 For more accurate and precise mea-surement of NAT sensitivity for viral genotypes, a newgeneration of pure nucleic acid standards needs to bedeveloped. Such standards can be quantified with moreaccurate and precise physicochemical methods that areindependent from molecular hybridization.20

Our study demonstrates that the sensitivity require-ments for NAT blood screening assays can be met by theGen-Probe TMA and AmpliScreen assays. However, thequality of performance of the AmpliScreen assay withMultiprep sample preparation needs to be optimized tohit a 100 geq per mL concentration of the VQC standards95 percent of the time. Because the AmpliScreen assay isperformed on 24 donation pools, the detection limit of5000 geq per mL per individual donation required by theFDA can be met with this assay. When the AmpliScreen

TABLE 5. The 95 and 50-percent detection limits (95% CIs) calculated by probit analysis on the reactivity ratesfound with different NAT assays on VQC HIV- RNA genotype B and genotype E standard dilutions

Assays LaboratoriesTestruns

Plasmainput (µL)

Amplificationinput (µL)


(95% CI)


(95% CI)

HIV RNA genotype BHIV AmpliScreen 1.5

(Cobas and MWP) 2 28 1000 250 126 (67-311) 7.1 (4.7-10.7)HIV Cobas

AmpliScreen 1.5 1* 24 1000 250 92 (49-238) 6.6 (4.4-10.1)EluHigh NucliSens-

HIV AmpliScreen 1.5 1* 24 2000 ∼735 37 (23-69) 4.6 (3.2-6.7)EluLow NucliSens-

HIV AmpliScreen 1.5 1* 8 2000 ∼1100 14 (6.4-49) 2.0 (1.0-3.8)Gen-Probe TMA 6 91 500 500 31 (20-52) 3.6 (2.6-5.0)EluLow NucliSens-HIV-1 QL

HIV RNA genotype E 1* 8 2000 220 123 (51-566) 11 (6-23)EluHigh NucliSens-HIV Ampliscreen 1.5 1* 24 2000 ∼735 26 (16-49) 3.3 (2.3-4.8)EluLow NucliSens-HIV Ampliscreen 1.5 1* 8 2000 ∼1100 16 (8-57) 2.3 (1.2-4.4)Gen-Probe TMA 6 89 500 500 33 (22-56) 3.9 (2.8-5.4)EluLow NucliSens-HIV-1 QL 1* 8 2000 220 147 (63-559) 13 (5-29)

* CLB.

LELIE ET AL.


or Amplicor assay are combined with a large-volume ex-traction method such as the NucliSens Extractor, the FDArequirement can also be easily met if pools of 48 or 96donations are screened.

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Fig. 3. Geometric S/CO ratios and range of the response val-

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type B reference panel 24 times in the AmpliScreen assay

with regular Multiprep sample preparation (A) and with

NucliSens extraction technology (B). The results are also

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56 test runs in the Gen-Probe TMA assay (C).

TABLE 6. Potency comparison of assays forHIV RNA*

Assays

Number of test runson genotype E standard/

Number of test runson genotype B standard

Potency ofgenotype E to

genotype B(95% CI)

EluHigh NucliSens-HIVAmpliScreen 1.5 24/24 1.40 (0.84-2.44)

EluLow NucliSens-HIV AmpliScreen 1.5 8/8 1.19 (0.50-3.11)

Gen-Probe TMA 89/91 0.92 (0.58-1.44)EluLow NucliSens-

HIV-1 QL 8/8 0.84 (0.23-2.33)

* The potency of assays for detecting the VQC genotype Estandard dilutions relative to the VQC genotype B standarddilutions is calculated in a parallel line model.



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LELIE ET AL.


sensitivity of hcv rna and hiv rna blood screening assays

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