pcr testing of plasma pools: from concept to reality

PCR Testing. of Plasma Pools: From Concept to. Reality

Peter Flanagan and, John Barbara

A NUMBER of factors might be considered to have contributed to the current drive to: the

~mplememation of genomic testing for hepatitis C virus (HCV) RNA within the field of transfusion medicine. The apparent urgency to establish effective systems aimed at the detection of seronegative, yet infectious, units is evident within the United States and in Europe. In both instances. concern initially relating to plasma destined for fractionation is now clearly impacting on blood services whose primary focus is the production of labile blood components. This review outlines the background to the development of current regulatory requirements and assesses the options that will enable transfusion services to ensure compliance, both the currently available option and~ those under development.

BACKGROUND

The safety ofmanufactured plasma derivatives is assured by a number of complementary approaches. These include donor selection, screening of individual donations for appropriate: serological markers, and the application of specific viral inactivation procedures during the manufacturing process. Together these have significantly improved the safety of these products and resulted in a high level of confidence with respect to viral safety The recent safety record in terms of lack of transmission of enveloped viruses in products often subjected to two independent specific viral inactivation pro- cesses is excellent. Solvent detergent and other specific inactivation technologies effectively de- stroy enveloped viruses, including HCV and Hu, man Immunodeficiency Virus (HIV). The requirement to introduce genomic screening for-these agents will not lead to improved safety for those products subjected to specific viral inactivation technologies. The cost of implementation will.

From the New Zealand Blood Service Auckland. New Zea- land: and the National Blood Service. Colindale Centre. Lon, don, England.

Address reprint requests to Peter Flanagan, MB. FRCP. FRCPath. New Zealand Blood Service. PO Box 2661L 169 Manukau Rd, Epsom, Auckland. New Zealand.

Copyright �9 1999 by W.B, Saunders Company 0887-7963/99/1303-000253.00/0

however, be very significant. It is thus highly unlikely that the requirement to undertake testing for HCV RNA for plasma would stand up: to a formal heatth economic evaluation. With nonenvel- oped viruses, the situation is different because of their resistance to solvent detergent-type technologies. Recent moves toward manufacture of high- purity products have resulted i'n a nuiiaber of episodes of transmission~ of hepatitis A infection (HAV)I and' increased awareness of the potential hazard of parvo~ims B t9 transmission:. 2 Genomic testing is a possible option to prevent trar~smissions of these agents.

During the early 1990s, however, not all prod, ucts were subjected to: a: specific viral inactivation step. In particular, a number of immunoglohulin products were ayailable whose manufacturing pro- cesses did not incorporate specific inactivation procedures. During 1'994, a number of cases of transmission of HCV infection by Gammagard (an intravenous immunoglobutin preparation produced by Baxter) were reported, 3 Initially introduced in �9 1986~ the manufacturing process for Gammagard did not include. ~ specific viral inactivation step. HCV transmissions appeared to coincide wi~h the introduction:of enhanced sensitivi:ty second-genera, tion HCV screening 4 assays. Presumab!y the exclu- sion of anti-HCV positive plasma units influenced the partitioning of complexed virus.during fraction~ ation. Regulatory authorities in. the United States and Europe were swift to: react. The United States FDA Centre for BiologiCs Evaluation and Research established a:requirement for testing finished preparations for HCV-RNA before release of those intra-~enous immunoglobutin preparations whose manufacture did not: include: a specific viral inactivation step,5 Within Germany, the Paul Ehflieh Institute established: a: requirement that plasma pool,s used: in the: manufacture of intravenous. immunog!obulin preparations that lacked a validated: virus inactivation procedure had to be shown tO not contain HCV RNA.6 The European Commis- gion:Committee for Proprietary Medicinal Products. (CPMP) adopted this same requirement for commu. nity-wide implementation; however; it was extended' to apply to any such.immunoglobulin preparation., irrespective of route of administration. 7 It was apparent, within Europe at least, that this was

164 Transfusion Medicine Reviews, Vo113, No 3 (July), 1.999: pp 164-176

PCR TESTING OF PLASMA POOLS 165

an initial response and that the implementation of more widespread genomic testing for products with or without viral inactivation steps would follow, as soon as effective and validated technologies were available. 8

CURRENT REGULATORY POSITIONS

In February 1998, the European Commission Commi~ttee for Proprietary Medicinal Products (CPMP) issued the final version of a guideline that identifies a requirement that from the first of July 1999 all fractionated plasma products issued within the Europ'ean Union must be derived from plasma pools that have been tested and found to be negative for HCV RNA using a validated polymerase 'chain reaction (PCR) technique. 9 The Guideline recommends the incorporation of a suitable working reagent (run control) in each run. This run control should be equivalent in HCV RNA content to100 IU/mL. In ;effect, the CPMP guideline will result in an extension to the identified batch release requirements for plasma derivatives, testing being the responsibility of laboratories identified by the National Control Authority; these laboratories are designated Official Medicines Control laboratories (OMCLs)I A strategy of pretesting of minipoois is recommended by the CPMR This approach is designed to avoid loss of a complete manufacturing pool and subsequent shortages of product.

The international nature of the plasma fractionation industry ensures that the impact of this guideline will be felt across the globe. This is amply confirmed by the rapidly developing litera- ture on this topic.

Within Europe, the remit of the CPMP is re- stricted to licensed medicinal products. It has no jurisdiction over blood components. Thus, no guidance was available from the CPMP on the management of blood components. The FDA within the United States was proactive and determined that although application of PCR to plasma start pools might be considered as an "in process control" measur e, the use of minipool testing would be considered as a donation screening test.l~ Measures also would need to be established to ensure that positive results coUld be traced to the level of an individual donation and hence donor. Implementa- tion of the process would be progressed through its Investigational New Drug program. An inevitable consequence of this approach was that Transfusion Services would need to consider the feasibility of

developing systems capable of providing timely results to allow labile component release to be secured on the basis of genomic testing. Thus. a measttre initially directed at improving safety of fractionated products had ted to the introduction of genome detection Systems for component release. Within the United States, a further driver can be identified. The FDA has previously signaled an intention to implement genomic testing for HIV once suitable technologies become available, with the likely aim Of automated single-donation testing systems.

Within Germany, the Paul Ehrlich Institute has responsibility for both licensed plasma derivatives and components produced within blood centers. Concern over the possible development ofdifferent standards of testing for plasma products and blood components led to consideration of the requirement for HCV RNA testing to be used as a release criterion for labile componentS. Undoubtedly influenced by early reports from the Red Cross Centre in Hagen Northern Westphalia, u a consultation process followed. From an initial consideration for testing for HCV, HIV. and hepatitis B virus (HBV) nucleic acids, the consultation process resulted in a requirement for the application of HCV RNA testing by PCR as a pre-release requirement for all blood components, both red cells a2 and platelets. 13 Test systems will be required to detect HCV RNA at a minimum of 5.000 IU/mL of individual donations and will need to be implemented throughout Germany by April 1999. In the absence of other guidance, it is likely that this level of sensitivity will be used elsewhere in Europe and beyond as the benchmark for transfusion services responsible for developing systems that will permit prospective component release. Table 1 summarizes the current regulatory position.

THE SCIENTIFIC BASIS FOR GENOME TESTING

Classically, virologists such as M.H. Adams 14 aimed to place their portion of biological science onto a firm, scientifically objective and quantifiable footing. To this end, the bacteriophages were extensively analyzed because they were subject to precise quantitative study and could be detected and assayed with a high degree of accuracy and reproducibility. The virology of animal infections was less amenable to precise quantitation because the assays depended on cell culture and animal

166 FLANAGAN AND BARBARA

Table 1. Current Regulatory Positions

Requirement for Requirement for Regulatory Authority Plasma Derivatives Blood Components

European Economic Community No mandated requirement (CPMP) excluding Germany

Germany PauI-Ehrlich Institute)

United States (FDA)

With effect from July 1, 1999, plasma derivatives released for clinical use

must have been produced from plasma pools that have tested negative in a validated PCR assay capable of routinely detecting 100 IU/mL HCV RNA

Requirement as per EEC

No mandated requirement. When minipool testing is undertaken,

systems must be developed that enable tracing of positive results to the individual donation level. Sys- tems require to be approved through the IND scheme.

With effect from April 1, 1999: Red cell and platelet components

should not be released until a negative NAT test result for HCV RNA is available. The test system should be validated and capable of detecting 5,000 IU/mL in an individual donation.

No mandated requirement. However, FDA has signaled an intention

to move to genomic testing for HIV once suitably validated technologies are available.

Abbreviations: CPMP, Committee for Proprietary Medicinal Products; EEC, European Economic Community; PCR, polymerase chain reaction; HCV, hepatitis C virus; NAT, nucleic acid amplification test; FDA, Food and Drug Administration (USA); IND, investigational new drug; HIV, human immunodeficiency virus.

inoculation. Such methods were patently impracti- cal in the context of mass, rapid pretransfusion screening of blood donations. Indeed, it was fortu- nate that the first virus for which blood services initiated routine screening, hepatitis B, produces a sufficient excess of one of its antigens (that on the surface of the virion--HBsAg) to enable effective detection of most infected potential blood donors. The other blood-borne transfusion-transmissible agents of major significance (HIV 1 and 2, HCV, and human T cell leukemia virus [HTLV]) because of their persistence, can be detected by the host's antibody response that they elicit (Fig 1). Indeed, a similar situation pertains to the anti-HBc elicited by HBV, although in most instances immune anti-HBs also will be present. However, with both antigen detection (where appropriate) and antibody assay, there is a period befo~}e production of viral antigens and subsequent seroconversion when virus may be present in the blood at infectious levels. Immedi- ately after infection, there is an "eclipse" phase when the virus uncoats and directs the synthesis of the proteins necessary for its replication and assem- bly. During this time, no infectious virus is recover- able in the blood so that the infectious phase of the "window period" is after eclipse and before seroconversion. This can be shown in chimpanzees

challenged with HIV at different points in the seroconversion window period of an experimen- tally infected chimpanzee (Busch, unpublished observations). For the first few days after infection, the virus cannot be transmitted (the "eclipse" phase). In these experiments, infectivity correlates with detection of viral genome by PCR or similar technologies. Virologists wo~'king in the field of transfusion medicine have long awaited the realiza- tion of sensitive techniques for direct detection of viral genome simply, rapidly, and efSciently in this window period without the requirement for relatively cumbersome and time-consuming cell culture assays. With the advent of the PCR assay came an in vitro technique that paralleled the sensitivity of biological infectivity testing but with greater objectivity, speed, and consistency in the absence of bio-assay variability. PCR and related techniques now allow detection of microbial infectivity after eclipse and immediately before "seroconversion."

Statistical Aspects

Viral particles in an inoculum, and the related numbers of genome copies detected with genome amplification tests, also referred to as nucleic acid amplification tests (NAT), can be considered as


marker

antigen

genome

antibody

•I ~ time months/years

time of infection

Fig 1. Generalized course of transfusion transmittable infections. This represents a schematic indication of the sequence of genome, antigen, and antibody generation. The timings, levels, and persistence of genome, antigen, and antibody vary for different agents and also may vary for a given agent in different individuals.

independently distributed material particles. This is generally the case unless viruses clump or aggluti- nate, for example, in the presence of specific antibodies. The equation describing the distribution of such independently distributed material particles in individual samples is the Poisson distribution. 15

sre-S

Pr= r! (1)

where s is the average number of particles per �9 sample; r is the actual number in a given sample; r!

is the factorial oft , that is, (r - 1) • (r - 2), etc.; Pr is the probability of having r particles in a given sample, that is, the expected frequency of samples containing r particles.

The frequency distribution of plaques on a series of cellular monolayers inoculated with equal vol- umes of serial dilutions of viral suspension, or the frequency distribution of bacterial colonies, follows the Poisson distribution. So also does the distribution of genome copy numbers at limiting dilutions of samples containing such genomes. Because of the Poisson distribution, when a suspension of viral genomes is dispensed into aliquots that should contain an average of one genome per aliquot, 37% of aliquots will contain no genomes, 37% will contain one, and the remaining 26% will

contain more than one. From this it also follows that in "infectious dose 50" determinations the dilution that contains on average one infectious dose is the one that infects 63% (and not 50%) of the targets. 16 The importance of the Poisson distribution in the context of standard preparations for minipool production is considered later.

Genome Levels During Window Periods

The level of viremia seen in HCV-infected individuals lies within the range 103 to 108 geq/mL, with a modal value of approximately 106 geq/mL.17 Before seroconversion levels of 107 geq/mL for HIV RNA, 108 for HCV RNA and 104 for HBV DNA have been reported (Lelie N, personal communication). In more detailed calculations based on PCR testing of commercially available seroconversion panels (Busch M and Kleinman S, personal communication), the length of the window periods for HIV, HCV, and HBV are 22, 70, and 56 days, respectively. When testing individual samples by NAT, these window periods would be shortened by 10 to 15, 41 to 60, and 6 to 15 days, respectively. Before genome detectability, the infection is in the "eclipse phase" and is unlikely to result in infectivity. Also, the genome copy doubling time is 1 day for HIV, 2 hours for HCV, and4 days for HBV, with


viral loads of 102 to 10 7 , 105 to 10 7 , and 102 to 10 4

geq/mL, respectively. More confidence regarding these kinds of data will be acquired when corrobo- rative results are provided by other research groups.

With data such as these, the projected benefits of strategies for NAT can be calculated and single- sample systems can be compared with a range of potential pooling strategies. Clearly, detection of HCV genomes during seroconversion is the best suited of the three agents (HIV, HCV, HBV) to pooling strategies because replication of virus, when it commences, is very rapid. This means that with HCV the period when the dilution factor from pooling might cause false-negativity is tikely to be very small.

THE TECHNOLOGY

An increasing range of genome amplification techniques has been developed since the PCR was first described. Details of the PCR technique have been reviewed previously, 1~ as have some of the other major approaches to sensitive nucleic acid detectionJ 9 All involve extraction or capture of nucleic acid, amplification, and detection: three distinct major phases.

Polymerase Chain Reaction

This technique mimics the exponential replication of DNA, using pairs of nucleotide primer sequences that anneal to regions that flank the target sequence to be amplified. By repeated cycles of prime r extension to complete the Complemen- tary nucleotide sequence and heating to denature the double strands of DNA into single strands with subsequent reannealling of primers, DNA synthesis can be multiply repeated. Because each cycle doubles the target sequence copy number, several million DNA copies can be produced in 20 to 30 amplification cycles. This large bulk of copies can be detected in a number of ways. The simplest are by electrophoretic site separation on agarose gels stained with ethidium bromide under ultraviolet light and by probes that specifically hybridize to internal amplified sequences. For RNA detection, DNA copies are prepared for amplification using a reverse transcriptase.

In 1991, Roche Laboratories (Somerville, NJ) purchased the patent rights to PCR from Cetus, and they have developed commercial assays for HCV RNA, HIV-1 RNA, HBV DNA, and CMV DNA in

plasma or serum. Roche offers either a 96-welt microplate format (or single tubes) for amplification and detection or an instrument (COBAS) for automated amplification and detection steps in batches of 24 samples. Overall assay time is up to 4 hours.

Nucleic Acid Sequence--Base Amplification and Transcription-Mediated Amplification

These two similar but separately patente~ techniques are single-step isothermal amplifications with a continuous cycle of reverse transcription and RNA transcription to replicate the RNA target. The process resembles that of RNA bacteriophage replication (Fig 2]. Organon Teknika (Boxtel, The Netherlands), who holds the patent on nucleic acid sequence-base amplification (NASBA), have a commercial system for sample processing, amplifi~ cation, and detection.

Gen-Probe markets their patented transcription- mediated amplification assay as a "one-tube" amplification and detection system, wkh only two enzymes ~reverse transcriptase and RNA polymerase) necessary for addition to the reaction mixture. An HIV/HCV "multiplex" RNA detection system is under clinical trials, suitable for testing plasma pools in a semi-automated format (Magenta, Gen Probe. San Diego, CA): trials of a fully automated system (Tigris. Gen Probe) for testing single samples are planned for 1999. Recently, an alfiance of Gen-Probe with Chiron (Emeryville. CA) (who hold the HCV genome patent) was announced. The impact that this alliance will have on HCV diagnos- tic kit manufacturers such as Roche remains to be determined as we learn more about the patent issues involved.

Ligase Chain Reaction

Ligase chain reaction is being developed commercially by Abbott Laboratories (Abbott Park. IL). It uses a thermo stable enzyme that covalently links (l!gates) the phosphate backbone of two adjoining nucleotide probes bound to a target nucleic acid. Repeated cycles of ligation and denaturation allow amplification analogous to PCR. Abbott Laborato- ries has license rights to PCR. with which ligase chain reaction instruments are compatible.


Fig 2. Nucleic acid sequence- base amplificatiOn, or NASBA, method,

viral 5' RNA

RNA/DNA ds hybrid

ss DNA ~- ,

ds DNA

100 to 1000 (-) strand

RNAs

(+) s t rand 3'

via RT & [primer + T7 promoter sequence]

(,) ; / W V .1111

(+i RN~lse H (removes RNA from a DNA/RNA duplex)

/WV ~3~ s e n s e l primer

RT

(') / W ~ (+)

T7 polymerase

then, each (-) RNA strand, via: RNA-DNA hyhrid.

ss DNA

CYCLIC

ds ; N A

OPTIONS FOR IMPLEMENTATION OF NUCLEIC ACID AMPLIFICATION TESTS FOR HEPATITIS

C VIRUS RNA

The primary driver for implementation is to ensure compliance with European regulatory requirements for fractionated plasma derivatives. Three possible approaches to implementation of NAT for HCV RNA (other than product testing) can be identified. These are testing of the manufacturing start pools, testing of minipools, and testing of individual donations. The challenge faced by the commercial sector, which primarily uses source plasma collected by apheresis, is different from those faced by the "not-for-profit sector." which is dependent predominantly on plasma recovered from whole blood donations. In this latter scenario, the additional responsibility for provision of labile components presents a number of logistical and medicolegal issues not encountered within the source plasma environment. In particular, the requirement for rapid identification of reactive donations and fast "turnaround times need to be ad- dressed.

Testing of Manufacturing Start Pools

The CPMP Guideline identifies that the decision on batch release of manufactured product: will reqmre demonstration that the plasma pool has

been tested for HCV RNA by a validated method of appropriate sensitivity. Release of a batch of product will require declaration of negative results by both the manufacturer and the OMCL. Sole reli- ance on testing of final pools poses a significant threat to manufacturers, because a positive result will lead to loss of all manufactured product relating to the pool. including intermediate frac- tions used in production of other batches.

The available data. based on both theoretical estimates and actual experience, suggest that the frequency of HCV "PCR-only" samples within the volunteer nonremunerated donor population would result in significant levels of product loss. Within the United Kingdom, using methods based on those described by Schreiber e ta l , 2~ the estimated frequency of HCV "PCR-only'" donations is 1 in 250.000. This equates to a frequency of 10 such donations each year (Barbara and Soldan. unpublished data). In England. before the recent introduction of precautionary measures aimed at reducing the theoretical risk of transmission of new variant Creutzfeldt-Jakob disease by plasma derivatives. 21 100 plasma pools derived from UK volunteer donors were fractionated every year. each containing 25.000 donations. Initial results of testing of plasma pools appear to confirm the predicted level of "PCR-only" donations, with 4 of 40 plasma


pools being considered to contain HCV RNA (Snape, unpublished data). The introduction of HCV RNA testing solely based on testing of plasma start pools would have resulted in the loss of 10% of pools, leading to inevitabl e shortages of manufactured product. The residual risk of HCV transmission, assumed here to equate to the prevalence of "PCR-only" donations, estimated within Germany, 22 is similar to that identified within the United Kingdom. A prevalence of 1 in 100,000 has been reported in volunteer donors from the United States. 2~ It is thus not surprising that plasma fractionators are attempting to develop strategies that will minimize plasma loss. In most instances, this has resulted in the development of systems based on minipool testing.

Testing of Minipools

This approach involves the creation of a pool of samples taken at the same time as the donation. The size of the pool can vary considerably, from 10 to several hundred donations. The sample can be derived either from the complete donation, in the case of source plasma collection, or from an additional blood sample. The minipools are created in such a way as to allow investigation of positive results to enable identification-of the single implicated donation (resolution). Thus, implementation of an effective minipool system will permit identification of individual positive donations that can be removed from the plasma inventory before construction of the manufacturing start pool. Testing of minipools provides the manufacturer with an additional margin of sensitivity over the OMCL, whose testing is based solely on a sample from the start pool. The likelihood of OMCLs detecting a positive start pool therefore is greatly reduced.

Testing of Single Donations

The development of systems that enable testing of individual donations might be considered to represent the ideal approach to implementation of NAT in the blood screening environment. Such systems should maximize sensitivity and enable results to be provided within a timescale similar to that currently achieved for standard serological screening, albeit at a high cost. Unfortunately, validated high-throughput technologies to support this approach are only now becoming available. Such systems will, however, require Food and Drug Administration Investigational New Drug

approval before general release. In practical terms, fully validated systems supporting single-donation testing are at least 2 years away, a timeframe that will not enable compliance with current CPMP requirements. Cost implications also need to be examined.

REQUIREMENTS OF AN EFFECTIVE MINIPOOL SYSTEM

The introduction of Quality Systems based on principles of current Good Manufacturing Practice has significantly improved confidence in the ability of test systems used within blood transfusion centers. Test systems used for screening of HCV RNA must aim to meet the same quality standards demanded of serological test systems. In addition, the application of PCR and related technologies as tools for screening of blood donations raises a number of issues further to those encountered in conventional testing systems, not least the pool creation itself, the requirement for positive tracking of individual donations during pool creation, and resolution of positive pools. Systems developed to undertake PCR testing within the blood bank need to be carefully designed, validated, and monitored to ensure that identified theoretical benefits are realized in routine practice. As with serological screening, the availability of robust automated commercial systems capable of producing consistent and accurate results with full information tracking should be considered as an essential prerequisite for introduction of NAT into a blood screening environment. Although fully automated and integrated systems that meet this requirement are not yet available, a number of laboratories have shown that modular systems based on commercially available technologies can be combined to achieve this goal. Table 2 identifies the key issues that should be considered when designing systems for use in large-scale NAT screening.

Table 2. Some Key Issues in Implementation of NAT for HCV RNA in the Blood Screening Environment

�9 HCV RNA stability in samples from donated blood �9 PCR inhibitors in samples �9 Development of pooling protocols �9 Pool size and system sensitivity �9 Interla boratory varia bility �9 False-positive results and cross-contamination �9 Confirmation, audit and donor management �9 In process control systems and standardization �9 Turnaround t ime--the impact on component release


Hepatitis C Virus RNA Stability in Stored Blood

The stability of HCV RNA during sample storage will be an important consideration for transfusion services developing NAT systems. Loss of detectable genome before separation of plasma has been identified as a concern. 23 Consideration will need to be given to the choice of anticoagulant used for sample collection, the temperature and length of storage before separation of plasma from the cellular components of blood, and also to the temperature and length of storage of separated plasma.

A number of studies have been reported. Unfor- tunately, the results are highly variable, including in one instance a finding that HCV RNA might remain stable in clotted blood for up to 24 hours? 4 Stramer et a125 spiked samples from two HCV seroconversion panels into whole blood containing ethylenediaminetetra-acetic acid (EDTA). 25 Al- though some sample variability was noted, HCV RNA was observed to be stable, with the projected time for 25% loss in log-transformed activity in whole blood exceeding 200 hours, even when stored at 25~ to 30~ Others (Tedder, Grant, and Kitchen, unpublished data) have found similar results. It appears reasonable to conclude that EDTA plasma is the sample of choice and that levels of HCV RNA will remain stable for up to 4 days, and possibly longer, under normal operational conditions.

The plasma preparation tube (PPT~), manufactured by Becton Dickinson (Grenoble, France), contains a solid EDTA anticoagulant with a gel plug. Centrifugation of the whole blood sample results in partitioning of plasma from the cells in

si tu. Systems such as this provide a number of operational benefits, including an ability to freeze samples in the primary collection tube without separation. This facilitates long distance transport- ability and also provides a system for long-term archive storage in the primary tube. However, less expensive alternative strategies are also workable.

Samples for long-term storage should be frozen at - 40~ or lower, higher temperatures having been reported to lead to loss of HCV RNA activity with time. 26

Polymerase Chain Reaction Inhibitors

The presence of inhibitors is a recognized potential problem of PCR testing. It has been suggested that pooling of samples will lead to an increased likelihood of PCR inhibitor activity. 2v This concern

has not been realized in practice. Centers reporting the results of minipool testing have not identified a significant level of inhibitory reactions. The Ger- man Red Cross Centre in Frankfurt has undertaken screening for HCV, HBV, and HIV for over a year using an in-house PCR system containing an internal inhibitor control. Results show that PCR inhibitors are identified in less than 1% of tests for HCV RNA. Valid results on samples with inhibitory activity in the initial test are generally obtained on repeat testing. 28

The likelihood of the presence of PCR inhibitors appears to be reduced with the use of more efficient nucleic acid extraction or capture techniques, particularly so with silica-based procedures popular in current commercial systems.

It is vital to ensure that NAT systems incorporate internal controls that show inhibitory reactions and validate negative results. Commercial test kits, such as the Roche Amplicor version 2.0 assay, include inhibitor and operator detection systems. Detection systems that monitor inhibitor activity only during the amplification stage of the reaction do not, however, control the whole process. Clel- land et a129 have suggested that a more effective control will involve addition of a noncompetitive marker virus before nucleic acid extraction, thus ensuring control of the whole PCR process. 29

System Sensitivity

The sensitivity requirements identified by the CPMP in Europe for plasma derivative production and the Paul Ehrlich Institute (PEI) in Germany represent significantly different challenges. This is not surprising because the aim of the CPMP is to reduce HCV RNA in plasma start pools to levels that will confidently be inactivated by the manufacturing process, whereas the aim of the PEI is to detect potentially infectious blood donations and prevent them from being transfused. The PEI requirement was determined after testing of a number of HCV seroconversion panels and analysis of HCV RNA levels in the "PCR-only" samples. 3~ A minimum detection limit of 5,000 IU/mL of individual donation was established. The CPMP requirement to detect 100 IU/mL of HCV RNA within the plasma start pool, when considered in the context of nucleic acid levels in individual donations, is significantly less exacting. For a 20,000-donation manufacturing pool, testing at this


level would detect only 2 • 10 6 IU/mL at individual donation level.

A number of factors determine the overall sensitivity of the minipool testing system; these are shown in Table 3. Sensitivity in this instance refers to the ability to detect HCV RNA at the individual donation level.

System sensitivity increases as the number of donations within the primary minip0o! decreases. Careful selection of the method uSed for nucleic acid extraction and of tha t used for amplification and detection also is important, as is the Volume of sample tha t can be exposed tO the extraction process.

The system that will be used for HCV NAT within the National Blood Service in England will combine the Qiagen (Hi!den, Germany) extraction system, using the Qiagen robotic processor (Bio Robot 9604, Qiagen), with the Roche Amplicor HCV version 2.0 assay using the automated COBAS system. Results of sensitivity testing using Roche Amplic0r 2.0 assay, following the probit analysis approach recommended by the PE I, identifies that whe n a po01 of 96 donations is used, the 95% detection limit will be 2,000 IU/mL in the donation, and for a 48-donation pool, 1,000 !U/mL (Harrison, unpublished data). Melsert et al, 31 using the Orga, non Technika Nuclisense extraction system combined with the modified Roche Amplicor assay, have reported similar results. These results Show that currently available technologies can enable compliance With the sensitivity limits identified by the PEI for labile components even when a 96- donation minipoo! is used. It seems likely that the final decision on the optimal pool size will be determined by logistical and operational concerns, principally relating to the management of component inventories.

Standards for NAT

Especially in the early phases of NAT development, there was cdnsiderable variation in test

Table 3. Factors Influencing Sensitivity of a Minipool System for Detection of HCV RNA

1. Number of donations within the minipool 2. Nucleic acid extraction system

Type of extraction Volume of sample for RNA extraction Efficiency of extraction process

3. Amplification and detection phase Assay sensitivity Genotype and variant detection efficiency

results both within and between different laboratories. 32 To standardize results for provision of consistent test performances of determined and reproduc- ible sensitivity, a working group was established under the auspices of the World Health Organiza- tion. This project, the Standardisation of Genome Amplification Technology, has led to the development of a number of working standards. These include a working standard developed by the United Kingdom National Institute for Biological Standardisation and ControP 3 and a similar'reagent (Pelipsy) provided by the Central Laboratories of the Dutch Red Cross. These standards have facili- tated comparative studies and an overall improve- ment of performance in NAT procedures. More recently, a World Health Organization International Standard for HCV RNA NAT has been developed. Working standards can be used as run controls; this will improve consistency of performance in NAT. The availability of an International Standard provides a mechanism for calibration, and comparison, of the various working reagents, thereby allowing more meaningful comparison of data produced from various sources. Most importantly, it will facilitate collaborative studies to clarify the relationship between Genome Equivalents (geq) and Inter- national units (IUs). The relationship is likely to vary for different working standards; 1 IU to 4 geq is an approximate figure for the National Institute for' Biological Standardization and Control work- lng standard. This figure will assist comparison for other data sources, pending the availability of results from definitive studies.

Pooling Approaches

Two basic approaches to pool construction can be defined, one based on the intersecting pool concept, the other on a sequential or multi-layered approach. Both approaches have been shown to produce satisfactory results and enable resolution of positive pools to single-donation level.

The intersecting pool allows resolution of a positive minipool in one step. The concept has been described by Mortimer 34 and used by the National Genetics Institute, which undertakes testing on behalf of a number of plasma fractionators. The National Genetics Institute system uses a pool of 512 donations constructed as a three-dimensional cube, of 8 • 8 • 8 donations. In effect, the 512-donation pool is made up of 24 subpools, each containing 64 donations, each donation entering three of the subpools. A single positive donation


thus will result in three positive subpools, the identity of the donation pinpointed by the point of intersection of the three results within the cube. A Tecan Genesis machine (Tecan AG Hornbrechiti- kon, Switzerland) is used to create the subpools using patented software. Pool construction is slow. Initial experience with this type of approach led to concern within the American Red Cross that resolution of a pool containing two or more positive samples would be problematic. 1~ The intersecting pool approach is, however, an elegant and operationally proven system that enables relatively large minipools to be prepared and subsequently analyzed in smaller subpools, when necessary. It is particularly attractive to the source plasma market, where there is no time constraint on resolution of positive results.

The sequential or multilayered pooling approach involves the creation of a series of subpoots of varying size. A primary pool of 480 donations, for example, contains five subpools of 96; final resolution to single-donation level involves a two- dimensional 8 • 12 matrix. Smaller primary pool sizes can be used based on 96, 48, or even 24 donations. /

This type of approach is being adopted by transfusion services aiming to use NAT as a pre-release requirement for labile components. The smaller primary pool size will maximize sensitivity and also minimize the impact of positive results on component inventories and timely release of components.

Management of Initially Reactive Minipools

Protocols will be needed to ensure effective management of minipools found to be reactive in the initial tests. These should comply with the principles used for serological screenin~ assays. In addition, a number of features are Specific to nucleic acid testing and will require consideration.

I n serological testing, the usual approach to investigation of initially reactive results will be to repeat the test in duplicate. Specimens giving reactive results in at least two of three tests will be considered posi'tive. A further test then will be undertaken on a specimen derived from the blood unit to confirm that the correct one has been identified and removed from the inventory.

One particular characteristic of nucleic acid testing requires consideration, namely, the impact of the Poisson distribution on the ability to detect samples close to the sensitivity limit of the test.

When the number of infectious units present within the sample is low, the number of infectious particles contained within a small sample will follow the Poisson distribution as described earlier. In some instances, the number of particles will be above and in others below the limit of detection. In such circumstances, repeated testing on a given sample will not necessarily give concordant results. In practical terms, an initially positive test might be repeatedly negative on further testing despite the sample containing low levels of HCV RNA. Al- though this is unlikely to be significant in the context of plasma pool testing, it might be important when testing is directed at eliminating individual seronegative yet infectious units from the blood supply. One way to overcome inconsisten- cies will be to undertake repeat testing on smaller minipools; that is, in the context of a 480-donation minipool, repeat testing will be undertaken on the five 96-donation pools. Negative results will be required in the 96-donation pool before components "are considered "safe." When 96 donations enter ihe primary pool, resolution can be undertaken u~ing a two-dimensional intersecting pool approach (using an 8 • 12 matrix). The reduction in dilution will lead to an increased concentration of viral nucleic acid in the smaller pool, thus reducing the impact of the sampling effect of the Poisson distribution. This type of approach was discussed at the European Plasma Fractionation Association meeting on NAT held in Amsterdam in June 1998 and was agreed to be the most secure approach to investigation of initially reactive minipools.

False-Positive Results

The nature of nucleic acid testing suggests that specificity will be good. Truly nonspecific reactions resulting from amplification of inappropriate nucleic acid will be uncommon. False-positive results, however, may occur. These might arise because of an error in the detection system but are most likely to result from cross-contamination. This can occur at any stage within the process. Initial experience confirms this to be the case, although, interestingly, the extent of the problem appears to vary significantly.

The initial results obtained within the Red Cross Centre in Northern Westphalia (Hagen) are of particular interest. 11 During 1996, this Centre com- menced nucleic acid testing of donations for HCV, HIV, and HBV. Pools of up to 600 donations were


prepared, serologically reactive donations being excluded. In-house PCR techniques were used. Eighty-eight HCV "RNA-only" donations were identified out of 890,000 donations tested. Three of the implicated donors subsequently developed HCV antibody, a rate consistent with theoretical estimates for HCV seroconversion (see earlier discus- sion). In a further 40 donations, HCV RNA was shown in fresh frozen plasma obtained from the donation or on testing of additional samples from the donor. These donors did not, however, subsequently develop HCV antibody. Platelets from some of these donors were transfused. Unfortu- nately, information on the HCV status of the recipients is not available. The apparently high prevalence of HCV "RNA-only" donations is of concern, and no satisfactory explanation for this finding has been offered. Other Centres, however, have not encountered similar problems. The Red Cross Centre in Frankfurt identified no HCV "RNA- only" donations out of 218,000, with an initial false-positive rate of 0.6%. Serological reactive donations were not excluded from the minipools, and HCV-antibody-positive donations were suc- cessfully identified by the system. 28

Management of Labile Components

A number of commercial fractionators have shown that minipool testing strategies will permit

identification of HCV "RNA-only" donations. 35,36 Some transfusion services are developing systems that will enable results of testing to be available in advance of issue of red cell and platelet components. However, few data are available that confidently show that such strategies will be effective, and most importantly, information identifying the impact on availability of short shelf-life components is currently lacking,

The ideal strategy for implementation of NAT will be one that enables results of testing to be available simultaneously with those of standard serological tests. Minipool testing involves four elements: first, preparation of the pools; second, extraction of nucleic acid; third, the amplification; and fourth, the detection of amplicons. Logistically, this is a much more complex challenge than that posed by serological tests. The Red Cross Centre in Frankfurt reports that results of nucleic acid testing are available simultaneously or shortty after the results of serological testing. However, this requires pooling of samples to be undertaken during the night to allow testing to be completed during

the normal working day. E~cen then, initially reactive results will require further investigation. In such circumstances, all components relating to donations included within the pool need to be held until definitive results are available, and this may take several days. The possibility that such delays will result in reduced availability of labile components has raised doubts about feasibility of using minipool strategies for this purposes

An alternative (interim) approach will be to continue to release labile components bas6~d solely on the results of serological testing, NAT results being used only to assure the safety of frozen plasma components, particularly those destined for fractionation. One likely consequence of this is that components derived from infectious donations will be transfused before the results of NAT are available. Strategies need to be developed to minimize the likelihood of such events, and clear procedures for recall of implicated components once results are known, and for notification of recipients transfused with implicated components will be required. The medicolegal implications of this approach are self- evident and will require the development of effective risk management strategies.

A decision to release fresh components in advance of NAT results being available raises a number of issues in relation to the informed consent process. The risk of transmission of HCV by components derived from donations giving negative results in serological testing will be very low. Should the potential recipient be informed of the possibility that they might be notified after the transfusion that further testing has indicated that the component may be infectious for HCV? Proto- cols for confirmation of early infection will be required, as will consideration of possible treat- ment with antiviral drugs. Education of clinical staff who take responsibility for obtaining informed consent will be necessary.

Greater experience with, and confidence in, minipool testing is necessary to enable development of optimal strategies for labile component management. It is unfortunate that regulhtory pres- sures based on concerns relating to plasma for fractionation will require concurrent validation and implementation of test systems. Data should, however, emerge that will enable standard setting in this and other controversial areas. The recent establish- ment of an Interagency Genome Amplification Testing Task Force within the United States is a


welcome development. 3s Key issues have already been identified, and recommendations are awaited.

FUTURE DIRECTIONS/OTHER AGENTS

Several strategic questions regarding NAT implementation can be identified:

�9 Testing single samples or pools of samples: if the latter, what pool size is optimal?

�9 Use of single virus or "multiplex" systems? �9 Which agents to screen for? With what technol-

ogy? (and will this be determined by patent?) �9 Will NAT replace serology for microbial

screening of blood donations? A number of additional viruses can be identified that might be detected by genomic techniques. If pools of samples are prepared, detection of "extra" viruses could be added to the system. Alternatively, various multiplex combinations of groups of RNA, or groups of DNA viruses, could be developed. However, the question of cost and benefit will require careful consideration. Candidate viruses for genomic screening other than HCV (for which in Europe there is a common mandatory requirement with regard to plasma pools for fractionation) include HIV-1, HBV, HAV, parvovirus B 19, and the recently described GBV-C 39 (a flavivirus-like virus) and TTV (a DNA virus of two distinct geno- types with similarities to parvovirus). 4~ The latter two agents highlight the potential dangers of an ability to screen donated blood by genomic methods. There is no clear evidence that these newly discovered viruses are pathogenic in humans. Deci- sions on the desirability of extending genomic screening beyond HCV must focus on wider issues than the ability of the system to identify viral genomic material. A framework will need to be developed to facilitate evidence-based decision making. Important questions will include: Is the agent pathogenic in humans? Does it pose any real risk to the safety of the blood supply? Will specificity issues entail unnecessary wastage of blood donations? The cost and effectiveness of introduc-

tion of more extensive testing will require very careful assessment. These issues should be treated with great caution. It will be particularly important to ensure that the impact of genomic screening for HCV, in terms of both cost and impact on timely availability of components, is fully understood before consideration is given to testing for additional agents by this type of technology.

Certainly, for the immediate future, NAT for HCV, HIV, or HBV will not be appropriate as a replacement for serological screening of blood. The latter (serology) has a proven track record of highly process-controlled and operationally reliable performance. Newer NAT systems will require validation to show their safety equivalence in these respects. The questions of sensitivity of pooled versus single- sample approaches is also very pertinent to the question of NAT replacing, serology. Even in the longer term, questions of genotype or variant detection sensitivity by NAT and the low genome levels at the tail end of carriage of HBV will require the exercise of great caution before moves to replace serological screening can be considered.

CONCLUDING REMARKS

Regulatory requirements relating to plasma destined for fractionation developed within Europe have signaled the arrival of molecular testing in the blood screening environment. Initially this requirement relates to testing for evidence of HCV, but it is likely that this will be extended to other agents within the next few years. Inevitably, a number of questions, and some concerns, have been raised, particularly so in respect of minipool testing strategies. These will be resolved as experience, and confidence, in the new technologies improves. A number of challenges can be identified that will need to be overcome before the full benefits of this new approach to testing can be delivered. Success- ful implementation will, however, improve the overall safety of blood products available for transfusion, albeit at extra cost.

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pcr testing of plasma pools: from concept to reality

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