PCR (polymerase chain reaction) methods detect viral nucleic acids directly, and therefore can identify recently infected donors who have not yet produced antibodies.
Hepatitis C (HCV), for example, is detectable by PCR around 1–2 weeks after the time of infection, whereas the levels of HCV antibodies, which is the immune system’s response to the infection, are not detectable until up to two months after the time of infection. This is called the window period, in which recently infected donors are not detected by conventional antibody-based assays.
Prior to preparation of the minipools, the plasma samples are placed in racks to enable automated de-capping and pooling.
Polymerase Chain Reaction (PCR) is a NAT-type of method, which is short for nucleic acid amplification technique, in which specific genetic material is amplified to reach detectable levels. In PCR, a certain kind of reagent (primers) is used to target a small but specific part of the virus-genome (deoxyribo-nucleic acid (DNA) or ribonucleic acid (RNA)) in question, and with the help of an enzyme, this small genomic area is amplified over and over again if the target is present. As soon as the small target is amplified, a third component of the PCR reaction (the probe), emits a signal that can be detected by the PCR instrumentation.
All plasma units are tested using antibody/antigen-based methods for the detection of human immunodeficiency virus (HIV), hepatitis C (HCV), and hepatitis B (HBV) at the time of donation. After that, all donations are PCR screened in minipools for detection of HIV, HCV, HBV, HAV, parvovirus B19 (B19V), and on occasion hepatitis E (HEV). Plasma from the USA is PCR tested in the USA due to FDA regulations. All untested plasma of non-USA origin is PCR tested in Stockholm. Our corporate PCR laboratory in Stockholm supplies all Octapharma production sites with individual donation level PCR results. The lab performs minipool testing of all plasma, unless it has been fully PCR-tested elsewhere. Manufacturing plasma pool scale testing is done either in Stockholm or at the Octapharma PCR laboratory in Frankfurt.
Minipool testing is not a regulatory requirement, but is rather an internal strategy to minimise the risk of contaminating a whole plasma pool for production, which would be a tremendous waste of raw material and loss of final product for patients. Minipool testing minimises the number of samples required to be analysed. Instead of testing 480 plasma samples individually, a 480- donation-pool is prepared that contains a small volume from each of the 480 samples. The 480-pool is analysed for detection of viruses by PCR and if a positive PCR result is obtained, the samples are resolved so that the individual, virus-containing plasma unit can be identified. The viruscontaining plasma units are then removed prior to production.
Our PCR laboratory in Stockholm consists of three groups, namely the PCR pooling team that performs the minipooling of samples from plasma units, the PCR analysis team that performs routine PCR analyses of minipool samples as well as plasma pool samples, and my team, the PCR R&D and support section that develops, validates, implements, and supports the PCR methods used in Stockholm. These three teams work both in parallel and as part of one workflow.
We are at the centre of many interesting questions. There is a fundamental curiosity which makes it enjoyable to explore methods and find ways to optimise them.
The barcodes of all plasma samples are scanned before the minipooling process starts, and the identities are handled within the LIMS-PCR system where the connection between the individual sample and corresponding minipool is made.
PCR pooling lab
A normal day in the PCR pooling lab starts with taking the PCR samples that arrived the day before out of the refrigerated room where they have been thawing. The sample tubes are put into racks, de-capped, and put into the minipooling robots, 480 samples at a time. The robot then aspirates a small volume from each sample, distributes it to 96 well microtiter plates and to another tube, the 96-pool tube. Small volumes from all of the 480 samples are thereby distributed in five 96-pools and corresponding microtiter plates. After that, yet another pooling is made from mixing a small volume from each of the five 96-pool samples into one tube, and a minipool containing a contribution from all the original 480 samples, a 480-pool, is made. The barcoded identities of all tubes and microtiter plates are hierarchically organised in the laboratory information management system (LIMS)-PCR system, which is a specialised LIMS platform designed to handle the complexity of minipools. Approximately 10–20 minipool runs are performed per day, which corresponds to the same number of 480-pools containing 5,000–10,000 individual plasma samples.
PCR analysis laboratory
Meanwhile, the PCR analysis laboratory performs nucleic acid extraction and PCR analysis of HIV, hepatitis C (HCV), hepatitis B (HBV), hepatitis A (HAV), parvovirus B19 (B19V), and on occasion also hepatitis E (HEV), on samples either from minipools or plasma pools. On a daily basis, 10–60 minipool samples, including 480- and 96-pools, are fully analysed with 5–6 PCR methods, and on a weekly basis 10–15 plasma pool samples are analysed with 4–5 PCR methods.
PCR R&D and support
The PCR R&D and support team provides the PCR analysis team with support related to the methods, interpretation of complex results, scientific advice, providing input on issues regarding virus safety and contamination, and also the reporting of PCR results. The R&D activities in my team consist mainly of developing new PCR methods for detection of various pathogens, both in plasma, serum, and other samples. The analytical methods must be re-developed from time to time e.g. to comply with regulatory demands, to allow for utilisation of new generations of instruments, to allow for a higher grade of automation etc. After a new method has been developed, it is validated according to current guidelines, meaning that several pre-defined parameters of the method are tested in a stringent manner, such as the determination of analytical sensitivity, evaluation of the method robustness, that all relevant genotypes are detected etc. If the method performance fulfils the pre-defined demands, the validation is approved. Before the method can be implemented, any possible regulatory impact is assessed by international drug regulatory affairs (IDRA). If this is the case, we prepare the documentation for authority submission, and then await regulatory clearance. Once authority approval is obtained, usually within a year, we implement the method in the PCR analysis group for routine testing of minipools and plasma pool samples. All these actions are handled in the change control system, to ensure proper documentation, assessment, responsibilities, and traceability.
The R&D and support team’s work is creative and often not routine, with many lively discussions. The very same curiosity that led me to my career in science, is what drives me here now at work. There is a fundamental curiosity which makes it enjoyable to explore methods and to find ways to optimise them. I enjoy the versatility of my role: one day I am discussing the most intrinsic genomic details of HCV with my team, the next day I am involved in a multi-site discussion on how HIV serology can be interpreted.
Working in our PCR laboratory, we are at the centre of many interesting questions, and it’s great to lead and be part of a team that delivers input to the regulatory aspects of PCR testing. My philosophy is: honour the donor and honour the patient. Every donated plasma unit counts, and thanks to a continuous, high-quality plasma supply and a strong quality organisation, we can provide safe and efficient life-saving treatments to patients around the world.