‘Sterility Testing of Blood Components and Advanced Therapy Medicinal Products’ (Munich, April 29, 2010) Organized by the DGTI Section ‘Safety in Hemotherapy’ - Abstracts

Experience with Bacterial Screening of Blood Products in the Netherlands

Dirk de Korte

Sanquin Blood Supply Foundation, Amsterdam, the Netherlands

Introduction: In a study to the actual bacterial contamination of whole blood units under the conditions as used in the Netherlands, it was shown that about 3 per 1000 units of whole blood resulted in a positive bacterial culture (de Korte et al., Transfusion 2001;41:815–818). Based on these results it was recognized that bacterial contamination of platelet concentrates (PC) from pooled buffy coats would be significant. Therefore, bacterial screening for platelet concentrates (PC) became mandatory in the Netherlands since October 2001. In another whole blood study, it was shown that diversion of the first volume of whole blood reduced significantly the bacterial contamination with about 40% (de Korte et.al. Vox Sang 2002;83:13–16). Therefore, during 2002 and 2003, a pilot study with diversion of the first blood volume during collection of whole blood units was performed to check the effect on the final platelet concentrates (PC) produced from pools of 5 buffy coats. No large problems by the new collection system were met in practice and a reduction of 50% initial positive cultures was shown for PC prepared from 5 pooled buffy coats. Based on these results, diversion became mandatory nationwide in the Netherlands starting July 1, 2004. Before the introduction of diversion the effect of an improved disinfection method of the donor arm was evaluated in the bacterial screening. However, the change from a single application of disinfection fluid to a two-step application of iso-propylalcohol 70% resulted only in 10% reduction of the amount of positive bacterial cultures. Therefore, it was decided to introduce diversion as a more effective intervention. The results of diversion on the screenings results of PC are evaluated.

Methods: After July, 2004, in the Netherlands, all whole blood and apheresis donations have to be performed with a blood collection system equipped with a diversion pouch to collect the first 20–30 ml (and subsequently used for test purposes). After preparation of PC from 5 pooled buffy coats, both an aerobic and a anaerobic bottle are inoculated with 7.5 ml PC each and cultured for 7 days in the BacT/Alert culture system (BioMerieux). Positive cultures are subcultured for confirmation and identification of the microorganisms. PC are released as ‘negative to date’ as soon as the culture has been started.

Results: In the period before implementation of diversion, in total 127,979 PC units were tested, with 1154 initial positive signals (0.90%). After implementation of diversion, in total more than 290,000 PC units were tested (until the end of 2009), with 1286 initial positive signals (0.44%). This means that a 50% reduction of the positive bacterial cultures could be obtained by the introduction of diversion as intervention. The percentage of initial positive units was comparable to the value obtained in the pilot study with 6749 units. Both the frequency of Coagulase Negative Staphylococci (0.27% to 0.13%) and Diphteroid rods (0.42% to 0.18%) were significantly decreased by the intervention studied. The frequency of Bacillus species, gram negative bacteria and other less frequent bacteria showed a negative trend. Initial results for the first two years were published (de Korte et al., Transfusion 2006;46:476–485) and confirmed in the subsequent years. In combination with the bacterial screening, which was preventing potential bacterially contaminated PC to be transfused, it was allowed to extend the shelf life of PC from 5 to 7 days in the Netherlands. In active surveillance during 2 years, it was found that for 160 cases of PC released as ‘negative to date’ which turned positive in the BacT/Alert culture after being transfused no adverse effects were found related to the possible bacterial contamination of the unit (Koopman et al. Vox Sang 2009;97:355–357). The hemovigilance data as reported (www.tripnet.nl) over the period 2004 till 2008 (after introduction of the diversion) showed only 2 serious adverse reactions with high imputability related to bacterial contamination of PC (about 250,000 PC transfused).

Conclusion: Bacterial screening was successfully implemented in 2001, followed by implementation during summer 2004 of diversion of whole blood during collection. The expected result of at least 50% reduction of the bacterial contamination of PC by this intervention was reached after introduction the diversion system in large scale for all collections in The Netherlands. Bacterial screening with release as ‘negative to date’ was shown to be a safe system, with no adverse effects after transfusion of units already released at the moment that the culture turned positive. The extension of shelf life from 5 to 7 days in combination with bacterial screening also showed to be safe, with over a period of 5.5 years after introduction of diversion a low degree of transfusion transmitted bacterial infections by PC, with no fatal cases.

The Reduction of Bacterial Transmission In England

Carl P. McDonald

National Bacteriology Laboratory, NHS Blood and Transplant, London, UK

Introduction: Bacterial contamination remains the major microbial cause of morbidity and mortality following transfusion. In the UK,38 cases of transmission of bacteria by transfusion have been reported, comprising of 10 fatalities. Platelet concentrates accounted for 84% of the transmissions.

Results: National Health Service Blood and Transplant (NHSBT) introduced the interventions of improved donor arm disinfection and diversion. Improved donor arm disinfection was introduced in apheresis donations in 2002, and for all donations in 2007. A reduction in bacterial contamination of 57% was shown in apheresis platelet concentrates and a 65% reduction in clinical cases was observed. The current national donor arm disinfection procedure consists of a single step method with a 30 second application and 30 second drying time, of 2% chlorhexidine gluconate and 75% isopropyl alcohol. Diversion was implemented nationally in 2003. This intervention gave a 66% reduction in bacterial contamination in pooled platelet concentrates, and a 76% reduction in clinical cases.

Diversion and improved donor arm disinfection have been shown to be highly effective interventions, but despite their introduction, transmission of bacteria by transfusion has still occurred from NHSBT blood products. Eight transmissions with three deaths were reported from 2007 to 2010. Bacterial screening will now be introduced on all NHSBT platelet concentrates in 2011 to further reduce the risk of bacteria transmission by transfusion. This will comprise of screening 268,336 platelet concentrates; 214,669 apheresis and 53,667 pooled. A single test will be undertaken and sampling will occur between 36 and 48 hours after collection. Platelet concentrate shelf life will be extended to 7 days.

Conclusion: Bacterial screening in conjunction with diversion and improved donor arm disinfection will further increase the safety of the blood supply.

Canadian Experience with Bacterial Screening in Platelet Concentrates

Sandra Ramirez-Arcos

Canadian Blood Services, Ottawa, Canada

Introduction: Bacterial contamination of blood products is still the most prevalent infectious risk in transfusion medicine despite the completion of a routine donor questionnaire and implementation of improved skin disinfection methods, first aliquot diversion, and bacterial detection methods. Platelet concentrates (PCs) are the blood product most likely to be contaminated due to their storage conditions.

Methods: Canadian Blood Services (CBS) implemented screening of apheresis PCs for bacterial contamination in March of 2004 and of buffy coat (BC) PCs in October of 2005. Platelet screening is performed between 24 and 30 h post-collection. During sampling, between 8 and 10 ml of PCs are inoculated into aerobic BacT/ALERT^® culture (BPA) bottles, which are placed in the BacT/ALERT^® 3D incubator for a maximum of 6 days until they are flagged as either positive or negative. Anaerobic cultures are not done at CBS. Once sampling is performed and other tests are completed, the PCs are put into inventory. Bacterial screening of PCs at CBS is not a pre-release test [1]. If a BPA culture is flagged as positive, the bottle is sent to an external microbiology laboratory for bacterial identification. If the PCs have been issued at the time the initial cultures are flagged as positives, the hospital blood bank is notified to ensure appropriate patient monitoring and testing. When positive PCs have not been transfused, they are removed from the hospital or blood center inventory and if possible, re-sampled for confirmatory cultures. Co-components are also removed from inventory and cultured if possible. Positive second (confirmatory) cultures are sent to CBS, R and D for bacterial identification. A confirmed (true) positive culture is reported when an initial test is confirmed during repeat culture from the blood product, a retention sample, and/or samples from the recipient. The same microorganism must be isolated from all samples. Indeterminate cultures are those when confirmatory tests cannot be performed or interpreted, and false negatives are documented if the initial test is negative but a remaining sample is found positive following testing of a product that has caused a post-transfusion reaction. The same microorganism should be isolated from the component and the recipient [1].

Results: As of March of 2010, 191,355 apheresis PCs have been tested at CBS and 268 (0.1%) initial positive cultures have been obtained. Out of these initial positives, 24 (8.9%) cultures have been confirmed as true positives and 49 (18.3%) were not confirmed as they were discarded or transfused. On average, this represents a 1 in 2,621 chance of bacterial contamination with current testing, including both true positives and suspected/true positives. Bacterial species identified from the confirmed cultures include the Gram-negative microorganisms Enterobacter cloacae and Escherichia coli, and the Gram-positive bacteria Staphylococcus aureus, Streptococcus spp and coagulase negative staphylococci (CoNS). One fatal false negative case was also reported due to transfusion of an apheresis PC contaminated with Serratia marcescens [2]. From October 2005 to March 2010, 181,196 BC platelet pools have been screened and 111 (0.06%) initial positive cultures have been reported. 24 (21.6%) out of the 111 initial positives were confirmed as true positives and 14 (12.6%) were indeterminate. This represents a 1 in 4,768 chance of bacterial contamination including both true and non-confirmed positive cultures. The true positive cultures were contaminated with Gram-positive organisms including CoNS, Streptococcus spp, Micrococcus spp, and Bacillus spp. A false-negative case involving the transfusion of a BC platelet pool was documented, which was found to be contaminated with group A Streptococcus.

Methods for Bacterial Detection in Blood Components

Michael Schmidt

German Red Cross, Institute of Transfusion Medicine and Immunohematology, Frankfurt, Germany

Introduction: Improvements in blood donor screening systems, e.g., the introduction of third and fourth generation antibody assays and nucleic acid testing (NAT)¹, have reduced the risks for the transmission of clinically relevant viral infections to far below the risks for the transmissions of bacterial infections. Therefore, bacterial contamination of blood products represents an ongoing challenge in Transfusion Medicine.

Methods/Results: The ideal screening test should have an extremely high diagnostic sensitivity, a short test time, and a high clinical efficiency. Methods can be grouped into the following three categories:

1. Bacterial detection systems used for routine screening of blood components: Most countries implemented culture methods like BacT/ALERT, BACTEC or Pall eBDS. These systems convinced with a very high analytical sensitivity (less than 1 CFU/ml). A disadvantage is a long incubation period up to seven days. Countries like the Netherlands or the USA which had long experience with blood donor screening for bacterial detection by BacT/ALERT reported about false negative results due to a sampling error. Based on the low bacterial concentration in the final product, the residual possibility that the sample volume processed in the culture system did not contain bacterial colonies was still present, although the final product was still contaminated. Under normal storage conditions, a few CFUs can grow and cause a severe septic reaction; this phenomenon can occur even if more of the product is transfused at the end of the shelf life.

2. Experimental bacterial-detection systems validated in spiking studies and feasible for routine screening of blood products: A generic bacterial detection by 16s DNA systems, by 23s DNA systems, by FACS systems or by the Scan system is reported in the literature. NAT systems included the challenge that PCR reagents might be contaminated with bacterial DNA. Therefore the analytical sensitivity is slightly reduced. All systems achieved sensitivities between 0.5 × 10¹ CFU/ml to 10⁵ CFU/ml. The benefit of these rapid bacterial detection systems is the short testing time, which allows a sampling collection immediately before releasing of blood products.

3. Upcoming detection systems that are only evaluated in spiking studies: New systems are the Pan Gen detection technology (Verax Biomedical Inc., Worcester, MA, USA), a CFDA system as described by Motoyama et al., a new non-invasive continuous O₂ measurement system, an automated enzyme-linked immune sorbent assay (ELISA) presented by Fleming at the AABB 2008, a ATP luminometry presented by Norton or the Bactiflow system. More clinical field trial data are still necessary for these systems.

Conclusions: The reduction of bacterial infections caused by blood products is still a major topic in Transfusion Medicine. A large number of countries have implemented bacterial screening systems for platelets into their routine screening program. Currently, only culture methods are used for routine bacterial screening. Those systems have a high analytical sensitivity, but residual risk still remains due to risks associated with sampling errors. Rapid bacterial detection system might cover this problem by collecting a sample volume at a late time period combined with a short testing time. More field trial data are eagerly awaited to calculate the efficiency of these new screening methods.

ISBT International Validation Study on Transfusion Relevant Bacteria Reference Strains (TRBRS)

Melanie Störmer¹, Julia Brachert¹, Dirk de Korte², Carl McDonald³, Silvano Wendel⁴, Erica M. Wood⁵, Antonio Arroyo⁶, Dana Devine⁷, Jay S. Epstein⁸, Christian Gabriel⁹, Cohava Gelber¹⁰, Ray Goodrich¹¹, Kay-Martin Hanschmann¹, David G. Heath¹², Michael R. Jacobs¹³, Shawn Keil¹¹, Bernd Lambrecht¹⁴, Cheuk-Kwong Lee¹⁵, Jan Marcelis², Susanne Marschner¹¹, Siobhan McGuane³, Marian McKee¹⁰, Thomas Mueller¹⁴, Tshilidzi Muthivhi¹⁶, Annika Pettersson¹⁷, Piotr Radziwon¹⁸, Sandra Ramirez-Arcos⁷, Henk W. Reesink¹⁹, Julieta Rojo⁶, Ineke Rood², Michael Schmidt²⁰, Christian K. Schneider¹, Erhard Seifried²⁰, Ute Sicker¹, Roslyn A. Yomtovian²¹, Thomas Montag¹

¹ Paul Ehrlich Institute, Federal Institute for Vaccines and Biomedicines, Division Microbial Safety, Langen, Germany (Presenter)

²Sanquin Blood Supply Foundation, Amsterdam, the Netherlands

³NHS Blood and Transplant, London, UK

⁴Hospital Sirio Libanês, São Paulo, Brazil

⁵Australian Red Cross Blood Service, Melbourne, Australia

⁶Centro Nacional de la Transfusion Sanguínea, México

⁷Canadian Blood Service, Ottawa, ON, Canada

⁸Food and Drug Administration, Rockville, MO, USA

⁹Austrian Red Cross, Blutzentrale Linz, Austria

¹⁰American Type Culture Collection, ATCC, Manassas, VA, USA

¹¹CaridianBCT Biotechnologies, Lakewood, CO, USA

¹²Walter Reed Army Medical Center, Washington DC, USA

¹³Department of Pathology, Case Western Reserve University, Cleveland, OH, USA

¹⁴German Red Cross, NSTOB, Springe, Germany

¹⁵Hong Kong Red Cross Blood Transfusion Service, Hong Kong, China

¹⁶South African National Blood Service, Weltevreden Park, South Africa

¹⁷VU University Medical Centre, Amsterdam, the Netherlands

¹⁸Regional Centre for Transfusion Medicine, Bialystok, Poland

¹⁹Academic Medical Center, Amsterdam, the Netherlands

²⁰German Red Cross, Frankfurt/M., Germany

²¹National VA Quality Scholar Fellow, Louis Stokes VA Medical Center, Cleveland, OH, USA

Introduction: Bacterial contamination of platelet concentrates (PCs) remains a persistent problem in transfusion. The Subgroup on Bacteria of the ISBT Working Party on Transfusion-Transmitted Infectious Diseases organised an international study on Tranfusion-Relevant Bacteria Reference Strains (TRBRS) to be used as a tool for development, validation and comparison of both bacteria screening and pathogen reduction methods. At present, no international transfusion-relevant bacterial strain panel exists for the investigation of blood component-related bacterial contamination.

Methods: Blinded TRBRS were prepared by the Paul Ehrlich Institute (PEI) and distributed, in collaboration with the American Type Culture Collection (ATCC), to 14 laboratories from 10 different countries. The deep-frozen bacteria suspensions were defined in identity and count. The participating laboratories were asked to enumerate, identify to species level, and to verify bacterial growth in PCs.

Results: The study was performed in response to the lack of bacterial standards reflecting species and strains relevant to the evaluation of blood components. The results of the study demonstrated the stability of the prepared TRBRS and consistency of results in a large number of transfusion laboratories all over the world. Therefore the TRBRS could be considered as a suitable tool in validation and assessment of methods for improvement of bacterial safety of blood components.

Conclusion: The study results were reported to the WHO Expert Committee Biological Standardisation (ECBS) for consideration and feedback regarding further steps required for development as international reference materials. It is intended to enlarge the number of organisms within the panel in the future.

PCR Detection of Bacterial Contaminations in Blood and Tissues

Christian Gabriel C., Katja Hofer, Martin Danzer

Red Cross Transfusion Service of Upper Austria, Linz, Austria

Introduction: The current gold standard in testing blood products on bacterial contaminants is the automated blood cultivation. Although the method is very reliable, the major constraint is the fact that blood products are commonly issued prior to the final testing result. Positive signals may lag some time and therefore it is not surprising, that blood products with bacterial contaminants may be transfused before they may be recalled [1, 2].

Methods: We developed a real time PCR-protocol with 7 hybridisation probes for the detection of bacterial 16s rDNA [3]. A broad range of bacteria may be detected with this approach in a sensitivity range between 5 and 15 CFU/ml. The sensitivity level is not uniform for all species and this points to the fact, that the extraction procedure is not producing equal amounts of DNA. On the other hand the use of CFU/ml in the standardisation of PCR procedures is misleading. This does not reflect the amount of DNA and therefore is not the correct approach to validate PCR assays in terms of sensitivity.

Results: In any case some facts must be seen with caution: enzymes are often contaminated with recombinant bacterial genomic material and any form of inactivation of enzymes leads to loss of activity of these enzymes. We screen batches of enzymes on background contamination, select enzyme batches with the lowest amount of bacterial contamination and use filtration procedures to purify selectively the bacterial DNA. Furthermore additional enzymes, like UNG, which may be usually added in PCR reactions are avoided. To improve the extraction efficiency of Gram-positive bacteria a specific extraction procedure may be added. PCR methods may be applicable in screening blood products prior to issue. This is especially important for platelets, which are issued within three days after donation. The sensitivity of automated blood cultures seems to be higher, but only in the case of a low level contamination, which generates a positive signal after 5–7 days. This may be clinically irrelevant, as it is well known, that platelets with a low shelf life generate better clinical effects.

Conclusion: The PCR method, although not as sensitive as the automated blood culture after prolonged incubation, proved to be safe and reliable enough to test platelets and due to the fact, that most apheresis platelets are issued within 24 hours in our institution the safety is high enough to use the PCR method in daily routine.

Microbial Safety of Advanced Therapy Medicinal Products (ATMPs) – Squaring the Circle

Thomas Montag, Utta Schurig, Ingo Spreitzer, Christian K. Schneider, Melanie Störmer

Paul Ehrlich Institute (PEI), Langen, Germany

Introduction: Guaranteeing microbial safety of advanced therapy medicinal products (ATMPs), reaching from hematopoietic stem cells up to chondrocytes for knee repairing presents a so far unsolved or at least, only partly solved problem. Bacterial contamination of parentally administered medicinal products have become rare events in the past few decades, which is basically due to the quality of the manufacturing processes in the pharmaceutical industry. In contrast, the manufacturing processes of cell based medicinal products show much less defined conditions.

Methods/Results: Sterility of source materials cannot be guaranteed and the hitherto known procedures for sterilization are, as a rule, not feasible. While sterile filtration is not possible due to the ratios of the orders of magnitude between bacteria and fungi on the one hand and animal cells on the other, heat treatment such as autoclaving or radioactive irradiation can damage or destroy the product. Thus, the sterility of the final product cannot be guaranteed. Considering the extremely short shelf life of many cell therapeutics, sometimes only a few hours, the results from established methods for sterility testing are often available too late. Moreover, sterility of a restricted sample does not guarantee sterility of the whole preparation. Small amounts of residual bacteria in the product will be overlooked because of sampling error and can grow up to enormous numbers during storage. In most cases, conventional methods for pyrogen testing are also not applicable for ATMPs. Considering these problems, there are some parallels in the warranty of microbial safety of cellular blood components and cell therapeutics. Therefore, the experiences collected in transfusion medicine in the past decades can be successfully used in a safe production of cell based medicinal products (e.g. donor interview, donor exclusion criteria like chronically persistent bacterial infections, closed system for production like blood bags). Considering the extremely short shelf life of much cell therapeutics there is a need for new principles in rapid bacteria detection and pyrogen testing. The requirement for scientific but also regulatory efforts is immense. There is a need for amendments to regulations and for establishment of new regulations as well as for advising the manufacturers how principles of asepsis in complex and complicated manufacturing procedures should be considered. In such a way the use of antibiotics in the manufacture of cell therapeutics, from the starting material to the finished product is still wide-spread. Antibiotic additives, however, do not protect the products, but may damage them, thus putting the patient at risk. In conclusion, a new understanding is required as there are fundamental differences to the approved and well defined manufacturing processes of conventional drugs.

Conclusion: Therefore the warranty of microbial safety of cell based medicinal products is a challenge for industry and authorities because as already indicated, microbial safety of a great number of ATMPs cannot currently be guaranteed. On the other hand, it is not compatible with medical ethics to deprive patients of the fascinating perspectives of advanced therapies. Therefore, we have no other choice but to face the squaring of the circle.