NEWS May 2016
Pre-to-Post Transplant Reach Allenex, the parent company of Olerup, has been acquired by CareDx. The acquisition was completed on April 14, 2016, following a successful tender offer for Allenex shares. The combined company has major operations and direct distribution channels in the US and Europe. Additional distribution agreements provide a global reach that covers five continents. Read more about CareDx, the acquisition and how it will positively impact the availability of new products and services in the future.
New product line and new team members A few words from Roswitha Keller General Manager, Olerup GmbH. ■ Page 2
Donor-derived cell-free DNA The use of biomarkers to help clinicians assess post-transplant risk presents an opportunity to improve long-term outcomes ■ Page 6
■ Page 3
HLA typing using Olerup SSP® kits and the QIAxcel® Advanced System
A full typing in 1h Read more about QTYPE® and how we can achieve an 11 loci typing in one hour using only a third of the potential of a 384 well plate ■ Page 5
SSP usually requires the separation of the amplification products on a standard agarose gel with gel documentation and manual data entry into the typing software. ■ Page 8
Why Olerup should be your top choice for SSP Olerup is dedicated to updating our primer mixes to achieve the best possible kit resolution ■ Page 9
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New product line and new team members Introduction of Olerup RT-PCR kits The innovative Olerup RT-PCR kits, called QTYPE® that many of our readers have been waiting for will shortly be available to our customers in USA, Europe, the Middle East and Asia. How can you order them? If your laboratory is Germany, Austria or in the BeNeLux countries, then you know that you can order them directly from Olerup GmbH in Vienna and if you are in the US, they will be available through our office in West Chester, PA. But if your laboratory is situated elsewhere did you know that Olerup GmbH has a very well established network of highly competent distributors who specialize in supplying products for the HLA and Blood Bank market? Olerup has more than 30 distributors worldwide and many of them have been supplying customers with Olerup products for more than 15 years. Our distributors continue to work with us because experience has shown them the high quality of the Olerup products. Additionally, they place great value on the fact that Olerup is the World’s number one SSP supplier, having reached and maintained this position by always supplying up-to-date top class products such as the new Olerup RT-PCR kits. The introduction of the new Olerup RT-PCR kits will once again provide HLA and Blood Bank laboratories with products that will greatly facilitate their typing work.
and Slovenia. Anke also supports the GmbH Distributors in parts of the EU. Last, but not least, Mr. Paul Kyle recently joined us as Regional Sales Manager for parts of Europe, the Middle East, Northern Africa as well as Asia. The three of them have worked in the HLA fields for up to 20 years and more. All three of them have a wealth of experience in the field of HLA typing and in particular in training and educating our customers how to make the best out of all the different Olerup products. The core team located in Vienna, consisting of the customer care department, marketing support and the finance department, all equally have their part in supporting the sales team, fulfilling and providing excellent customer service and adhering to customer needs in a professional and timely manner. Olerup + CareDx Finally, if you haven’t heard already, Olerup has entered into a new collaboration with CareDx (http://www.caredx.com) and we would like to share with you the excitement we feel about the opportunities that this will open up, not only in the field of pre- but in particular also in the field of advanced post-transplant diagnostics. You can read more about this collaboration, what it brings to Olerup and our possibilities to serve our customers with even more exciting new products and services in the very near future in this issue of Olerup News.
Allenex Acquired by CareDx The parent company of Olerup, Allenex AB, has been acquired by CareDx, Inc. CareDx is a San Francisco headquartered molecular diagnostics company focused on the discovery, development and commercialization of clinically differentiated, high-value diagnostic surveillance solutions for transplant patients.
commercialization of molecular diagnostics. Both companies are actively working on integration to create a seamless experience for providers and patients alike. As developments continue, we will be sure to notify the Olerup community through Olerup News, press releases and other publications.
CareDx completed the acquisition of Allenex on April 14, 2016, following a successful tender offer for Allenex shares in Sweden. The combined company has major operations and direct distribution channels in the US and Europe. Additional distribution agreements provide a global reach across five continents.
“This combination creates a focused diagnostics company throughout the pre-to-posttransplant continuum,” said Peter Maag, President and CEO of CareDx. “Leading up to the merger, we have found an equally dedicated team in Allenex to advance innovation and care in this tremendously challenging field of medicine. Our combined resources will accelerate the development of new tools for the personalized management of transplant patients in an exciting genomic era. We wel-
The management teams of Allenex and CareDx bring decades of experience in transplantation science, genomic discovery and the
come Allenex employees and their partners worldwide to the team.” For more information please visit www.allenex.com and www.caredx.com
Peter Maag President and CEO at CareDx
Olerup and venues 2016 / 2017 Olerup products and representatives can be seen throughout 2016 and 2017 at the following meetings, conferences and exhibitions. We look forward meeting you.
MEET US AT
Apart from the SSP and RT-PCR kits Olerup also supplies SBT products and it may interest our readers to know that the world-renowned Stefan Morsch Foundation in Birkenfeld, Germany – one of the biggest registry labs worldwide – is one of our most important reference labs for the SBT Resolver product line in Europe. Olerup in Vienna Who makes up Olerup’s team in Vienna? Ms. Ines Müller is our Key Account Manager for Germany and parts of Austria while her colleague, Dr. Anke Steiner, is the Key Account Manager for the Netherlands, Austria,
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Roswitha Keller General Manager, Olerup GmbH
East-West Immunogenetics Conference: April 21st – 23rd Wroclaw, Poland
BSHI September 13th – 14th Oxford, UK
EFI May 11th – 14th Kos, Greece
ASHI September 26th – 30th St. Louis, USA
Birkenfelder Tage June 1st – 3rd Birkenfeld, Germany
Eurotransplant September 29th – 30th Leiden, Netherlands
DGI / DGTI September 7th – 10th Nürnberg, Germany
Arab Health January 31st – February 2nd, 2017, Dubai, UAE
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The QTYPE Real Time PCR System – the quickest way to a full typing. ®
Utilizing a third of the potential With 384 total wells and four colours, we have a total of 1,536 potential reaction wells. As one channel is reserved for the internal amplification control, three channels can be used for HLA-specific reactions. This gives a potential of 1,152 HLA-specific reactions in a 384 well plate. A lot of additional space to grow into!
SCORE™6 Workflow
Patient • Enter patient information • Enter sample information
Test • Associate samples with tests • Tests can be QTYPE® or Olerup SSP® kits • Import QTYPE® or QIAxcel® data
Analysis • Analysis is performed automatically • Review data • Authorise results
Report • Assemble report • Print or save report
ACCUTYPER® Real Time PCR Instrument Once the QTYPE® test has been set up, it is time to place it into an instrument. QTYPE® will be compatible with the most popular real time cyclers currently available on the market. In addition, Olerup will provide the ACCUTYPER®, an affordable yet fully-featured real time PCR instrument. It has a simple interchangeable 96 and 384-well block which can be switched in less than 1 minute and can excite and detect reactions in up to 4 channels. The ACCUTYPER® run for QTYPE® analysis can be launched directly through SCORE™6, making a seemless, streamlined and integrated workflow. Once the run is completed on the ACCUTYPER®, the results will be directly imported into SCORE™ and analysed. The instrument also comes delivered with an open research software which can be used for other assays - whether they are probe based, end stage or melt curve. For users with other real time PCR instruments, the workflow is also simple. The instrument is controlled from the manufacturer’s software and at the end of the run the data should be exported. SCORE™6 can then import this data and automatically interpret the genotype. SCORE™6 Interpretation Software The HLA typing software for analysis of Olerup SSP® results you know so well has been upgraded and improved. In addition
to a face-lift, the underlying database technology has been upgraded in SCORE™6 for improved reliability. The workflow has been modified slightly to include 5 screens: Home, Patient, Test, Analysis and Report. All typing information from your previous version of SCORE™ can be easily imported into SCORE™6 to ensure continuity of your typing information. All existing tools and options from SCORE™5 can be found in SCORE™6, and many more. A full typing in an hour Currently a run with QTYPE® 11 on a Roche Lightcycler is 43 minutes and when adding time for preparing the plate and the analysis of the result we have a full 11 loci typing in around an hour”, says Ben Passey, Development Manager at Olerup.
PREPARE
QTYPE HLA Typing Kits Format wise, the QTYPE® HLA Typing Kits resemble our SSP products since they contain pre-dispensed, dried reaction mixes in each well. Since QTYPE® uses hydrolysis probe technology, these kits also contain a sequence-specific reporter probe in each well. Due to the presence of SSP primers and a hydrolysis probe, each reaction uses three cis-located polymorphic regions to determine ®
specificity. Clearly, this increases the resolution power of each individual mix. Included in the kit is a hot-start master mix which contains the necessary components for PCR, meaning you just need to add your DNA and dispense the mixture across the plate. Only 3 µg of DNA is required for a full low to intermediate resolution 11 loci typing. Seal the plate using the included optically-clear sealing sheet, briefly centrifuge and place it in the real-time cycler. Data will be collected in real time, eliminating primer dimers and PCR artefacts with no waiting for end point melt curves.
10 min
RUN
Multiplexing Power - Hydrolysis Probes The Olerup QTYPE® HLA typing kits use the hydrolysis probe technology, a powerful and flexible multiplexing tool which allows us to grow with the HLA database. Briefly, hydrolysis probes contains a fluorescent re-
port molecule on one end and a fluorescence quencher molecule on the other end. When on the probe, they are in such close contact that all fluorescence emitted by the reporter is absorbed by the quencher molecule, resulting in no net signal. The probe is sequence specific and is designed to bind between the two PCR primers. During the extension step, the PCR polymerase cleaves the hydrolysis probe, freeing the fluorescent probe into the supernatant. With each cycle, the real time instrument automatically monitors the accumulation of fluorescence over time.
<50 min ANALYSE
Olerup SSP® is proud to present the QTYPE® Real Time PCR System enabling rapid and precise HLA typing at a low to intermediate resolution for samples that require a fast turnaround time. The QTYPE® Real Time PCR system consists of three components QTYPE® HLA Typing Kits, the ACCUTYPER® Real Time PCR Instrument and SCORE™6 interpretation software. A seemless integration between the kit, instrument and intrepretation software will streamline the laboratory workflow. The QTYPE® HLA Typing Kits and SCORE™6 can be used with the Roche LightCycler 480 II. Work is ongoing to validate additional real time PCR instruments, such as the ViiA7 and QuantStudio 6 & 7.
5 min
• Prepare DNA/Master Mix • Dispense plate • Seal plate • Vortex and centrifge • Set up instrument (automatic on ACCUTYPER®) • Insert plate • Run • Transfer raw data (automatic on ACCUTYPER®) • Automatic data analysis • Review results
Ben Passey Development Manager, Olerup SSP® AB
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Donor-Derived Cell-Free DNA (dd-cfDNA): An Emerging Biomarker of Allograft Health in Single Solid Organ Transplant Recipients A significant objective of post-transplant care is to avoid rejection while minimizing unwanted side effects from immunosuppression therapies. The use of biomarkers to help clinicians assess post-transplant risk presents an opportunity to improve long-term outcomes1,2.
Solid Organ Transplant
The approach by CareDx enables identification of allograft DNA without requiring independent genotyping of the donor and the recipient.
Continuing to lead the way in sequencing based typing
Data fully compatible with Assign™ SBT sequence analysis software that includes analysis of non-coding regions. SBT Resolver™ Amplicon Coverage map 5’UTR Exon Intron Exon Intron Exon Intron 1 1 2 2 3 3
Exon Intron Exon Intron Exon Intron Exon Intron Exon 4 4 5 5 6 6 7 7 8
Quiescence
Active Injury
Figure 1. AlloSure measures the percent of donor- derived cell-free DNA in the total cell-free DNA present in blood from a solid organ transplant recipient.
Class II updates: : Complete sequencing of all exon 3 allele groups in addition to exon 2 : Complete coverage of exon 2 and exon 3
AlloSure: A Clinical-Grade dd-cfDNA Test CareDx has developed AlloSure, a proprietary analytically validated test using Next-Generation Sequencing (NGS), to quantify dd-cfDNA present in the blood of solid transplant recipients.
: Complete coverage of exon 1, 3, 4 and 5 in addition to exon 2
cation and sequencing of 266 regions of the human genome. Using novel computational analysis, the fraction of donor-specific DNA is calculated. A detailed description of this process is available7.
AlloSure utilizes targeted amplification of single nucleotide polymorphisms (SNPs) to identify non-recipient (donor) DNA in the blood of transplant patients. This involves the extraction of cfDNA from stabilized plasma (Streck BCT), followed by amplifi-
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0197 Please note that SBT Resolver™ HLA-DRB3, DRB4, DRB5 and B57 are for Research Use Only.
Designed, developed and manufactured by Conexio Designed, developed and manufactured by:
Qarad bvba Cipalstraat 3 B-2440 Geel Belgium
www.olerup.com SBT-150828B-1
Donor-Derived Cell-Free DNA (dd-cfDNA) and Transplantation Numerous studies using various quantitative methods have shown that allograft DNA is detectable as a fraction of total cfDNA in a recipient’s plasma4,5. This can be called dd-cfDNA. Next-Generation Sequencing (NGS) has been used as a tool for the detection of dd-cfDNA. The hypothesis that acute rejection leads�to the elevation of dd-cfDNA is supported by several publications6.
Conexio’s sequencing based typing kits for HLA Class I and Class II genes, covering all the required NMDP null alleles.
dd-cfDNA in Blood
Cell-Free DNA (cfDNA) While DNA is most commonly found within cells, there are trace amounts of DNA that can be found circulating in an individual’s bloodstream. This DNA is a consequence of release from cells and is commonly referred to as circulating or cell-free DNA (cfDNA). cfDNA as a biomarker has been used to detect pathologies in other areas of healthcare such as oncology and prenatal testing. cfDNA and Solid Organ Transplantation The presence of allograft donor-specific DNA circulating in the blood stream of transplant recipients is thought to result from organ injury and was first detected utilizing polymerase chain reaction (PCR) in gender mismatched transplant recipients3. By applying the latest in genomic technology, clinicians may be able to use cfDNA as a biomarker in solid organ transplantation.
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Pioneering since 2002
www.olerup.com
References 1. Starling et al. Multicenter Analysis of Immune Biomarkers and Heart Transplant Outcomes: Results of the Clinical Trials in Organ Transplantation-05 Study. Am J Transplant 2015 Aug 10 [Epub ahead of print]. 2. Gielis et al. Cell-Free DNA: An Upcoming Biomarker in Transplantation. Am J Transplant 2015 Oct; 15(10):2541–51. 3. Lo et al. Presence of donor-specific DNA in plasma of kidney and liver transplant recipients. Lancet 1998 May 2; 351 (9112): 1329–30. 4. Snyder et al. Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci USA. 2011 Apr 12; 108 (15):6229–34.
5. De Vlaminck et al. Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci Transl Med. 2014 Jun 18; 6 (241): 241–77. 6. De Vlaminck et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc Natl Acad Sci USA. 112(43):13336 (2015). 7. Woodward et al., Proficiency Testing for and Surveillance of Heart Transplant Health Using Donor-Derived Cell-Free DNA. J Mol Diag 17:859. 2015.
0197 Please note that SBT Resolver™ HLA-DRB3, DRB4, DRB5 and B57 are for Research Use Only.
EC REP Qarad BVBA, Cipalstraat 3, B-2440 Geel, Belgium
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HLA typing using Olerup ® ® SSP kits and the QIAxcel Advanced System SSP usually requires the separation of the amplification products on a standard agarose gel with gel documentation and manual data entry into the typing software. The QIAxcel® Advanced instrument is a capillary electrophoresis system that enables automated separation and analysis of up to 96 samples in about 24 minutes. The generated data can then be directly transferred to the Olerup version of Helmberg-SCORE™ software. The QIAxcel®Advanced system therefore enables a highly automated and fast result generation with minimal hands-on time and no exposure to toxic gel stains like ethidium bromide.
Olerup and Qiagen have recently agreed upon a new Joint Improvement Program of the QIAxcel® Advanced System for Olerup SSP® data generation and automatic data transferal into the Olerup version of Helmberg-SCORE™. The program encompasses the development of optimized QIAxcel® analysis settings for Olerup SSP® fragments and the design of a new pre-made process profile for customers using Olerup SSP® with the QIAxcel®. The companies will also publish a step by step guide on the complete procedure covering setting up the QIAxcel®, optimized SSP fragment analysis in the Screengel®
software, and import of results into Helmberg-SCORE™. Qiagen and Olerup agreed to closely work together to further improve their support of new and existing customers using Olerup SSP® with the QIAxcel® Advanced.
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Why Olerup should be your top choice for SSP Olerup is dedicated to updating our primer mixes to achieve the best possible kit resolution Olerup SSP® AB does not just supply the same SSP kits year after year. We provide SSP kits that are up-to-date with the recently released allele database at the time of batch manufacturing. Up-to-date kits, of course, means more than just updating kit documentation. It is about updating the actual primer mixes to improve resolution of the kit to address newly assigned alleles. This approach is the Olerup
approach. It demands continuous investments into the product line, a dedicated production lab, as well as significant pre-production primer mix optimization. Additionally, updating kits for new alleles is not only adding new wells and expanding the kit formats. Much work is put into multiplexing in order to keep the product formats
as slim as possible. Table 1 below reflects our recent work in this area. New batches that were produced during 2015 included 4307 unique primer mixes. Of these, 22% were new designs, either through multiplexing within previously existing designs (19.6%) or through adding new wells to the kits (2.4%).
Table 1. Outlines the number of primer mixes (wells) that have been produced and updated for new alleles across the complete Olerup SSP® product line during 2015.
Olerup SSP® allele updating work in numbers across the product line, 2015 Total number of produced and unique primer mixes (wells)
4307
Out of which: Multiplexing instances for resolution of new alleles
764 (19,6%)
Added wells
113 (2,4%)
Sum of changes on primer mix level for allele updating
877 (22%)
We take great pride at Olerup SSP® AB in our work to continuously update the primer mixes prior to batch production. This allows us to provide SSP trays that are up-to-date in the truest sense. Over the years, the increasing number of assigned alleles has generated many challenges to the manufacturers of HLA typing assays. We have adopted strategies to address these challenges and are straightforward in providing customers with information about our improvements and changes.
QiAxcel® data and corresponding images can easily be imported into SCORE™ and are shown side by side to assist reviewing.
Flexible solutions for SSP low resolution typing In 2012, Olerup SSP® AB launched the expanded versions of the single locus low resolution trays, giving our customers the choice between the classical screening/combi formats and the expanded single-locus formats with increased resolution. In 2015 we can confirm that our decision to move in this direction was a good one. The outcome is increasingly popular low resolution kits with more redundancy, greater resolution, and increased resistance to ambiguities.
A high resolution line which keeps up The updating strategy for our high res kits has also changed over the years. To be able to accommodate the large sets of newly assigned alleles, Olerup has decided to focus high resolution kit updating on confirmed alleles, which are defined by the IMGT/HLA database as an HLA allele that has been sequenced by more than a single laboratory or from
multiple sources. This status is updated for each new database. We consider all nulls, as well as polymorphisms outside of the region encoding the peptide binding domain.
Currently, the HLA-A, B, C, DRB1, DQB1 and DPB1 high resolution kits are updated according to this strategy prior to each batch production. The Olerup SSP® Add-ons - Time saving solutions to meet requirements As a result of new null allele requirements in 2015 by NMDP for unrelated HSCT, Olerup SSP® AB has just recently expanded the “Add-on” line to also include kits specifically targeted these nulls. The result is a product portfolio of small SSP kits for smart solutions covering all null allele testing required by the NMDP.
Anna Hedlund, Head of R & D at Olerup SSP®
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Olerup - Global distribution to HLA laboratories At Olerup, we strive to continuously develop and improve our web services. We believe that information and news of our products and companies should be easily accessible for users. If you have not recently visited our web pages, please do so and see how they can be of value for you. Olerup web pages are easiest to access through www.olerup.com, the portal to our
distribution network. Among other information, you can find news about our companies, see what meetings Olerup will attend and, of course, get further information about our different products.
has been working with us for many years and are well-established partners to HLA laboratories in their respective region/country. Nevertheless, every year we add new distributors to our global network.
Distributor contact information is also easily accessible at the web page. Olerup is currently represented in more than 50 countries around the world. The majority of our distributors
A Tool for Enhanced Risk Assessment
Improving outcomes in kidney transplantation: Non-HLA antibodies and Enhanced Clinical Risk Assessment The XM-ONE® transplantation cross match is used prior kidney transplantation, to detect non-HLA antibodies against the donor’s endothelia. Results from the first clinical trial using XM-ONE®, published in February 2009 in the medical journal Transplantation1, demonstrated that XM-ONE® could predict rejection reactions in patients that were approved for transplants using test methods that are considered standard. Later publications have shown that XM-ONE® is a valuable complement to traditional antibody testing prior organ transplantation.
CONTACT US USA
GERMANY/AUSTRALIA/BENELUX
Olerup Inc Orders and support: info.us@olerup.com
Olerup GmbH Orders and support: support-at@olerup.com
Tel: +1-877-OLERUP1 Fax: +1-610-344-7989
Tel: +43-1-710 15 00 00 Fax: +43-1-710 15 00 10
901 S. Bolmar St., Suite R West Chester, PA 19382
Lowengasse 47/6 1030 Vienna, Austria
NORDIC (DENMARK, FINLAND, ICELAND, NORWAY, SWEDEN AND THE BALTICS)
OTHER REGIONS
XM-ONE® is CE marked for sales in Europe and has been cleared by the FDA for sales in the U.S. Read more about XM-ONE® at www.absorber.se Breimer, M, Rydberg, L, Jackson, AM et al, Multicenter evaluation of a novel endothelial cell crossmatch test in kidney transplantation, Transplantation 2009, 87(4):549-556.
To place your order please use the Nordic Web Shop or contact our sales team through info@olerup.com
Olerup product lines are in other regions made available through our network of specialized distributors. To find your distributor please go to http://www.olerup.com/distributors/
Tel: +46-8-50893900 Fax: +46-8-7178818 Franzéngatan 5 112 51 Stockholm, Sweden
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OLE-160425A-1
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Introducing QTYPE
®
Speed and Precision in Real Time • QTYPE® is a robust and easy-to-use alternative for rapid HLA typing. • Use of hydrolysis probes provides the multiplexing power needed to handle future allele expansion. • Data collected in true real time eliminates primer dimers and PCR artefacts with no waiting for end point melt curves. • Seamless analysis is performed by our proven SCORE™ software.
QT-160425A-1
For Research Use Only.
Manufactured by:
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www.olerup-ssp.com
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