ISSUE 2, September 2016
OLERUP QTYPE – a full typing within one hour
QTYPE – a new standard for speed and resolution
Read more about QTYPE and how we can achieve an 11 loci typing within one hour using only a third of the potential of a 384 well plate. ■ Page 6
■ Page 2
®
A few words from Gordon Hill, General Manager at Olerup Inc.
CareDx® – Your Partner in Transplant Care At this year’s annual meeting you will meet your old Olerup colleagues in a different format.
■ Page 3
AlloMap® – Personalizing Care for Heart Transplant Patients
AlloSure , a new tool for monitoring transplant patients ®
Read more about CareDx’s new service for transplant patients. AlloSure is a clinical-grade proprietary NGS based test to detect donor-derived cell-free DNA (dd-cfDNA) in solid organ transplant recipients.
■ Page 8
AlloMap is a non-invasive blood test that uses genomic technologies to identify the absence of cardiac rejection.
■ Page 4
Olerup should be your top choice for SSP® Olerup is dedicated to updating our primer mixes to achieve the best possible kit resolution.
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CareDx news SEPTEMBER 2016
Advancing the pre- to post-transplant continuum It is with great pleasure that we bring you the first installment of CareDx news. In April 2016, CareDx, Inc. based in Brisbane, CA acquired Olerup’s parent company Allenex AB. Both companies have well-
established core products in transplantation, the combination of which creates a focused diagnostics company covering the pre- topost transplant continuum. I encourage you to turn the pages and learn more about our
new venture together as we join forces to bring you new, innovative transplant diagnostics.
QTYPE – a new standard for speed and resolution We are excited to launch QTYPE 11 and our new, sleeker SCORE™ 6 software. With a run time of just 43 minutes on the Roche LightCycler 480 II®, and full HLA typing of 11 loci within one hour, QTYPE will set a new standard for speed in your laboratory.
We sincerely hope you have an opportunity to come visit us at ASHI booth #302 and invite you to join us during our Lunch Symposium on:
Our multiplexed TaqMan approach provides the resolving power necessary to combat the persistent description of new alleles. Even with using only 1/3 of the plate capacity, QTYPE resolves the majority of single antigen bead alleles.
DATE: Wednesday, September 28, 2016 TIME: 12:30 pm – 2:30 pm LOCATION: Regency E, Second Floor at the Hyatt Regency St. Louis at the Arch
Best regards, Gordon Hill
CareDx and venues 2016 / 2017 CareDx 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 EVENT
DATE
LOCATION
AIBT
October 6th – 8th
L’Aquila, Italy
Arab Health
Jan 31 – Feb 2
Dubai, UAE
ISHLT
April 5 – 8
San Diego, USA
EFI & DGI Joint Meeting
May 30 – June 2
Mannheim, Germany
American Transplant Congress
April 29 – May 3
Chicago, USA
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CareDx news SEPTEMBER 2016
CareDx launches QTYPE at ASHI At this year’s annual meeting you will meet your old Olerup colleagues in a different format. In April this year CareDx acquired Allenex and the Olerup portfolio. CareDx, Inc., headquartered in Brisbane, California, is a global molecular diagnostics company focused on the discovery, development and commercialization of clinically differentiated, high-value diagnostic solutions for transplant patients. By adding the Olerup products to the portfolio CareDx is able to provide solutions within both pre- and post-transplant care. Following the acquisition, CareDx now has major operations and direct distribution channels in the US and Europe. Additional agreements provide a global reach across five continents.
The management team at CareDx has added Anders Karlsson, previous CEO of Allenex and Olerup, as the Chief International Business Officer. This global organization brings together decades of experience in transplantation science, genomic discovery and the commercialization of molecular diagnostics. The combined resources will accelerate the development of new tools for the personalized management of transplant patients in an exciting genomic era. Please turn to page 8 to read more about one example, AlloSure and how this test, focusing on donor derived cell-free DNA, can aid clinicians monitoring transplant patients.
and available on the Roche LightCycler 480 II. Data will be presented by Dr. John Lunz from Gift of Hope, Itasca, IL. As part of the integration of two organizations, the company has changed its tagline to “Your Partner in Transplant Care”. This reinforces CareDx as a patient-focused company offering solutions across transplantation. We look forward to working with existing and new customers in this exciting field. For more information about us and our products and services please visit us at www.caredx.com
This meeting in St Louis is also the launch meeting of QTYPE. The product is validated
HLA typing using Olerup SSP kits and the QIAxcel Advanced System
QIAxcel data and corresponding images can easily be imported into SCORE and are shown side by side to assist reviewing.
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™ 5 software. The QIAxcelAdvanced 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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CareDx news SEPTEMBER 2016
AlloMap – Personalizing Care for Heart Transplant Patients AlloMap is a non-invasive blood test that uses genomic technologies to identify the absence of cardiac rejection. When used in conjunction with standard clinical assessments, AlloMap may help identify patients with stable allograft function who have a low probability of moderate to severe acute cellular rejection (ACR) at the time of testing. AlloMap is a panel of 20 gene assays, 11 informative and 9 used for normalization
and/or quality control, which produces gene expression data used in the calculation of an AlloMap test score – an integer ranging from 0 to 40. Compared with patients in the same post-transplant period, the lower the score, the lower the probability of acute cellular rejection at the time of testing. AlloMap testing is performed at the CLIA-certified and CAP accredited clinical laboratory at the CareDx headquarters based in Brisbane, California. Since
January 2016, AlloMap testing is also available throughout Europe via our commercial partner, Diaxonhit. AlloMap was developed to fill a need for a non-invasive surveillance solution to assess the risk of transplant rejection. It has been the flagship product of CareDx since 2005. Since inception, CareDx has achieved a number of milestones with its core technology:
The AlloMap Testing Process Sample collection and preparation The AlloMap test requires a blood sample obtained by routine phlebotomy and additional processing steps that enable the extraction and stabilization of RNA from peripheral blood mononuclear cells (PBMCs). After blood is collected, it is centrifuged to isolate the PBMCs. Further processing of the PBMCs releases the RNA from the cells and preserves it to ensure the recovery of high quality RNA for testing. The preserved sample is shipped together with the completed test requisition form to the clinical laboratory at CareDx. Step 1. Patient’s blood sample is drawn at a local lab. Step 2. The blood sample is prepared and sent via overnight courier to the clinical laboratory at CareDx in Brisbane, California. Step 3. In most cases, the blood sample is shipped to CareDx on the same day as your patient’s lab. The sample generally arrives at the clinical laboratory at CareDx the next morning. Step 4. Once the blood sample arrives, complex gene expression information is extracted and analyzed. Step 5. Gene expression information is translated into clinically actionable information and reported to the clinican within 1 to 2 days after blood sample is received. Step 6. The clinican uses the AlloMap score, along with other standard clinical assessments, to evaluate the patient’s probability of rejection. Step 7. Patient is notified of results.
AlloMap testing process at the Clinical Laboratory at CareDx
data to assess the quality of all of the testing process. These include:
The testing procedure involves sequential steps beginning with purification of RNA from the sample received and finishing with the reporting of the AlloMap test score to the clinician. The clinical laboratory at CareDx has optimized and standardized the performance of the AlloMap test processes. Comprehensive quality control ensures the reliability of the gene expression measurements used in the calculation of the AlloMap test score.
• Gene-specific measurement ranges
Testing procedure After purification, RNA is reverse transcribed into complementary DNA (cDNA), which is added to each of 60 wells containing gene-specific primers and probes. The expression of each gene is then measured by amplification and fluorescence detection using instruments that are configured for standard quantitative real-time polymerase chain reaction (qRT-PCR). Quality control and normalization The relative expression of the quality control genes used in AlloMap testing provides the
• Efficiency of the qRT-PCR • Precision • Accuracy and consistency Generation of the AlloMap Test Score A proprietary mathematical algorithm combines the measured expression values for each gene into a single integer value between 0 and 40 that is reported as the AlloMap test score. The clinician uses this score in the overall assessment of the probability of rejection at the time of testing. Reporting results The clinical laboratory at CareDx reports the AlloMap test score to the ordering physician within 1 to 2 business days after receipt of the sample at its Brisbane, CA facility. Upon receipt of the test report from CareDx by fax, the physician interprets the report as part of the patient’s overall clinical assessment.
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CareDx news SEPTEMBER 2016
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 in 2016 up to now included 2,898 unique primer mixes. Of these, 23% were new formulations, either through multiplexing (20,8%) or through adding new wells to the kits (2,2%).
Olerup SSP allele updating work in numbers across the product line, Jan – Sep 2016 Total number of produced and unique primer mixes (wells)
2 898
Out of which: Multiplexing instances for resolution of new alleles Added wells
63 (2,2%)
Sum of changes on primer mix level for allele updating
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. 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 icreasingly popular low resolution kits with more redundancy, greater resolution, and increased resistance to ambiguities.
603 (20,8%) 666 (23%)
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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CareDx news SEPTEMBER 2016
The Olerup QTYPE RT PCR System – Now The Olerup QTYPE system enables speed and precision in HLA typing at a low to intermediate resolution for samples that require a fast turnaround time. The Olerup QTYPE RT PCR system consists of the QTYPE HLA typing kit and the SCORE 6 interpretation software. It is currently validated on the Roche LightCycler 480 II RT PCR instrument. Once the run is completed on the LightCycler, the raw data can be easily imported into the interpretation software for a rapid analysis. Start to finish in one 1 hour. Work will start shortly to validate additional real time PCR instruments, such as the ViiA-7 and QuantStudio 6 & 7.
Multiplexing Power – Hydrolysis Probes The Olerup QTYPE HLA typing kits use hydrolysis probe technology, a powerful and flexible multiplexing tool which allows us to grow with the HLA database. Briefly, hydrolysis probes enhance our kits as the allele database grows a fluorescent report 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.
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
A complete workflow offering speed and precision in real time QTYPE HLA typing kit KIT CONTENTS – 384 well plate containing pre-aliquoted, dried primers and hydrolysis probes
10 min
45 min
– Mastermix including Taq polymerase
• Prepare DNA/Master Mix
• Set up instrument
– Tubes to make the reaction mix
• Dispense plate
• Insert plate
– Optically clear adhesive sealing sheet
• Seal plate
• Run
– Lot-specific kit information
• Centrifuge
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CareDx news SEPTEMBER 2016
available on Roche LightCycler 480 II 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 5 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.
Utilizing a third of the potential With 384 total wells and four colours, we have a total of 1,536 potential reactions. 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. As an example, QTYPE 11 have HLA-specific reactions in its current format.
SCORE 6 Interpretation Software A new version of SCORE has been developed specifically for analysis of real-time data generated from the QTYPE kit. The software has received a face-lift and the underlying database technology has been upgraded in SCORE 6 for improved reliability. The workflow includes 5 screens: Home, Patient, Test, Analysis and Report. It is straightforward to enter sample information, import real-time data and obtain the analysed results. A full typing within an hour Currently a run with QTYPE 11 on a Roche LightCycler 480 II 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 less than an hour”, says Ben Passey, Development Manager at Olerup.
Ben Passey Development Manager, Olerup SSP AB
MULTIPLEXING – 384 wells and 4 colours allowing 1,536 individually detected reactions – One color reserved for internal amplification control leaving 1,152 reactions available for HLA-specific reactions
5 min • Transfer data • Automatic data analysis • Review results
– Capacity to continue to develop and upgrade the kits with new database releases
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CareDx news SEPTEMBER 2016
AlloSure: measuring the percent of Donor-Derived Cell-Free DNA in solid organ transplant recipients CareDx is continuing to expand testing services in transplant patients. AlloSure is a clinical-grade proprietary Next-Generation Sequencing (NGS) based test to detect donorderived cell-free DNA (dd-cfDNA) in solid organ transplant recipients.
Solid Organ Transplant
dd-cfDNA in Blood
AlloSure testing is conducted at the CareDx CLIA-CAP-accredited clinical laboratory in Brisbane, CA. Cell-free DNA (cfDNA) has been used as a biological marker in other areas of healthcare such as prenatal testing and oncology. There is potential utility for cfDNA as a tool to inform on allograft injury and surveillance across solid organ transplantation.
Quiescence
Information on AlloSure has been presented at several conferences and most recently as an oral presentation at the American Conference of Clinical Chemistry. The analytical validation of AlloSure included all internal processes from sample accessioning to results reporting. The steps taken by CareDx for the analytical validation of AlloSure meet the recommended standards of several laboratory guideline groups, including the CLIA / CAP Guidelines. Further studies have been designed for both clinical validation and utility. Studies with AlloSure have observed that circulating dd-cfDNA levels are higher at the time of acute rejection compared to levels found in non–rejecting patients. A trend of rising dd-cfDNA ~1 month prior to biopsy-based diagnosis of rejection has also been noted. In kidney transplantation, currently available methods for diagnosing acute rejection are limited to either non-specific tests or invasive
Active Injury
procedures. DART — Circulating DonorDerived Cell-Free DNA in blood for diagnosing Acute Rejection in Kidney Transplant Recipients — is a nationwide study with 14 transplant centers across the United States. This study started in 2015 and completed enrollment in June, 2016. The study objective is to correlate levels of dd-cfDNA to active rejection in kidney transplant recipients and to review kidney function as measured in post transplant testing. Clinical
analysis is underway to review the use of AlloSure for both diagnosis and surveillance of allograft health. Further information on AlloSure can be found at the CareDx booth at ASHI and at allosure.com
References 1. Christopherson, C et al “Analytical Validation of a Clinical-grade Next-Generation Sequencing Assay for Assessing Allograft Injury in Solid Organ Transplant Recipients” AACC 2016 Poster presentation B-365 August 3 2016. 2. Grskovic M et al “Donor-derived Cell-free DNA in Plasma Increases with Rejection and Decreases after Treatment in Kidney Transplant Recipients” Poster SA-PO1124, Late-Breakers, American Society of Nephrology, November 2015.
3. Kobashigawa et al “Initial Analysis of the Donor-Derived Cell-Free DNA Outcomes AlloMap Registry (D-OAR) Study in Heart Transplant Recipients Undergoing Surveillance for Rejection” ISHLT April 28 2016.
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CareDx news SEPTEMBER 2016
Conexio’s sequencing based typing kits for HLA Class I and Class II genes, covering all the required NMDP null alleles.
Continuing to lead the way in Data fully compatible with Assign™ SBT sequence analysis software that includes analysis of non-coding regions. sequencing based typing 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
Class II updates: : Complete sequencing of all exon 3 allele groups in addition to exon 2 : Complete coverage of exon 2 and exon 3 : Complete coverage of exon 1, 3, 4 and 5 in addition to exon 2
Distributed by
0197 Please note that SBT Resolver™ HLA-DRB3, DRB4, DRB5 and B57 are for Research Use Only.
Qarad bvba Cipalstraat 3 B-2440 Geel Belgium
www.olerup.com
Designed, developed and manufactured by Conexio
conexio-genomics.com
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Pioneering since 2002
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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
SBT-150828B-1
www.olerup.com
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CareDx news SEPTEMBER 2016
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.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
1 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.
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CareDx news SEPTEMBER 2016
Olerup – Continuing to be a global distributor to HLA laboratories At Olerup, we strive to continuously develop and improve our web services. The recent acquisition by CareDx of the parental company Allenex will in no way change our way of working together with the transplant community. However you might notice some disturbance during the autumn due to web work. In the end of the year we will launch our new web page, CareDx.com. This will be the place where you will find information
of all our products and services. We believe that information and news of our products, services and company should be easily accessible for users. You can also find out at what meetings CareDx will be present and, of course, get further information about our different products.
represented in more than 50 countries around the world. The majority of our distributors 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. We are currently
CONTACT US USA
GERMANY/AUSTRALIA/BENELUX
Olerup Inc Orders and support: info.us@caredx.com
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Olerup product lines are in other regions made available through our network of specialized distributors. To find your distributor please go to www.olerup.com/distributors/
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Introducing QTYPE Speed and Precision in Real Time • Olerup QTYPE is a robust and easy-to-use alternative for rapid HLA typing within one hour. • 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 SCORE 6 software. For Research Use Only.
OLE160920A-1
Now available on the Roche LightCycler 480 II
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