Wake Forest HLA Lab presents the issues and challenges that provoked thoughts of changing from SSP to SBT (Sequence-based typing), reveals lessons learned during implementation and shares their personal experience of working with Life Technologies during the installation of their 3500 XL Genetic Analyzer. Visit the Life Technologies website to learn more about HLA Typing. http://owl.li/eedmi

1. Sequence-based HLA Typing
-or-
How I stopped worrying and started loving sequencing

2. Overview
• Why we felt it was time for SBT
• How we evaluated SBT
• Our experiences with Life Technologies
Wake Forest Baptist Medical Center

3. Lab Background
• Wake Forest HLA Lab opened for business in
early 1970’s
• Went to Molecular Typing for Class II in 1997
• By 1999 doing all typings (Class I & II) by
molecular methods
Wake Forest Baptist Medical Center

4. Programs Supported
• Solid Organ Program:
• Renal transplant Program
• Living Related/Unrelated transplants
• Deceased Donor transplants
• Pancreas transplant program
• Cardiac transplant program
• Disease Association & Pharmacogenomics
testing
Wake Forest Baptist Medical Center

5. Programs Supported
• 2 Bone Marrow Transplant Programs:
• Wake Forest /Baptist Medical Center
• An adult living related transplant program
• An adult un-related living transplant
program
• Carolinas Medical Center
• A pediatric living related transplant
program
• A pediatric living un-related transplant
program
Wake Forest Baptist Medical Center

6. Annual Throughput – 2011
• 2489 typings done by 6 techs (does not include
antibody screening tests)
• 255 hi resolution typings by SSP (bone marrow
patients & donors)
• 1505 low resolution typings by SSO (kidney,
kidney/pancreas, pancreas, cardiac patients)
• 138 low resolution imported deceased donor typings
by SSO
• 66 low resolution local deceased donor typings by
SSP (after hours testing)
• 236 low resolution non-transplant clinical typings by
SSO
Wake Forest Baptist Medical Center

7. Technologies Currently Implemented
• SSO (Sequence Specific Oligonucleotide)
typing for low resolution
• Solid organ & disease association patients
• SSP (Sequence Specific Primers) typing for low
– hi resolution } BEING PHASED OUT
• Some renal patients/donors
• Some disease association patients
• Bone marrow patients/some donors
Wake Forest Baptist Medical Center

8. Technologies Currently Implemented
• Real-time PCR for low resolution typing
• Deceased donor typing and as needed for
backup situations
• SBT (Sequence Bases Typing) for hi resolution
• All bone marrow patients
• All NMDP donors (including donors from
other registries)
• Potentially matched living related donors
Wake Forest Baptist Medical Center

9. Issues & Challenges that
Provoked Thoughts of Changing
SSP was becoming grossly inadequate
Wake Forest Baptist Medical Center

10. “SSP is a flawed
system to begin with
because of the need to
change kits in keeping
with changes in
numbers of alleles.”
Dr. Michael Gautreaux
Wake Forest Baptist Medical Center

11. Issues and Challenges
• Running Gels
• Ethidium bromide Biohazardous &
• Agarose Expensive Disposal
• Dropouts = Repeats
• Power Source = Shock Hazard
• Technical Issues
• Missing 1 well can mean costly repeats
• TIME!!!!!!
Wake Forest Baptist Medical Center

12. Issues and Challenges
• Price per test **
• SSP +/- $1600 for Class I & II
• SBT +/- $1000 for Class I & II
**what’s charged to the patient not our actual cost
Wake Forest Baptist Medical Center

13. Number of alleles keeps increasing
• Number of alleles
keeps increasing
• 2000: 750 Class I
525 Class II
• 2005: 1200 Class I
750 Class II
• 2011: 5400 Class I
1600 Class II
Wake Forest Baptist Medical Center

14. Evaluation Criteria and Results
Our List of “Must Haves”
Wake Forest Baptist Medical Center

15. Evaluation Criteria
• Something that didn’t take up the whole lab
• Something easy to maintain
• Something user-friendly
• Something that was less labor intensive
• Something allowing less materials to be used
• Something offering good customer support
• Something with proven reliability
Wake Forest Baptist Medical Center

16. Results: Small Footprint
3500 XL Genetic Analyzer
Wake Forest Baptist Medical Center

17. Results: Easy to Maintain Instrument
• The 3500 XL “Dashboard”
makes it almost idiot proof
• Tells you what needs to
be done and when
• WIZARDS give you a
guided step-by-step
of how to do things
• Tells you how long (in
days and samples) you
have left on your
reagents
Wake Forest Baptist Medical Center

18. Results: User Friendly Software
• uTYPE® is very
straightforward analysis
software (seems too easy
to be true---no seriously)
• uTYPE® even suggests
how to resolve any
ambiguities, most often
listing several Z primers to
choose from (making it
nice that you don’t have to
have all those Z primers in
house)
Wake Forest Baptist Medical Center

19. Results: Less Labor Intensive
• Cut time for 6 typings
• 2+ full days using SSP
• 1 day + couple hours using SBT
• Increasing productivity
• Updating the Library
• 1 – 2 days to update SSP worksheets and
software
• Minutes to do SBT software on 6 computers
Wake Forest Baptist Medical Center

20. Results: Less Resources/ Materials Used
• Cut tech time for hi resolution typing drastically
• Hands on SSP (for 1 sample): 2 hours
• Hands on SBT (for 1 sample): 1.25 hours
• Drastically decreased volume of DNA
• Cutting costs all throughout the lab
• Cut out 90% agarose consumption
• Cut out 90% ethidium bromide consumption
• Nice having the ability to stretch your reagents
Wake Forest Baptist Medical Center

21. Results: Good Customer Support
Just doesn’t get any better
than “our” Keith & Melissa
Keith Kurutz Melissa Bowman
Field Application Specialist SE Account Manager
Keith.Kurutz@lifetech.com Melissa.Bowman@lifetech.com
Wake Forest Baptist Medical Center

22. Results: Proven Reliability
• As for Life Technologies:
• Confident in the company:
• With 10,000+ employees
• Servicing 160+ countries
• Supporting 50,000+ products
• Confident in support staff
• In person, on-site
• Telephone
• Online
• Always keeping up with the latest technology
“We are a science company.”
Wake Forest Baptist Medical Center

23. Results: Proven Reliability
• As for their products:
• Been a longstanding customer because
good products
• If an issue is raised, it’s addressed (no ifs
ands or buts)
Wake Forest Baptist Medical Center

24. Implementation Process &
Lessons Learned
…..and still Learning
Wake Forest Baptist Medical Center

25. SBT Implementation Timeline
October, 1
Mid-August 2011 Late January
2011 •Put into routine
2012
•Intensive week- use--started with •Added patients
long training NMDP donors to routine testing
September Late December Late March
2011 2011 2012
•Validation packet •Trained first tech •Trained final tech
started and
submitted
Timeline from first training to
implementation into routine clinical use:
Less than 60 days
Wake Forest Baptist Medical Center

26. Lessons Learned
• Intensive training process
• Good Technique is CRITICAL
• Lots of pipetting
• Reliable/well maintained equipment is a must
• Must be the calm in the midst of a storm
Wake Forest Baptist Medical Center

27. Working with Life Technologies
MOST AMAZING CUSTOMER SUPPORT!!!!!
• Willing to work with you in order to do
what’s best for your lab
• Still learning little tricks and techniques
Front Loading
Allele Comparator
Wake Forest Baptist Medical Center

28. Front Loading 1. Ambiguity
Solver
2. Alleles in
Question &
Exons
4. Z- Primers
3. All of the
to Resolve
Various
Ambiguities
Possible
Combinations
Wake Forest Baptist Medical Center

29. Allele Comparator
1. Allele Comparator
2. Alleles in
Question 3. Differences
shown in RED
Wake Forest Baptist Medical Center

30. SPECIAL Thanks To Some SPECIAL People:
Dr. Michael Gautreaux
Kimberly Beane, CHT
Patti Shew
Sharlie Brown, CHS
Joanna
Fulcher
Jennie Stewart, CHS
Andy Schroll, CHT
David Kiger, CHS
Tabitha Peake, CHT
Wake Forest Baptist Medical Center

31. Wake Forest/HLA Immunogenetics Staff
Wake Forest Baptist Medical Center