case study: sequence-based hla typing - how i stopped worrying and started loving sequencing

Sequence-based HLA Typing -or-

How I stopped worrying and started loving sequencing

Wake Forest Baptist Medical Center

Overview

• Why we felt it was time for SBT

• How we evaluated SBT

• Our experiences with Life Technologies


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


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


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


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


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


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


Issues & Challenges that

Provoked Thoughts of Changing

SSP was becoming grossly inadequate


“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


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!!!!!!


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


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


Evaluation Criteria and Results

Our List of “Must Haves”


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


Results: Small Footprint

3500 XL Genetic Analyzer


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


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)


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


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


Results: Good Customer Support

Just doesn’t get any better than “our” Keith & Melissa

Keith Kurutz Field Application Specialist

[email protected]

Melissa Bowman SE Account Manager

[email protected]


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.”


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)


Implementation Process &

Lessons Learned …..and still Learning


SBT Implementation Timeline

Mid-August 2011

•Intensive week-long training

September 2011

•Validation packet started and submitted

October, 1 2011

•Put into routine use--started with NMDP donors

Late December 2011

•Trained first tech

Late January 2012

•Added patients to routine testing

Late March 2012

•Trained final tech

Timeline from first training to implementation into routine clinical use:

Less than 60 days


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


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


Front Loading

1. Ambiguity Solver

2. Alleles in Question &

Exons

3. All of the Various Possible

Combinations

4. Z- Primers to Resolve

Ambiguities


Allele Comparator

1. Allele Comparator

2. Alleles in Question 3. Differences

shown in RED


SPECIAL Thanks To Some SPECIAL People:

Sharlie Brown, CHS

Dr. Michael Gautreaux

Patti Shew

Andy Schroll, CHT

Joanna Fulcher

David Kiger, CHS Tabitha Peake, CHT

Kimberly Beane, CHT

Jennie Stewart, CHS


Wake Forest/HLA Immunogenetics Staff

case study: sequence-based hla typing - how i stopped worrying and started loving sequencing

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