Laboratory Validation. Chapter 16
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1 Laboratory Validation Chapter 16
2 Importance The DNA profile is used to convict or free a suspect It must be perfect! No mistakes like we have in regular laboratories all the time Also DNA evidence must be accepted within the court and believed by the jury Therefore procedures and specific labs need to be validated
3 DNA profiles We have gone through the steps: Isolating and extracting DNA Amplifying the sample with PCR Determining genotypes Producing a DNA profile is highly technical Needs to be performed by qualified and trusted lab personnel Produce accurate and trusted profiles
4 Two topics: These are often interchanged: Quality Assurance (QA) Provide adequate confidence in the product that the DNA profile is correct Done through repeated testing Quality Control (QC) Activities that maintain the quality of the product on a day to day basis
5 Terms Semi - Twice per Semi-annually = every 6 months Bi - Every two Bi-annually = every two years
6 Validation Terms Demonstrating the procedure is truly working in the hands of the lab personnel Robust Successful results are obtained a high percentage of the time Reliable Results that accurately reflect the sample Reproducible Same results are obtained every time
7 Proficiency test Terms Evaluation of the lab s performance Tests are performed every 6 months Standard Operating Protocols (SOP) The lab s procedures (manual) Should also represent what analysts actually do every day with every sample Laboratory accreditation Successful completion of testing or inspection by an accrediting body
8 Accreditation Requires the lab demonstrates and maintains good lab procedures Requires: Self testing within the lab Fulfilling applications and procedures Passing the proficiency tests Maintaining accreditation Court may request proof of accreditation or proficiency test results
9 Blind Proficiency Testing Purpose of the proficiency testing is to prove that the lab and the specific technician are following correct SOP in all situations If the personnel do not know they are being tested then it is known as a blind proficiency test If analysts are aware they are being tested they may behave different than normally
10 Four Models of Blind Testing 1. Blind/Law Enforcement The local law enforcement disguises the test as a real sample 2. Blind/Conduit Lab The lab has no idea they are being tested by their accreditation body 3. Blind/Analyst The lab knows, but the analyst is blind 4. Random audit Open testing, done at random times
11 Advantages to Blind Testing The analyst and/or lab are being tested without their knowledge Therefore test should be an accurate representation of their normal work Also the threat of blind testing means that the analyst will not know when they are being tested Therefore perhaps will always be on guard
12 Drawbacks to Blind Testing Cost The effort to submit a fake sample without the lab knowing costs more than open testing It requires deception Perhaps not a good side effect of accuracy Protection of the donor sample s ID If the analyst is blind they will enter the DNA profile of an innocent person into the database how to remove that profile?
13 Proficiency Test Results Results are classified into the following: 1. No errors made 2. Mixture was not detected 3. Error in typing that was known by lab Reported along with DNA profile 4. Error in typing that was unknown The error rate can be calculated Error rates around 0.5% or less are desired
14 Organizations All of the following organizations ensure the proficiency testing is performed properly: TWGDAM or SWGDAM DNA Advisory Board American Society of Crime Lab Directors National Forensic Science Training Center College of American Pathologists Cellmark Diagnostics Collaborative Testing Services, Inc Others
15 Validation Defense lawyers rarely challenge the science behind DNA testing any longer Now they challenge the lab procedures or personnel Need to validate: All procedures All personnel In order to guarantee results for court
16 Validation Two types of validation needs to be performed: 1. Development Validation Testing and validation of new methodologies As technologies change and new methods are developed by researchers 2. Internal Validation Testing and validation of established procedures within laboratory s environment
17 Development Validation Standard Specimens Proof that samples derived from different tissues of the same person will produce the same DNA profile Example blood vs. semen Consistency Reproducibility between different labs is proven DNA profiles only useful if they can be compared within a complete trust
18 Developmental Validation Population Studies Samples grouped by ethnicity are genotyped Determine allele frequencies for major ethnic groups Vital for determining probability of given DNA profile Reproducibility Dried samples are proven to produce the same profile as liquid samples
19 Development Validation Mixed Specimen Studies Ability of procedures to determine a mixture is present Also to distinguish the genotypes of each sample within the mixture Environmental Studies Known samples are environmentally stressed (Exposure to UV, humidity, temp fluctuations) Determine what levels of degradation still produce accurate DNA profiles
20 Developmental Validation Matrix Studies Known samples are contaminated with common substances (Denim dyes, soil, etc) Prove procedures will work in presence of contaminates Non-probative evidence Validation done on real samples from closed cases Prove procedures work with real DNA samples
21 Development Validation Non-human Studies Determine is non-human DNA present in sample will interfere with DNA profile Prove procedure can identify human DNA Minimum sample A dilution series of known DNA samples are tested Determine the range of DNA concentrations that will be effectively genotyped with given procedures
22 Publication of Results Whenever new technology or methodology is introduced Must be validated first Then once it is validated results need to be published Peer reviewed journals This is vital so that methods will be accepted in courtrooms and by juries
23 ASTM and American Academy of Forensic Sciences Springer-Verlag Elsevier Science Elsevier Science Figure 16.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition 2005 Elsevier Academic Press
24 Internal Validation Precision Studies Determine the base pair size ranges for all STR alleles in allelic ladder Prove they peaks always within a narrow range (precision) Stutter Studies Percentage of stutter bands is calculated Upper heights of stutter bands is used to determine mixture calculation procedure
25 Internal Validation Heterozygous peak heights Genotype a known heterozygote and determine the difference in peak heights Help determine mixture calculation procedure Annealing Temperature Studies Genotype with annealing temp that is two degrees above or two degrees below recommended temp Find the range that the PCR machine can be off by without changing the results
26 Internal Validation PCR Condition Studies Optimize number of cycles Optimize temperatures for each step Test if adding or removing certain reagents will improve or change DNA profile Because all labs have slightly different conditions and machines Also proves that SOP of the lab will produce expected DNA profiles
27 Inter Laboratory Validation Validation must be done to prove that different labs will produce the same DNA profile for the same individual Also validate how genotypes are reported How repeat units are counted, labeled, etc Now commercial kits are available for 13 CODIS STR s Much easier to compare results between labs even different countries
28 Reference Samples Reference samples are available for purchase This way lab can test procedures Then check against known genotypes Available for: RFLP markers STR markers Mitochondrial DNA Y chromosome STR s
29 Commercial Kits Originally all labs have to develop their own PCR mixes They needed to determine: PCR conditions Temps, length of time Concentrations of reagents and primers Ways to optimize multiple markers together Prepare their own allelic ladders
30 Commercial kits Currently: Forensic Lab buys one kit Contains all 13 markers optimized together in one reaction Everything needed for PCR mix All lab needs to do is purify DNA Add correct amount of DNA to kit Run tube(s) through validated conditions
31 Currently Validation of kits Validation is the job of the company producing the commercial kit Not only does each kit need to work Each kit needs to be almost identical Companies provide: Certificate of Analysis To prove to a court that a given kit s lot number has been validated properly
32 New Methods There are new methodologies and technologies being developed constantly Some interesting ones to note: Portable devices Take to crime scene High throughput DNA profiles The thousand dollar genome (sequencing) Automated lab procedures (robotics)
33 Any Questions? Read Research Paper Read Chapter 18 Skipping 17
34 Guest Lecturer Next class we will have a Forensic Toxicologist giving a guest lecture One thing he ll be covering is genotyping a specific enzyme that processes toxic substances in the body Seems that people have different abilities to process toxins depending on their genotype Read research paper Be prepared to ask him specific questions
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