I. Purpose: To provide basis for ensuring quality testing using mitochondrial DNA (mtDNA) sequence analysis for identification of old skeletal remains for the Department of Defense; government or government-contracted laboratories must comply with the following provisions for DoD laboratory approval:

II. Provisions:

1. Laboratory Accreditation: A. College of American Pathologists (General and Molecular Pathology sections; or B. American Society of Crime Laboratory Directors - Laboratory Accreditation Board.

2. Standards for MTDNA Sequencing of Skeletal Remains to supplement basic accreditation requirements: [in some standards these requirements are found in one or both of the accreditation requirements of ASCLD or the CAP, but are reiterated to ensure requirement compliance.]

A. Facilities:

1) Space dedicated to skeletal'. remains processing.

2) Access to the laboratory must be controlled and limited.

3) Separate pre- and post-PCR areas.

4) In pre-PCR area, separate areas for extraction and PCR set up.

5) Dedicated equipment (marked) for amplification room and dedicated storage for PCR product.

6) Air handling appropriate for prevention of sample contamination.

7) A minimum of 4 linear feet of bench space per analyst during processing in both pre- and post-PCR areas or sufficient space to prevent sample contamination when more than one analyst is working.

B. Personnel:

1) DNA Analysts (caseworkers) have primary responsibility for analyses performed and

must be capable of defending their work --n court; they may be assisted by other analysts, molecular biologists, technicians and technologists in the performance of their work.

2) DNA Analysts must have a Baccalaureate degree or higher; a Masters degree in forensic science, biochemistry, or molecular biology or certification in medical technology 's preferred.

3) DNA Analysts must have documentation of training and at least six months experience in DNA analysis, as well as experience in mtDNA sequencing of degraded DNA from aged bone specimens, to include demonstrated competency in performing such analysis.

4) Technical Director of the laboratory must:

a) hold a doctorate-level graduate degree.

b) have at least one year postgraduate experience in molecular biology.

c) a member in good standing of the American Academy of Forensic Sciences.

5) Technical Director and DNA Analysts must participate in continuing education in-house and out-of-house yearly. Training and education must be documented.

C. External proficiency testing: Every analyst must undergo proficiency testing twice a year, at least one of which must be an external proficiency test and every lab must undergo blind proficiency testing twice a year. Every deficiency must be explained and corrective action must be demonstrated; approval withdrawn until demonstrated competency after incorrect determinations and reapproved after documentation of further applicable training, reasonable investigation of cause, documentation of corrective action, and demonstration of proficiency to the Quality Assurance oversight Committee.

1) Open: College of American Pathologists, Parentage Survey (PI).

2) Blind: Duplicate samples will be sent twice a year by CILHI to the laboratory as a blind proficiency test. Exact sequence match is anticipated, although the amount of sequence may differ.

D. Written internal quality assurance program:

1) Documentation of deficiencies and corrective action.

2) Use of reagent blanks during extraction procedure.

3) Use of negative control, positive control, and reagent blank in each amplification reaction.

4) Evaluation of negative control, positive control, and reagent blank in product eels.

5) written guidelines for acting on positive and negative control results out of specification.

6) Use of National Institute of Standards and Technology (NIST) specified positive human control.

7) All results must be read independently by a second qualified individual.

8) All reported sequences must be confirmed by duplicate testing or preferably by sequencing of the reverse strand.

E. Chain of custody:

1) Use of DA Form 4137 form for external chain of custody.

2) Must maintain internal and external chain of custody in accordance with Army Regulation AR190-45.

3) Each sample must be labelled with a unique identifier.

4) Casework specimens must be stored under proper seal.

F. Case documentation in single case file and available to the government to include:

1) transportation slips.

2) telephone conversation notes.

3) chain of custody forms.

4) photographs of evidence and appropriate data.

5) case notes sufficient to explain and support analysis.

6) raw data.

7) quality control results.

8) reagent and control lot numbers.

G. Standard Operating Protocols:

1) Protocols must be written and followed for specimen handling, testing procedures, and interpretation.

2) Analytical protocols must be validated.

3) Amplification cycles limited to 40 or less per amplification, second round of amplifications acceptable.

4) Quality control and assurance must be documented and incorporated into standard operating protocols.

5) Written protocols must be submitted, approved, and filed by the Quality Assurance Oversight Committee before use in casework.

H. Reagents:

1) Documented quality control of production, acquisition, and use of all critical reagents.

2) Water:

a) NCCLS Type I water.

b) 0.1 ppm metal ions (including zinc, iron, lead, and magnesium), monitored weekly.

3) PCR Primers:

a) 95% purity

b) tested for function

c) tested for human mtDNA contaminants

4) Polymerase:

a) licensed for PCR.

b) tested for function.

I. Approved report format:

1) Signed by analyst and certifying official, if not the analyst (i.e. technical director, program manager, corporate official, or other designated person).

2) CILHI and laboratory case number must be incorporated into the final report.

3) AFIP directed format/rules.

4) Terminology: a) reference to Anderson sequence. [Nature Vol. 290, pp. 457-465, 9 April 19811 b) use of common nomenclature. (Wilson, et al, Crime Laboratory Digest, Vol. 20, pp. 68-77, Oct 1993)

5) Sequence information must be in format compatible with AFDIL computer system.

J. Analysis:

1) Unknown bone samples must be routinely sequenced before putative reference samples. (except in the creation of a database)

2) Sequenced results must be independently analyzed by a second individual.

3) Statistical analysis by 'counting' method (frequency of observations/population].

K. Database for use in statistical analysis:

1) Database sequences must be confirmed by duplicate testing or preferably by sequencing of the reverse strand.

2) Database sequences must be made public.

3) Database sequence must include at a minimum:

a) Hypervariable Region I: 16024 - 16365.

b) Hypervariable Region 11: 73 - 340.

III. Quality Monitoring

1. Inspections: AFDIL, Civilian Expert Consultant, in the case of a contract lab, the Contract Officer's Representative, if available. Performed semi-annually for the first two years and annually thereafter. Reports to be filed with the Quality Assurance Committee.

2. Copies of all accreditation materials and correspondence with the accrediting organizations filed with the Quality Assurance Oversight Committee.

IV. Quality Assurance Oversight Committee

1. Function: Programmatic and technical oversight of quality assurance program, monitors execution and compliance, grants DoD approval, reviews accreditation materials, monitors proficiency testing results, oversees inspection reports, approves protocols, resolves technical disputes, assures satisfaction of military concerns; does not take action but reviews records and recommends corrective action.

2. Structure: The committee should be a subcommittee of the AFIP Scientific Advisory Board (SAB), composed of five (5) committee members appointed by the AFIP Director: a member of the SAB (chair), a DoD DNA Registry Representative, and three Technical Consultants.

3. Meeting Schedule: Annually and on ad hoc basis when necessary to resolve issues.

4. This meeting will be funded by the AFIP.

The Assistant Secretary of Defense for Health Affairs has ultimate responsibility for quality assurance of DNA testing performed for identification.