Mitotyping Technologies
Quality Manual
Version 1.1
Issued 6/1/01
Approved________ Date________
2
Table of Contents
Introduction
3
I.
Quality Manual Organization
6
II.
Goals and Objectives
6
III.
Organization and Management
6
IV.
Personnel Qualifications and Training
6
V.
Facilities
7
A. Security
B. Contamination
7
7
VI.
Evidence Control
9
VII.
Validation
9
VIII.
Analytical Procedures
10
A.
B.
C.
D.
10
10
10
10
Introduction
Reagents
Critical Reagents
Reference Standards
IX.
Equipment Calibration and Maintenance
10
X.
Proficiency Testing
11
XI.
Corrective Action
11
XII.
Reports
11
XIII.
Review
11
XIV. Safety
11
XV.
11
Audits
XVI. Subcontractor of Analytical Testing
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Introduction
This Quality Manual has been prepared under the guidelines of the American Society of
Crime Laboratory Director/Laboratory Accreditation Board (ASCLD/LAB). Where
appropriate, references have been made to the written Standard Operating Procedures of
Mitotyping Technologies which contain, besides the primary information for this quality
manual, supplementary information regarding administration, case management,
laboratory protocols, and interpretational guidelines.
Specific quality manual guidelines (standard 1.4.2.1) of the ASCLD/LAB are addressed
in the list below:

A quality policy statement including objectives and commitments by
management. A quality policy statement will be found incorporated in the Mission
Statement within the Standard Operating Procedures (Preface).

The organization and management structure of the laboratory, its place in any
parent organization, and relevant organizational charts. A description of
company organization will be found in the Standard Operating Procedures under the
heading “Organizational Structure”, accompanied by a chart outlining relationships
among personnel and general titles and responsibilities.

The relationships and responsibilities of management, technical operations, and
support services in implementing the quality system. The company is small and
has few personnel and high level of interaction among staff. The Laboratory Director
is also the Technical Manager, and supervises the Quality Manager. The quality
manual is prepared with input from both the Technical Manager and Quality
Manager. The Quality Manager has the responsibility of implementing the quality
manual requirements and determining if personnel are implementing and fulfilling all
the requirements in day-to-day operations.

Job descriptions, education, and up-to-date training records of laboratory staff.
Job descriptions and educational requirements for staff are described in the sections
titled “Personnel Job Descriptions” and “Personnel Qualifications” in the Standard
Operating Procedures. Training records are kept in the Training Notebook in the
main office, and are updated as needed after training or new hires. This notebook
also contains up-to-date academic transcripts for all staff. In addition, this quality
manual addresses personnel qualifications and training in Section IV.

Control and maintenance of documentation of case records and procedure
manuals. Control and maintenance of documentation of case records is described in
Chapter 6, “Sample Storage, Data Collection, and Record Keeping” of the Standard
Operating Procedures. Procedures are included as Chapter 7, “General Laboratory
Procedures”, in the Standard Operating Procedures, while Chapter 3, “Preparation of
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Standard Operating Procedures” describes the preparation and updating of the SOP
by personnel.

The laboratory’s procedures for ensuring that measurements are traceable to
appropriate standards, where available. These procedures are referred to in
Sections VII (D), and IX of this quality manual and in the sections titled “Control
and Reference Materials” and “Equipment and Instrumentation” of the Standard
Operating Procedures..

The type and extent of examinations conducted by the laboratory. Mitotyping
Technologies is exclusively involved in forensic mitochondrial DNA analysis of
biological materials. The analysis extends to working with minimal and degraded
specimens as well as more abundant materials.

Validation and verification of test procedures used. This is described in the
sections titled “Internal Validation Studies” and “Proficiency Testing” in the Standard
Operating Procedures, and in Section VII of this quality manual. Documentation of
Validation Studies is provided in Volumes 1-3 of the Validation Notebooks. Ongoing
external proficiency testing is recorded in the Proficiency Testing Notebooks.

Handling evidence items. This is described in the section titled “Evidence Control
and Storage of Samples” in the Standard Operating Procedures, and in Section VI of
this quality manual.

Major equipment and reference measurement standards used. This is described
in the sections titled “Controls and Reference Materials” and “Equipment and
Instrumentation” in the Standard Operating Procedures, and in Sections VIII,
Analytical Procedures, and IX, Equipment Calibration and Maintenance, in this
quality manual.

Calibration and maintenance of equipment. This is provided in the section titled
“Equipment and Instrumentation” of the Standard Operating Procedures and in
Section IX of this quality manual. Actual documentation of equipment maintenance
is provided in the Equipment Notebook.

Verification practices for ensuring continuing competence of examiners
including interlaboratory comparisons, proficiency testing programs, and
internal quality control schemes (e.g., technical peer review). Proficiency testing
is discussed in the section titled “Proficiency Testing” in the Standard Operating
Procedures and in Section X of this quality manual. External proficiency testing is
done through the College of American Pathologists on the 180 day schedule required
by DAB. Documentation of interlaboratory exchanges is available within the
Validation Notebooks as well as in several notebooks containing research data
developed in exchanges with NIST and AFDIL. Internal quality control schemes are
discussed in the section titled “Technical Review/Audits” in the Standard Operating
Procedures.
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
Gaining feedback and taking corrective action whenever analytical discrepancies
are detected. This is addressed in the section titled “Corrective Action” in the
Standard Operating Procedures.

Monitoring court testimony to ensure the reporting of scientific findings in an
unbiased and effective manner. Monitoring of court testimony is addressed in the
section titled “Examiner Testimony” of the Standard Operating Procedures. Actual
records collected in the monitoring of testimony are available in the Testimony
Notebook.

Laboratory protocol permitting and departures from documented policies and
procedures. A general statement regarding flexibility of operational practices is
given in Chapter 3 of the Standard Operating Procedures. The policy requires that
departures from standard practices be noted in the case file.

Dealing with complaints. Complaints will be handled by the Laboratory Director
with input from the Quality Manager. A full investigation into complaints will be
carried out with full documentation and disclosure.

Disclosure of information. The policy on this is discussed in the section titled
“Security and Confidentiality” in the Standard Operating Procedures.

Audits and quality system review. This is discussed in the section titled “Technical
Reviews/Audits” in the Standard Operating Procedures, and in Section XV of this
quality manual.
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I. Quality Manual Organization
The quality manual consists of sections that address the current DAB standards. In
general, the quality manual addresses quality control and assurance issues already
covered in the same detail in the Standard Operating Procedures. However, the quality
manual provides a single concentrated resource for personnel to consult about the
standards in place that must be followed to assure quality results in forensic
mitochondrial DNA analysis in this laboratory.
In addition, the following documentation which continuously traces quality control
management is available for examination:
1) Technician Training Notebook (office)
2) Reagent Notebook (main lab)
3) Validation Notebooks (office)
4) Proficiency Notebooks (office)
5) Safety Notebook (main lab)
6) Yield Gel Duplicate Photo Notebook (main lab)
7) ABI 310 Sequencer Sample Sheet Notebook (main lab)
8) PCR Schedule Notebook (main lab)
9) Equipment Notebook (main lab)
10) Audit Records/Technical Reviews (office)
11) Testimony Notebook (office)
II. Goals and Objectives
The overall goals and objectives of Mitotyping Technologies are addressed in the
company’s Mission Statement in the Standard Operating Procedures. The goals of the
quality control and assurance program are to maintain and constantly improve accuracy
and consistency in the work product by setting high standards for all areas which might
influence case outcomes, as delineated in the following sections.
III. Organization and Management
The organization and management structure of the laboratory are described and
diagrammed in the section of the Standard Operating Procedures titled “Organizational
Structure”.
IV.
Personnel Qualifications and Training
Personnel qualifications for all staff may be found in the section of the Standard
Operating Procedures titled “Personnel Qualifications”, which states requirements for
hiring. Comprehensive job descriptions for each staff position may be found in the
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section of the Standard Operating Procedures titled “Personnel Job Descriptions”.
Training is addressed in the section titled “Technician Training”. Documentation of the
actual training periods for personnel can be found in the Training Notebook. This
notebook also contains current academic transcripts for each staff member.
In addition, the laboratory has a process for monitoring the court testimony of each
examiner. The Standard Operating Procedures, in the section titled “Examiner
Testimony”, describes this process. A section titled “Continuing Education” addresses
the ongoing opportunities for staff members to advance their training and expertise.
V. Facilities
A. Security
Security for the building and laboratory are discussed at length in the Standard Operating
Procedures under the heading “Security and Confidentiality”.
B. Contamination
The monitoring and control of contamination are of utmost importance and concern to the
laboratory carrying out DNA testing, and an extra level of caution is required in a
mitochondrial DNA facility. For all samples, pre- and post-PCR processing is separated
in the laboratory. Extraction and PCR of questioned samples or minimal DNA known
samples is done in the clean room (see below). Extraction and PCR of abundant known
samples is done in the known sample room off the main lab, which is reserved
exclusively for post-PCR processing. The Standard Operating Procedures address, in
numerous locations, the quality measures in place to monitor and prevent contamination,
and these quality measures are collected from the SOP below:

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Questioned casework samples are stored in one of two places: 1) the refrigerator in
the clean room (hairs or stains), or 2) the freezer in the clean room (bones). Bone
packages will either be labeled with a biohazard label or enclosed in a biohazard bag.
Known reference samples will be stored only in the main lab refrigerator in a clean
ziploc bag labeled with biohazard label, case number, date, and sealed across the
closure with signed and dated evidence tape. Extraction products from questioned
samples are stored in a case-numbered box in the clean room refrigerator (freezer
part). No PCR products will be stored in this refrigerator/freezer whatsoever. The
PCR products generated from these samples will be stored in the main lab refrigerator
only, appropriately labeled. Extraction products from known samples and their PCR
products are stored in the main lab refrigerator until sequenced, in a separate box
from the questioned sample PCR products.
In general, all samples, including reference samples, will be extracted and proceed
through PCR individually, accompanied by their extraction reagent blanks. For the
purposes of cycle sequencing and analysis of sequencing reactions on the ABI 310,
samples from different cases may be run on the machines at the same time. At this
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point in the analysis, sufficient DNA is present in an individual cycle sequencing
sample to type without concerns about cross contamination. Analysis of questioned
and known samples will always take place separated by time and space. Prior to
DNA extraction and PCR amplification setup, questioned and known samples will
never be handled in the same room of the laboratory.
In a mitochondrial DNA lab, there is ongoing consideration of sample type and
robusticity. On occasion, it is necessary to extract DNA from a known hair or bone
when blood and saliva are not available. Because of contamination concerns, samples
of this type must be extracted in the clean room, even though the samples are known
reference samples. When this is necessary, the questioned samples for the case will
have been finished prior to working with the known.
Pre-amplification and post-amplification procedures on all questioned and known
samples are done in separate areas of the lab. Amplification is completed on
questioned samples before DNA extraction and amplification begin on known
samples. PCR setup on minimal DNA samples is done in the dedicated PCR hood in
the Clean Room, while PCR setup for abundant DNA samples is done in the known
sample room within the Main Lab. Contamination prevention includes some or all of
the following as necessary: use of lab coat, face shield, and disposable gloves (two
pairs; tape the first pair to the lab coat), set-up under dead-space hood, periodic
replacement of bench paper and working on large disposable Kimwipes during stages
of the case, ultraviolet radiation of pipettes, autoclaving pipette tips and tubes, and
preparation and use of autoclaved/dedicated reagents, and use of aerosol barrier tips.
When required, preliminary PCRs are done to test cleanliness of reagents and
equipment prior to beginning a new case. Besides the samples, PCR runs include the
extraction reagent blank and always include a PCR negative control to check for
contamination in reagents and equipment, and a PCR positive control to be sure the
PCR reaction has worked. The positive control DNA is taken from storage and added
to the positive control tube last, after the questioned sample tube, reagent blank tube,
and negative PCR control tube have been closed. The positive control sample is
K562.
Cycle sequencing will be performed with the primers used for the first round of PCR,
to generate both strands from the PCR reaction for sequencing. Cycle sequencing
will be done on the sample and reagent blank products produced in the first round of
PCR, with the exception of PCR reactions with a positive (contaminated) PCR
negative control sample. These entire PCR reactions will be discarded.
Cycle sequencing will be performed with L-strand primers on each reagent blank
PCR product in order to detect contamination which might not be apparent on a yield
gel photo. Since this strand would reflect the presence of any contaminant present in
that entire PCR, it is adequate for screening the PCR for contamination. If there is
product in this sequencing run, indicating the presence of a contaminant, the other
strand may then be sequenced in an attempt to identify the source of the
contamination.
Critical reagents (Isoquick kits, BSA, K562, primers, Taq polymerase, L2 and L6
buffers, stain extraction buffer, sequencing reagents [Big Dyes] and size fractionated
silica) will be tested on non-probative known samples prior to use on evidentiary
materials. These test records will be kept in the Reagent Notebook.
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BSA, primers and dNTPs will be made up in dilution under the hood designated for
PCR setup in the clean room. Stock solutions of primers and aliquots of BSA,
primers, and dNTPs will be kept in the clean room freezer; when they are needed in
the main lab they will be transferred as necessary. Sigma ultrapure tissue culture
water will be periodically aliquotted into sterile tubes and stored in the labeled box in
the freezer in the clean room. Aliquots will be prepared under the hood designated
for PCR. These aliquots will be used for extractions and PCR.
The common autoclave will be used to sterilize those solutions that are used to make
up some critical reagents, for example, SEB, L2 and L6. See the appendix of
reagents in the Standard Operating Procedures for details on these recipes. All
instruments such as forceps or scissors that are used to handle questioned samples
will also be washed, individually wrapped in foil, marked with autoclave tape, and
autoclaved.
Sigma ultrapure tissue culture water will be used universally for protocols requiring
sterile deionized and distilled water. Non-sterile deionized distilled water will be
used from the Nanopure system in the laboratory for washing glassware and Waring
blender chambers. A final rinse of the Waring blender chambers will be done with
Sigma water.
Pipettors will be calibrated and cleaned once per year on-site by a licensed tester.
Records on re-calibration will be maintained in the Equipment Notebook. In the
interim, pipettes can be partially disassembled and cleaned with 10% bleach and 70%
ethanol. Those pipettors dedicated for extraction and PCR will be left in the hoods
and exposed to UV light between samples.
Biohazard and fume hoods will be cleaned thoroughly once per month with 10%
bleach and 70% ethanol. New bench paper will be placed down at this time. Lab
benches used for running PCR agarose gels will be wiped down with bleach weekly.
The ultraviolet lights in the hoods will be turned on for at least one-half hour after
each and every use of the hood. Sample blocks of the PCR machine will be bleached
periodically. Large Kimwipes will be used as a work surface in the hood and then
replaced between samples, and the hood will be UV-irradiated between samples.
VI. Evidence Control
The Standard Operating Procedures contains policy about the handling of evidence in the
section titled “Evidence Control and Storage of Samples” in Chapter 5.
VII.Validation
Validation studies were undertaken prior to initiation of case work and is ongoing where
required to assure that changes to laboratory protocols result in preservation of accurate
analysis. Validation studies are addressed in the Standard Operating Procedures in the
section titled “Internal Validation Studies”. Documentation of validation studies may be
found in Volumes 1-3 of the Validation Notebooks.
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VIII. Analytical Procedures
A. Introduction
The Standard Operating Procedures contain a chapter titled “General Laboratory
Procedures” that describes all portions of the mitochondrial DNA testing carried out in
the laboratory. A later chapter titled “Report Preparation” describes interpretational
guidelines and final report. References to the scientific methods being used in the
laboratory are included in Appendix B.
B. Reagents
All reagents that are used in mitochondrial DNA testing are shown, at first reference, in
bold letters within the text of the Analytical Procedures. These may be looked up in
Appendix A of the Standard Operating Procedures which provides information about
reagent preparation, catalog numbers and vendor identifications for every purchased
reagent.
Further information about reagent handling may be found in the section titled “Reagents
and Solutions” in the Standard Operating Procedures.
C. Critical Reagents
Critical reagents are listed in the section titled “Reagents and Solutions” in the Standard
Operating Procedures. These reagents require testing before use on probative samples,
and the results of these tests are archived in the Reagent Notebook.
D. Reference Standards
The requirement for periodic testing of reference standards, and the descriptions of these
reference standards, are stated in the section titled “Control and Reference Materials” in
the Standard Operating Procedures.
IX. Equipment Calibration and Maintenance
Equipment calibration and maintenance are addressed in the section titled “Equipment
and Instrumentation” of the Standard Operating Procedures. This section identifies
critical equipment and the required maintenance that is to be performed according to the
yearly schedule on this equipment. Documentation on this maintenance is kept in the
Equipment Notebook.
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X. Proficiency Testing
Proficiency testing is required to meet DAB guidelines, and Mitotyping Technologies
undertakes proficiency testing on the 180-day schedule from the College of American
Pathologists. The Standard Operating Procedures section titled “Proficiency Testing”
documents the requirements for proficiency testing and reporting. Proficiency test results
and reports are stored in the Proficiency Testing Records notebook.
XI. Corrective Action
Policy on corrective action in the event of testing discrepancy during either casework or
proficiency testing is described in the Standard Operating Procedures in the section titled
“Corrective Action”.
XII. Reports
The Standard Operating Procedures contain a chapter titled “Report Preparation” which
documents the required contents of a final report at the completion of testing. This
section also describes interpretational guidelines which must be followed by examiners
when analyzing data.
XIII. Review
Case review, both technical and administrative, is required at the conclusion of a case. A
description of this review may be found in the section titled “Case Review” in the Report
Preparation chapter of the Standard Operating Procedures. An additional annual review
of ten randomly selected case files is required and is described in the section titled
Technical Reviews/Audits.
XIV.Safety
Required safety protocols are described in the Standard Operating Procedures, Chapter 2,
“Health and Safety”. Documentation of safety audits and reagent MSDS may be found in
the Safety Notebook.
XV. Monitoring Court Testimony
Requirements for monitoring the court testimony of examiners are described in the
Standard Operating Procedures under the section heading “Examiner Testimony” in
Chapter 4.
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XV. Audits
An annual DAB audit is required and this requirement is documented in the Standard
Operating Procedures in the section titled “Technical Reviews/Audits”. Audit records are
found in the Technical Review/Audits Notebook.
XVI. Subcontractor of Analytical Testing
Any laboratory that has been subcontracted must also comply with DAB guidelines. This
requirement is also documented in the Standard Operating Procedures under the section
titled “Subcontractor Compliance with DAB Guidelines”. The laboratory will require
certification of compliance with these guidelines and review the data received from the
subcontractor to verify its integrity.
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