Houston Forensic Science Center Forensic Analysis Division Forensic Biology. Biology Training Manual

Transcription

1 Houston Forensic Science Center Forensic Analysis Division Forensic Biology Biology Training Manual FAD-BIO-TM.2 Page 1 of 16 Issued by Technical Leader

2 Table of Contents Section 1 Introduction 2 Safety and Contamination Control 3 Identification of Blood and Species Origin 4 Semen Identification 5 Identification of Male DNA through Quantification 6 Report Writing and Testimony 7 Case Internship Program 8 Reading Assignments FAD-BIO-TM.2 Page 2 of 16 Issued by Technical Leader

3 1 Introduction Purpose and Scope The purpose of this manual is to provide a consistent training program for the analysis of forensic evidence by the Biology Section at the Houston Forensic Science Center. This program provides individuals with the theoretical background and the working knowledge to conduct independent casework analysis and effective expert witness testimony in the field of body fluid identification. Heavy emphasis shall be placed on quality assurance of all tests performed, data integrity via thorough documentation, and excellence in obtaining consistent and congruous results. The training program detailed in this document provides the following: Theoretical knowledge of the principles of body fluid identification testing. Working knowledge of the principles and practices of serological theories such as antigen-antibody reaction, as they relate to the forensic analysis of body fluids. The ability to provide independent, accurate, and consistent forensic analysis on forensic case material. The ability to provide effective expert witness testimony that includes, but is not limited to, the presentation of body fluid identification technology and the defense of analytical conclusions. Goals To develop a basic understanding of the methodology and theory of chemical, microscopic, and immunological testing procedures used to identify blood and potential human origin. To develop a basic understanding of the methodology and theory of chemical, microscopic, and immunological testing procedures used to identify semen. FAD-BIO-TM.2 Page 3 of 16 Issued by Technical Leader

4 To develop a basic understanding of the theory and methodology of the detection of male DNA via real time PCR quantification. To develop skills that will allow the trainee to independently and successfully analyze forensic samples. To become familiar with the sensitivity and limitations of the procedures used. To develop an understanding of contamination issues and the steps necessary to avoid contamination. To understand the use of controls during each procedure. To become familiar with and understand the function of any buffers, solutions, or reagents used. To become familiar with all documentation required. Requirements for Qualification Prerequisites: Individuals must have a Bachelors degree in Biology, Molecular Biology, Biochemistry, Genetics or a closely related field. Must have successfully completed courses in biochemistry, genetics, molecular biology or other subjects providing a basic understanding of forensic DNA analysis, as well as course or training in statistics and/or population genetics as it applies to forensic DNA analysis. Competency Test: Individuals must pass competency test(s). These tests are to determine the trainee s ability to consistently analyze body fluids from a variety of sources. Competency tests will include blood, semen, mixed fluids, and simulated cases, as appropriate, given the technique(s) in question. Written Examination: A written examination shall examine the trainee s understanding of the theoretical and working knowledge of body fluid identification tests and how they are applied in the laboratory. Mock Trial: A mock trial shall be used to determine the trainee s ability to provide effective expert witness testimony. FAD-BIO-TM.2 Page 4 of 16 Issued by Technical Leader

5 Instructions for Training Administered through laboratory director using the resources of the Houston Forensic Science Center. This program is designed to provide each trainee with the theoretical background and working knowledge to reliably analyze forensic material utilizing body fluid identification tests. Every topic listed in this manual is important; a deficiency in one area can lead to the failure of a successful analysis and/or defense of the analysis in a court of law. Quality assurance guidelines should be followed for every sample processed in the training program. This will ensure that each trainee maintains a high degree of concentration and awareness during the performance of his/her training, encouraging the learning of good habits that will be applied to actual case work. The order of topics listed in this manual is not necessarily the order in which the tests will be performed. It may be necessary to learn and perform some techniques out of order. Additionally, individuals may be trained in and obtain competency for individual techniques. Pertinent scientific literature and technical manuals should be made available to the trainee. All potential safety hazards to the trainee must be explained BEFORE performing a task that may involve that particular safety hazard. Instructions for the Trainee The trainee is required to keep files on all work completed. These files should include, but not be limited to, worksheets and copies of reports issued for cases in which they participate. The trainee will maintain a notebook documenting all testing conducted during their training period. These files and notebooks should be checked periodically by the Quality Manager and/or Supervisor. The trainee will document weekly what they read or did as part of their training that week. Documentation Protocol Notebook: FAD-BIO-TM.2 Page 5 of 16 Issued by Technical Leader

6 The Biology Training Manual shall be made available to each analyst. The analyst shall not deviate from any procedure without permission from the Section Supervisor. Any deviation (purposely or by mistake) from the protocol shall be thoroughly documented on the worksheet at the time of occurrence. 2 Safety and Contamination Control Objectives: 1. To instruct the trainee on the organization of the Houston Forensic Science Center including rules and regulations. 2. To introduce the trainee to the Houston Forensic Science Center Forensic Analysis Division and to provide basic instruction on all sections comprising the lab and the analyses performed in those sections. 3. To make the trainee aware of the potential hazards that exist in the laboratory. 4. To obtain a working knowledge of factors and conditions that influence the deterioration of evidence as it relates to packaging, handling, and storage conditions and time. 5. To develop an understanding of the necessity for detailed comprehensive notes and adequate labeling of evidential materials. 6. To gain a thorough understanding of Evidence Handling Procedures regarding receiving, identifying, and handling of evidence, as well as specific guidelines for handling biological evidence in the Biology Unit. Guidelines: The trainer or Laboratory Safety Officer will instruct the trainee on the following potential hazards: Infectious Agent Viral agents, including HIV and Hepatitis Bacteria, including sexually transmitted diseases Fungi Parasites Hazardous Materials FAD-BIO-TM.2 Page 6 of 16 Issued by Technical Leader

7 Caustic Agents (Acids and Bases) Carcinogens/Mutagens Organic Chemicals Electrical Hazards Shock from any pieces of electrical equipment Burn Hazards Autoclaves Sterilizers Bunsen burners Hot Plates Eye Damage Alternate light source Ultraviolet (UV) light Laboratory Safety Procedures Prior to the commencement of training, the Laboratory Safety Officer must train individuals in laboratory safety. Various manuals are provided that must be followed to ensure safety of all laboratory personnel. The following manuals are to be used for reference and guidance for laboratory safety: MSDS Notebook, Biology Training Manual, various safety videos including Bloodborne Pathogens, and the Houston Forensic Science Center Safety Manual. The trainee will also be briefed on the fire evacuation plan for the laboratory. It is the responsibility of the trainer to alert the trainee to safety hazards specific to the laboratory. The trainee will be taken throughout the laboratory and shown areas of interest to his/her work. He/she will also be provided with a written job description, and an organizational chart. The DNA Unit uses Polymerase Chain Reaction (PCR) Technology, which allows very small amounts of DNA to be amplified over a million times. Because of the sensitivity of this technique, contamination control is a very serious issue that must be emphasized and practiced with every sample, starting with the identification of the fluids present on evidence. The Evidence Handling Procedures must be strictly followed. All items used in the identification, transfer and isolation of forensic samples must be free of contaminate DNA. Gloves must be worn at all times while handling samples. This is to protect both the analyst and the sample. FAD-BIO-TM.2 Page 7 of 16 Issued by Technical Leader

8 A fresh pipette tip shall be used for each transfer of sample to be used for fluid identification and subsequent DNA analysis. Special precautions must be taken to ensure that the bench and surrounding areas have been properly decontaminated. Scissors, tweezers, and other instruments used for cutting or extraction shall be sterilized in between each sample. The preparation of the known and unknown samples shall be separated by time. Tasks: Obtain and read handbooks and documentation pertinent to new employees Obtain and understand the Houston Forensic Science Center organizational chart. FAD-BIO-TM.2 Page 8 of 16 Issued by Technical Leader

9 3 Identification of Blood and Species Origin Objective: Guidelines: To develop a basic understanding of the methodology and theory of chemical, microscopic, and immunological testing procedures used to identify blood and determine human origin. To understand the chemical reactions involved in phenolphthalein and luminol tests. To understand the principle of immunology involved in antigen-antibody reactions. Tasks: Practical tests 1. Test phenolphthalein and luminol for false positives. Use at least 10 of the following: potassium dichromate, copper, copper sulfate, garlic, potatoes, carrots, dried vegetables, flour, whole wheat flour, milk, fresh fruits (pulp and juice) including lemon, grapefruit, oranges, bananas and watermelons, horse radish, lead oxides in paint, iodine, urine, turnips, eggs, Coca Cola, saliva, sweat, feces, tears, coffee, tobacco, whiskey, tomato juice, iron, rust, nickel, potassium ferrocyanide, sodium cobaltinirite, manganese dioxide, artichoke, cabbage, onion, dandelion root, and bleach 2. Perform serial dilutions up to 1:1,000,000 of human blood to determine sensitivity of phenolphthalein and luminol. 3. Test phenolphthalein and the ABAcard HemaTrace test for crossreactivity to at least 5 animals/primates, if available. 4. Perform serial dilutions up to 1:1,000,000 of human blood to determine sensitivity of the ABAcard HemaTrace test. FAD-BIO-TM.2 Page 9 of 16 Issued by Technical Leader

10 4 Semen Identification Objective: To understand the methodology and theory of testing procedures used to identify semen. Guidelines: To develop a basic understanding of the methodology and theory of chemical, microscopic, and immunological testing procedures used in the identification of semen. To become knowledgeable of sperm cell morphology and proficient at identifying human sperm. To develop a working knowledge of the stereo and compound microscopes. Tasks: Practical tests 1. Test human and animal semen, blood, urine, saliva, feces, sweat, and semen free vaginal swabs for p30 using the Seratec PSA (p30) test. 2. Perform serial dilutions up to 1:1,000,000 of semen to determine limits of sensitivity with One step AP method and the Seratec PSA (p30) test. 3. Test at least 10 of the following items for false positives with the One step AP method: Human milk, alpha-naphthol, human liver, human urine, human kidney, red blood cells, almonds, cauliflower, Brussels sprouts, clover, garlic, turnips, rice bran, raisins, mango fruit, ginger, figs, dates, animal semen, semen free vaginal fluid (multiple individuals, if possible) 4. Test body fluids (listed above and tissue samples) with bright flashlight, alternate light source, and UV light. 5. Compare human and animal sperm using the Christmas Tree stain. 6. Extract sperm cells from swabs that contain blood and/or feces mixed with semen. Stain slide using the Christmas Tree stain and observe for the presence of sperm under a microscope. FAD-BIO-TM.2 Page 10 of 16 Issued by Technical Leader

11 5 Identification of Male DNA through Quantification Objective: To understand the methodology and theory of testing procedures used in the detection of male DNA via qpcr. Guidelines: Tasks: To develop a basic understanding of the methodology and theory of Chelex DNA extraction. To develop a basic understanding of the theory of real-time PCR quantification. To develop a working knowledge of the Plexor HY quantification kit. To develop a working knowledge of the AB 7500 Real-Time PCR System and its software. To develop a working knowledge of the Plexor Analysis Software-Forensic program. Practical Tests 1. Perform at least three Chelex extractions. Each extraction should contain at least ten samples. 2. Perform at least three Plexor HY quantifications. DNA Standards should be prepared new for at least three quantifications. FAD-BIO-TM.2 Page 11 of 16 Issued by Technical Leader

12 6 Report Writing and Testimony Objective: To develop the skills necessary to effectively report body fluid identification results and provide expert testimony about those results in a court of law. Guidelines: Tasks: To develop a working knowledge of the terminology and the presentation of analysis and results. To become skilled at expressing written and oral results simply, concisely, and accurately. Practical tests 1. Conduct analysis and draft a report on a proficiency or mock case. 2. Draft reports on cases previously analyzed. 3. Attend court and observe experienced examiners testify. 4. Successfully complete a mock trial. FAD-BIO-TM.2 Page 12 of 16 Issued by Technical Leader

13 7 Case Internship Program Objective: Guidelines: Tasks: Once the trainee completes his/her competency tests, the trainee is placed in a case internship program under experienced serologists. The purpose of this internship is to allow the newly qualified examiner to work cases, but under the close supervision of an experienced analyst. As techniques are added to an experienced analyst s repertoire, he/she is not required to reenter the case internship program. The newly qualified examiner will work cases and sign his/her own reports while under the close supervision of experienced examiners for a minimum of two months. The newly qualified examiner, the experienced examiners, and the Biology Supervisor will all decide when the analyst is ready for unsupervised casework analysis. The newly qualified examiner will conduct the entire analysis of each case, prepare the notes, and write the report. The experienced examiner will observe all results, initial the notes and evidence, and review all work conducted. The experienced examiner will constructively critique the work conducted by the newly qualified examiner and ask the new examiner questions about the case that could be expected in court. The new examiner will sign their own reports; the experienced examiner will document their role as the supervising examiner via signature on the report and/or review sheet. The new examiner as well as the experienced examiner will initial all documents in the case folder. The newly qualified examiner will perform practice technical reviews on a minimum of 10 cases, after which the experienced analyst will perform the actual technical review. The experienced examiner will then compare the two reviews, and give feedback to the newly qualified examiner in order to gain experience in performing technical reviews. This phase of the case internship program will last no less than two months. The newly qualified examiner may be signed off to perform casework analysis/write reports and technically review cases at two separate times. FAD-BIO-TM.2 Page 13 of 16 Issued by Technical Leader

14 8 Assigned Readings Crime Scene Evidence Preservation: Fisher, Barry A.J., "Introduction", Techniques of Crime Scene Investigation, 7 th edition, 2004, pp Saferstein, R., PhD., "Physical Evidence", Criminalistics, An Introduction to Forensic Science, 9 th edition, 2007, pp Warrington, Dick, "DNA Collection and Packaging", Forensic Magazine, Vol. 6, No. 2, pp Blood Identification: Abacus Diagnostics, Inc., "OneStep ABAcard HemaTrace for the Forensic Identification of Human Blood", 2000, (product insert). DeForest, Peter R., Gaensslen, R. E., Lee, Henry C., "Blood", Forensic Science An Introduction to Criminalistics, 1983, pp Fisher, Barry A.J., "Blood and Other Biological Evidence", Techniques of Crime Scene Investigation, 7 th edition, 2004, pp Lee, Henry C., "Identification and Grouping of Bloodstains", Forensic Science Handbook, Vol. I, 1982, pp Lytle, L. T., PhD., and Hedgecock, D. G., "Chemiluminescence in the Visualization of Forensic Bloodstains", Journal of Forensic Science (JFS), Vol. 23, 1978, pp Saferstein, R., PhD., "Forensic Serology", Criminalistics, An Introduction to Forensic Science, 9 th edition, 2007, pp Seeley, et al., "Cardiovascular System: Blood", Anatomy and Physiology, 2 nd edition, 1992, pp Shaler, Robert C., PhD., "Modern Forensic Biology", Forensic Science Handbook, Vol. I, 2002, pp Semen Identification: Seratec, In-vitro diagnostic test for professional forensic use for the detection of seminal fluid by the semi-quantitative determination of PSA (Prostate-specific antigen), 2009, (product insert). FAD-BIO-TM.2 Page 14 of 16 Issued by Technical Leader

15 Bechtel, F. S., "Immunological Methods for Seminal Fluid Identification", Proceedings of a Forensic Science Symposium on the Analysis of Sexual Assault Evidence, 1983, pp Baechtel, F. S., "The Identification and Individualization of Semen Stains", Forensic Science Handbook, Vol. II, 1988, pp Chang, Thomas S. K., "Seminal Cytology", Proceedings of a Forensic Science Symposium on the Analysis of Sexual Assault Evidence, 1983, pp Davies, Anne and Wilson, Elizabeth, "The Persistence of Seminal Constituents in the Human Vagina", Forensic Science, Vol. 3, 1974, pp DeForest, Peter R., Gaensslen, R. E., Lee, Henry C., "Body Fluids", Forensic Science An Introduction to Criminalistics, 1983, pp Eckert, William G., "Serology and DNA Typing", Introduction to Forensic Sciences, 2 nd edition, 1992, pp Fisher, Barry A.J., "Investigating Sexual Assault and Domestic Abuse Crimes", Techniques of Crime Scene Investigation, 7 th edition, 2004, pp Gaensslen, R. E., "Identification of Semen and Vaginal Secretions", Sourcebook in Forensic Serology, Immunology, and Biochemistry, NIJ, 1983, pp , Jones, Edwin L. Jr., "The Identification of Semen and Other Body Fluids", Forensic Science Handbook, Vol. II, 2005, pp Kind, S. S., "The Acid Phosphatase Test", Methods of Forensic Science, Vol. 3, 1964, pp Saferstein, R., PhD., "Forensic Serology", Criminalistics, An Introduction to Forensic Science, 9 th edition, 2007, pp Shaler, Robert C., PhD., "Modern Forensic Biology", Forensic Science Handbook, Vol. I, 2002, pp Willott, G. M. and Allard, J. E., "Spermatozoa- Their Persistence after Sexual Intercourse", Forensic Science International, Vol. 19, 1982, pp Wilson, E. F., "Sperm's Morphologic Survival after 16 Days in the Vagina of a Dead Body", Journal of Forensic Science (JFS), Vol. 19, 1974, pp FAD-BIO-TM.2 Page 15 of 16 Issued by Technical Leader

16 Detection of Male DNA via Quantification: Chelex 100 as a Medium for Simple Extraction of DNA for PCR-Based Typing from Forensic Material. BioTechniques Vol. 10, No.4 (1991) Plexor HY System for the Applied Biosystems 7500 and 7500 FAST Real-Time PCR Systems, User Manual, Part# TM293 Houston Police Department Crime Laboratory Chelex-Plexor Validation Screening SOP # 13: Extraction of DNA through the use of Chelex Extraction Screening SOP #14: Plexor HY Quantification DNA SOP #8: DNA Quantification Courtroom Testimony: Eckert, William G., "Scientific Evidence in Court", Introduction to Forensic Sciences, 2 nd edition, 1992, pp Kahn, Lisa, J.D. and Feldstern, Daniel, J.D., "How to Succeed as an Expert Witness", GenePrint, 1998, pp Sapir, Gil I., J.D., MSc., "Legal Aspects of Forensic Science", Forensic Science Handbook, Vol. I, 2002, pp FAD-BIO-TM.2 Page 16 of 16 Issued by Technical Leader