1. IN THE NAME OF GOD

2. بسمه تعالی آزمایشگاه مرجع سلامت Sample & Sampling for Toxicological Analysis Seyed Ali Nazeri nazeriali2002@yahoo.com

3. INTRODUCTION At least six different test groups are required to be analyzed to exclude even the most commonly encountered poisons.

4. Group 1: Gases Group 2: Volatile substances Group 3: Metals Group 4: Drugs Group 5: Pesticides Group 6: Miscellaneous substances Group Tests

5. TOXICOLOGICAL ANALYSIS OF TISSUE Collect biological samples from organs and tissues. A forensic toxicologist cannot simply look for the presence of a toxin or drug in a body, she must understand how the body processes these molecules. Toxicological analysis must start as soon as possible after a person’s death.

6. SAMPLE & SAMPLING FOR TOXICOLOGICAL ANALYSISA

7. Issues in Specimen Collection Selection – Multiple, varied sites of collection Collection – Appropriate method of collection – Adequate volumes for analysis Storage and handling

8. Types of samples The specimens available for analysis may be numerous, they include: Biological Samples Non-Biological Samples

9. Postmortem biological samples 1- Stomach Content (all available) 2- Blood (20-30 mL, 10 mL) 3- Urine (all available) 4- Liver (250 gr) 5- Kidney 6- Vitreous Humor (all available) 7- Bile (all available) 8- Muscle (10 cm 3 )

10. Biological Specimens Urine Qualitative - the presence of a drug in the urine of an individual indicates that some time prior to death the drug or poison was present in the blood of the individual. Stomach contents Stomach contents may contain unabsorbed poisons, tablets, capsules, caplets which may be intact and visible. These can be removed from the stomach contents, and identified.

11. Urine Testing: The Most Commonly Used Drug Test

12. Bile Since it is produced by the liver, it is similar in respect to the liver samples in that concentrations of drugs in the bile are usually greater than concentrations in the blood. Liver According to it’s structure, which contains metabolizing enzymes and fat, It is the best tissue sample for toxicological analysis

13. Vitreous humor Vitreous humor is in a protected position behind the lens of the eye. Because of this protected position, it is isolated from putrefactive processes, from charring and from trauma. Vitreous fluid is less susceptible to these effects, particularly because it is likely to be free from microorganisms

14. Blood The potential for postmortem ethanol formation complicates the interpretation of ethanol-positive results. Sodium fluoride is the most commonly used preservative for postmortem specimens. Sodium fluoride protects blood from postmortem changes such as bacterial production of ethanol or other alcohols. It also helps to protect other labile drugs such as cocaine, nitrazepam and clonazepam from degradation.

15. Fluoride and enolase activity The fluoride ion is seemingly effective in inhibiting the activity of several kinds of enzymes, such as enolase a component in the glycolytic pathway, and is important for the action of yeasts, fungi and many micro-organisms responsible for fermentation.

16. Hair Since hair grows at a predictable rate (generally 1 cm/month) can be used to provide a historical record of drug or poison exposure. Procedure is to chop a hair sample into 1 cm increments and analyze them separately to “track” drug exposure over a long period of time.

17. Biological samples in alive persons Urine and blood samples should be collected as soon as possible in suspected opioid and alcohol abuse.

18. Non-biological Specimens

19. Non-biological submissions Used to direct analysis of biologicals May indicate the nature of substances that may have been ingested, inhaled or injected Examples: – Containers found at the scene – Syringes – Unidentified tablets or liquids

20. Tablets Ampoules Powders Capsules Drugs of abuse Syringes Food & Beverage Residues Non biological samples

24. Storage and Handling

25. Identification of samples – Continuity – Contents Specimens delivered to lab without delay Specimens should be analyzed as soon as possible Storage areas should be secure Proper specimen handling

26. Storage and Handling Not feasible to analyze specimens immediately Sample should be in well-sealed container Sample containers must be sterile Use of preservatives and anti-coagulants Refrigeration vs. Freezing – Both inhibit bacterial action; esp. freezing – Freezing results in  prep time – Freeze-thaw cycle may promote breakdown

27. Storage of Samples Preservative – Sodium fluoride Anti-coagulants – Sodium citrate – Potassium oxalate – EDTA – Heparin Anticoagulants are not really necessary in postmortem blood samples since the blood is hemolyzed.

28. Sample transfer and storage Evidence collected from the victim of a suspected DFC must be properly sealed and secured. Biological specimens should be stored at 2-8 °C to help prevent degradation. Specimens should be transported refrigerated to the laboratory as quickly as possible; in any event minimizing the time they are kept at temperatures above 25 °C.

29. Sample transfer and storage Whole blood Blood should be collected in addition to urine, preferably within 48 hours of the alleged incident. Blood sampling should be performed with disposable syringes; the use of ethanol or other solvents with volatile fractions should be avoided in skin disinfection. At least two 5 ml samples should be collected in blood tubes containing compounds such as NaF and potassium oxalate (recommended concentrations for NaF 2.5 g/l and potassium oxalate 2 g/l) to prevent degradation and clotting with one sample used for analyses and the other retained in case of a need for a defence analysis. The blood samples should be refrigerated (at 2-8°C) as soon as possible. If it is not possible to conduct analysis within 24 hours, it is advisable to preserve the sample by storage in a freezer (after separating the plasma). Furthermore, it is advisable that sample residues are stored in a freezer (–18 °C) in case further analyses are requested at a later date. In cases where blood plasma may need to be separated by centrifugation from blood cells prior to analysis, the separation should be done before the freezing of whole blood. It should be noted that the timeframes provided for detection of drugs in urine and blood are general guidelines and that many drugs will no longer be detectable in conventional samples such as urine, four or five days after ingestion

30. Sample transfer and storage Head hair In cases of late reporting of the alleged assault or if chronic exposure to a drug must be assessed, head hair should be collected at least four weeks after the alleged assault. At least two hair samples (thickness of a pencil) should be cut as close to the scalp as possible (see the figure 1 in annex 5). It is very important that hair is sampled in a strict manner by properly trained personnel. A sampling protocol is given in annex 5. In cases of a shaven head, pubic, axillary, torso or leg hair may also be collected for analysis, although the interpretation of the quantitative results in these cases remains very difficult. When segmental analysis is not possible (if only axillary, torso or leg hair are available), analysis could be eventually limited to a qualitative analysis, because the growth rate is not well established, as happens in head hair. Consequently, a positive result in these type of hair samples indicates that the alleged victim consumed the compound at any time, but not necessarily at the time of the assault. Hair samples should be stored at room temperature, in a dry environment protected from light

31. Sample transfer and storage Other biological samples In some cases, vomit from the scene of the alleged assault or from the clothes of the complainant may be a useful specimen. If a drug is not fully absorbed before vomiting occurs, the drug may be detected at relatively high amounts in a vomit stain. If collected, vomit or a vomit stain should be stored preferably frozen.

32. Sample transfer and storage Biological samples and sampling Urine Urine should be collected in any case in which the complainant reports within the first 120 hours (5 days) after the alleged assault. While in reality many of the drugs listed in annex 1 may have been eliminated from urine in less than 120 hours, a few may remain at low concentration. A minimum of 50 ml of urine should be collected in at least two sterile containers (no preservative needed) and stored at 2-8 °C. If it is not possible to analyse samples within 24 hours, it is advisable to store the samples in a freezer (–18 °C). Unused samples should be stored in a freezer in case further analysis is requested for at last 12 months

33. Sample transfer and storage Other samples If the scene of the alleged assault is searched, cups, drinking glasses, bottles, containers and liquids that may contain residues of drugs should be collected and submitted for analysis. Other items of evidence that could prove useful to the investigation include plates, foods, pharmaceutical products and prescriptions for medicines. Photographic/video (cameras, video recorders) and electronic evidence from computers may also prove useful to the case as there have been several instances when the perpetrator(s) have recorded the assault. For trace evidence, clothing, sheets and bedding, sexual devices, condoms, etc. should be collected with the classical precautionary measures for DNA analysis. Police and crime scene investigators are normally trained to collect such evidence. Care should be taken that scene residues are packed individually in order to avoid cross- contamination of biological samples, especially if volatile compounds are implicated in the alleged assault

34. گردهمایی کارشناس مسئولین آزمایشگاههای بهداشتی دانشگاههای علوم پزشکی کشور 10- 9 آبان 94