IN THE NAME OF GOD

بسمه تعالی وزارت بهداشت درمان و آموزش پزشکی آزمایشگاه مرجع سلامت Sampling, Sample transfer and storage for Toxicological Analysis Seyed Ali Nazeri

 Introduction  Group Tests  Types of samples  Sample transfer and storage  Introduction  Group Tests  Types of samples  Sample transfer and storage

 Toxicological analysis must start as soon as possible after a …  At least six different test groups are required to be analyzed to exclude even the most commonly encountered poisons. Introduction

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

SAMPLE & SAMPLING FOR TOXICOLOGICAL ANALYSISA

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

Biological samples in alive persons Urine and blood samples should be collected as soon as possible in suspected drugs and alcohol ….

Postmortem biological samples 1- Stomach Content (all available) 2- Blood (20-30 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 )

Non-biological Specimens

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

Sample transfer and storage

Evidence collected, 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.

Sample transfer and storage  Urine Urine should be collected in any case in which the complainant reports within the first 120 hours (5 days) after the alleged assault. 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

Sample transfer and storage  Whole blood Blood should be collected in addition to urine, preferably within 48 hours of the alleged incident. The use of ethanol or other solvents with volatile fractions should be avoided in skin disinfection. At least two 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.

Urine Testing: The Most Commonly Used Drug Test

Sample transfer and storage Whole blood 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..

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. It is very important that hair is sampled in a strict manner by properly trained personnel. 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. Hair samples should be stored at room temperature, in a dry environment protected from light.

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.

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 … etc. should be collected with the classical precautionary measures for DNA analysis.

References : 1- Guidelines for the Forencic Analysis of Drug, UNITED NATIONS, Clarck `s Analysis of Drug and Poison, Pharmaceutical Press Third Edition, Published P0wer Point,Maryam Akhgari, Pharm D., Ph.D Assistant professor of Pharmacology and Toxicology

گردهمایی کارشناس مسئولین آزمایشگاههای بهداشتی دانشگاههای علوم پزشکی کشور آبان 94

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.

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

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

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.

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.

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.

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

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.