Forensic Medicine & Applied Toxicology-3

Category: Education

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A third in a series of presentations meant for senior medical students of Moi University School of Medicine


Presentation Transcript

Forensic Medicine & Applied Toxicology:

Forensic Medicine & Applied Toxicology Samples and Sampling Technique for Specimen Collection By Dr. Willis Ochieng Toxicologist

Integrity of Samples:

Sample contamination can lead to successful legal defence in many trials Avoid the unnecessary sample contamination by collecting biological fluids using new or chemically clean sampling instruments Most forensic sampling of liquids is done by hypodermic syringes The sampler must – Be adequately clean Make use of appropriate needle gauges and lengths for the correct collection of specimen samples Is recommended that one needle and syringe should be used per specimen taken In laboratories with insufficient funds, it may be necessary to reuse these materials Soake for a minimum of 30 minutes in a disinfectant, e.g., 10% solution of household bleach ( Jik ) in water (0.5% w/v sodium hypochlorite in water), followed by washing with a non ionic detergent and rinsing with large quantities of clean water The autopsy and toxicology staff must maintain the cleanliness of the specimen container as they collect the specimen by avoidance of spillage* Integrity of Samples 2 07/08/2014

Possible Clinical Forensic Samples-1 :

Samples Evidence type Sampling materials Comments Blood DNA profiling and blood grouping EDTA blood bottle Fridge Blood Alcohol and drug analysis Blood bottle with preservative and anti coagulant Freezer for solvent residues, otherwise fridge. Urine Alcohol and drug analysis Bottle with preservative Fridge Possible Clinical Forensic Samples-1 3 07/08/2014

Possible Clinical Forensic Samples-2:

Samples Evidence type Sampling materials Comments Saliva Semen and ABO/secretor blood grouping Plain bottle Freezer Mouth swab Semen Plain sterile cotton wool swab Freezer Skin swabs Semen, saliva, blood, lubricants Plain sterile cotton wool swabs Freezer Loose hairs Hair comparison Secure sample bag Room temperature Possible Clinical Forensic Samples-2 4 07/08/2014

Possible Clinical Forensic Samples-3:

Samples Evidence type Sampling materials Comments Matted head or pubic hair Semen Secure sample bag Freezer External and internal vaginal swabs Semen, blood, lubricants Plain sterile cotton wool swab Freezer External and internal anal swabs Semen, lubricants Plain sterile cotton wool swab Freezer Tampon/sanitary towel Semen Secure sample bag Freezer Fingernail cuttings Visible blood , skin, tissue, fibres Secure sample bag Freezer Clothing worn at the time of the assault/abuse Blood, semen saliva, vaginal fluid, lubricants. Paper bags Room temperature Possible Clinical Forensic Samples-3 5 07/08/2014

Possible Autopsy Toxicology Specimens-1 :

Samples Evidence type Sampling materials Comments Blood, Heart Always. For toxicology screen Glass Identify source. Preserve with 2% sodium fluoride and potassium oxalate. Reserve an aliquot without preservative, if possible. Blood, peripheral For complete toxicology Testing Glass Identify source. Use femoral or subclavian blood if possible Blood clot Trauma cases. May reveal the pre-trauma drug status. Glass Useful for ethanol analysis. Urine Always. For alcohol and drug analysis Glass Submit any quantity, even if < 1 ml, for immunoassay screening. Blood, Heart Always. For toxicology screen Glass Identify source. Preserve with 2% sodium fluoride and potassium oxalate. Reserve an aliquot without preservative, if possible. Possible Autopsy Toxicology Specimens-1 6 07/08/2014

Possible Autopsy Toxicology Specimens-2:

Samples Evidence Type Sampling Material Comments Bile Always. Toxicology screening. Glass Tie off gall-bladder to reduce contamination. Collect prior to liver. Vitreous humour Always. Toxicology screening. Glass Combine fluid from both eyes into a single tube. Gastric contents For complete toxicology testing Glass Tie off stomach to reduce contamination of other viscera. Note total volume Liver Always. Toxicology screening. Plastic Identify source. Deep right lobe preferred. Fridge. Kidney Metals, ethylene glycol. Plastic Fridge Possible Autopsy Toxicology Specimens-2 7 07/08/2014

Possible Autopsy Toxicology Specimens-3:

Samples Evidence Type Sampling Material Comments Spleen CO, CN Plastic Very useful when blood not available in fire deaths. Brain, fat Lipophilic drugs Plastic Brain may be especially useful in infant drug deaths. Lung Volatile poisons Plastic. Collect in sealed container. Collect tracheal air as well. Meconium Foetal poisoning Plastic For in utero toxic exposure Insect larvae Drug screening Suitable container Useful in badly decomposed bodies Possible Autopsy Toxicology Specimens-3 Soil Hair Toxicology screening Drug history Suitable container Suitable container Useful in exhumed bodies . Identify proximal and distal ends 8 07/08/2014

Criterion for Selection of Post-mortem Specimens in poisoning:

Criterion for Selection of Post-mortem Specimens in poisoning

Post-mortem Specimens in poisoning:

Any tissue or sample is accessible at post-mortem . The type and number of specimens chosen is however dependent on several factors - Institutional (laboratory) policy Instrument sensitivity Case history Specimen availability in a given case Because the autopsy allows a one-time opportunity to collect as many specimens as may be needed, it is suggested that as many specimens be obtained as is necessary and practical The hope of implicating therapeutically used poisons in hospital deaths, diminish with the survival time in the hospital prior to death Under no circumstances should the clinical specimens be relied on as interpretation may not be based on hospital determined data Many a time, the laboratory encounters cases of decomposed, skeletonised or embalmed cases The possibilities for specimens in some of these cases are limited only by availability 10 Post-mortem Specimens in poisoning 07/08/2014

Routine & Commonly Sampled Specimens Blood & its superiority:

Specimen of choice for detecting, quantifying and interpreting poison concentrations in post-mortem toxicology Most meaningful interpretation & laboratory data are generated from blood analyses Higher drug concentrations than the recorded therapeutic levels is not always and obvious indicator of intoxication Finding higher parent drug to metabolite ratio, one is more likely to conclude or suspect that the intoxication was acute Pharmacokinetic parameters gradually change during toxicogenesis Pharmacokinetic parameters convert into toxicokinetic parameters with entirely different half-life, elimination or absorption constants, etc . At death, interpretation becomes more difficult and important in cases where poison analytes known to undergo post-mortem redistribution are determined to be present in a heart blood specimen Laboratories perform initial toxicological tests on cardiac blood, reserving the peripheral blood specimens for cases where additional data is needed for interpretation Clinically drug levels are measured in either plasma, serum or erythrocytes, forensically the use of whole blood is preferred Measure the drug concentration in two or more independent samples , since the value of a result from a single sample will always be limited 11 Routine & Commonly Sampled Specimens Blood & its superiority 07/08/2014

Samples & Sampling Urine :

Urine contains products of metabolism or detoxification process in the liver and elsewhere and has the greatest potential to provide the qualitative ante-mortem poison-exposure information Urine matrix is generally devoid of circulating large molecular weight compounds due to the renal filtration process Immunoassays and non-instrumental spot tests can be performed directly with it for the analysis of certain drug classes Detection times for poisons in urine can vary from 24 hrs to as long as a month , depending on the poison Positive identification of poisons in urine indicates past poison use, but does not indicate when or how much poison was ingested In order to interpret the content of exposure, blood is tested for the poisons found in the urine In cases where death has occurred rapidly due to poison ingestion, negative urine poison findings may be consistent if blood poison concentrations are very high 12 Samples & Sampling Urine 07/08/2014

Samples & Sampling Bile :

Analytical value of bile is not very different from that of urine It contains principally products of metabolism of drugs & poisons The qualitative finding of the presence of poison or metabolites in bile is important for documentation of historical exposures to specific agents and chronic poisons-use history Generally, poisons of greater molecular weight are usually excreted into bile following phase II conjugation Opiates particularly morphine have high concentrations into bile compared to blood and as a result it has been used for years as a specimen for opiates Many drugs and poisons accumulate at concentrations higher than those in blood making bile a useful specimen for the analysis of a wide variety of drugs and conventional poisons and their metabolites 13 Samples & Sampling Bile 07/08/2014

Samples & Sampling Vitreous Humour :

Vitreous humour may accumulate some substances more than aqueous humour When the death scene is unclear, vitreous humour can provide some insights, provided that the other autopsy findings are also taken into consideration By virtue of its protected environment inside the eye, vitreous humour is less subject to contamination and bacterial decomposition It may be used to distinguish ante-mortem drug and more specifically alcohol ingestion from post-mortem alcohol formation Because vitreous humour is contained in a peripheral compartment, there is a delay in both the uptake of drugs and alcohol into this fluid . Since drugs take a long time to equilibrate into vitreous humour, they equally take long to be excreted from it This makes it a tissue that may reflect a closer ante-mortem poison concentration The eye has limitations to its use as analytical specimen Only a few drugs may have tropism for the eyes based on their physicochemical characteristics Despite these concerns, vitreous humour is the best specimen from which to evaluate post-mortem digoxin concentrations Even if a particular poison’s predilection is the eyes, the analysis of femoral blood in addition to vitreous humour is still recommended for an appropriate interpretation of toxicity 14 Samples & Sampling Vitreous Humour 07/08/2014

Samples & Sampling Gastric Contents :

Gastrointestinal tract is a common and the suicidal route of exposure to both drugs and poisons It is the major route of drug administration and, therefore, a major compartment for the investigation of a potential poisoning Observation of a large quantity of the parent drug in the gastric content, relative to a prescription dose, is indicative of an oral drug overdose Presence of a reasonable large amount of drug does not mean it is the cause of death Usually the equivalent of five or more dosage units in the stomach is a better criterion for reflecting on the possibility of suicide In order to increase accuracy or quality of data, it is important to make a record of the total volume of gastric contents present in the decedent in order to calculate the total amount of the drugs present in the stomach The best thing is taking the entire specimen to the laboratory for the staff there to mix the contents and do their own aliquoting The use of the sense of smell as analytical tool can both be useful and dangerous Cyanide ingestion produces stomach contents with the characteristic odour usually said to be similar or close to that of bitter almonds 15 Samples & Sampling Gastric Contents 07/08/2014

Samples & Sampling Tissues :

Tissues commonly collected for post-mortem analysis include liver, kidney, brain, lung and spleen These tissues provide the best and most useful ground with which to interpret blood findings It is easier to work with tissues than body fluids Analysis of tissue may be done more appropriately than analysis of biological fluids for some poisons Liver provides the computations of fatal ratios needed in the search for cause of death due to poisoning In heavy metal poisoning, kidney is a very useful specimen as heavy metals concentrate in it Spleen , an organ rich in blood, is useful for the analysis of compounds which bind to haemoglobin, such as carbon monoxide and cyanide In fire deaths where extensive charring is present, spleen may be the only useful specimen available to perform toxicological assays Lung tissue is particularly useful in cases where inhalation of volatile substances, such as solvents is suspected Brain , due to its high fat content accumulate lipophilic substances, such as chlorinated hydrocarbons, organophosphate pesticides, and other organic poisons Brain is in a protected environment and tends to be more resistant to post-mortem decomposition Concentrations of poisons in tissues should not be expressed per volume of homogenate 16 Samples & Sampling Tissues 07/08/2014

Samples & Sampling Hair:

Telogen hairs are one of the most common useful evidence findings at crime scenes and/or on homicide victims Occasionally, the microscopic characterisation of the found hair is the only physical evidence association to a victim or suspect Hair can be characterised by mitochondrial DNA methods Suitable for the analysis of heavy metals Limitation - Traditional microscopic comparison techniques cannot state with certainty that a found hair originated from a particular individual Results of hair testing for drugs of abuse are consistent with autopsy toxicology reports It is a specimen from which chronic drug use may be determined Drugs have been identified in hair including drugs of abuse and various therapeutic agents It is a non-invasive specimen for assessing patient compliance Hair specimen is useful in extremely decomposed or skeletonised cases where no other specimens remain 17 Samples & Sampling Hair 07/08/2014

Samples & Sampling Bone and Bone Marrow:

Use of bone marrow as a specimen is not common though important in trauma cases Because marrow is protected by bone, the highly vascularised tissues may be the only one free from contamination when contamination of blood specimens is suspected Process of decomposition may transform the bone marrow from spongy red marrow to a brown viscous liquid or paste-like substance This paste may still be useful for qualitative analysis of poisons 18 Samples & Sampling Bone and Bone Marrow 07/08/2014

Samples & Sampling Skeletal Muscle :

Skeletal muscle is not part of routine specimens in many laboratories despite the fact that it meets the criteria of an ideal forensic specimen It is relatively homogeneous Almost always available Not easily contaminated Skeletal muscles is useful in cases where drugs suspected of undergoing post-mortem release are detected in the heart blood Extremity muscle is recommended Femoral muscle is a specimen of choice for roughly predicting the ratio of free to total drug in blood even when blood specimens are not available. Why? Because the femoral muscle is relatively spared of both post-mortem diffusion of drugs and bacterial invasion It is often well preserved despite advanced decomposition of other tissue 19 Samples & Sampling Skeletal Muscle 07/08/2014

Samples & Sampling Meconium :

No laboratory uses faecal specimen in post-mortem analysis of poisons Meconium is used to determine foetal drug exposure Meconium analysis is performed- To assess addictive drug exposure in new-borns so that treatment may begin early after birth To determine drug exposure in stillborn infants Meconium is not a homogenous specimen because it forms layers in the intestine as it is being deposited 20 Samples & Sampling Meconium 07/08/2014

Samples & Sampling Entomological Specimen:

Medicolegal questions can be answered by close observation of larval development Presence of insects and their developmental pattern in decomposing bodies provide important forensic information Insects lay eggs in the tissue and give rise to greedy larvae Larvae contain chemical substances which were in the deceased body When sampled, may provide indication of either presence or absence of the poison in the body Homogenised fly larvae of Calliphorid genus (blowfly) are useful Depending on temperature, larvae may be present as soon as one to two days after death Drawbacks are— Rapid diminution of larval drug concentration which does not allow long term storage Larvae lose significantly drug concentration within one day of being transferred from their food source 21 Samples & Sampling Entomological Specimen 07/08/2014

Samples & Sampling Miscellaneous Specimens:

Any item with which a body or bodily fluid has been in contact is a potential candidate for the identification of drugs or poisons Examples include- Tracheal air Blood stains on clothing Soil samples collected at the site of a skeleton or decomposed body Cremation ash 22 Samples & Sampling Miscellaneous Specimens 07/08/2014

Samples & Sampling Non-biological specimens:

Any material found at the scene of death , is a potential toxicological evidence however small it may appear to be Since non-biological materials are evidence, conventional forensic laboratories may not have capability of analysing them It is better if these are sent to the crime laboratory rather than toxicology laboratory 23 Samples & Sampling Non-biological specimens 07/08/2014

Technique for Specimen Collection :

Technique for Specimen Collection

Technique for Specimen Collection Blood-1 :

The correct technique & procedure must be used to maintain the integrity of a particular blood specimen Autolytic processes cause disintegration and increasing permeability of the physiological and anatomical barriers leading to changes in substance concentrations A nalyses done with only one blood specimen from one source does not provide a correct result which is easily interpreted Post-mortem blood specimens from two sites, heart and peripheral , should be collected at every autopsy If a blood sample is needed in a case which does not require autopsy, then only peripheral blood should be collected Heart blood specimens should be taken by needle aspiration using a suitable hypodermic syringe Is of little importance in post-mortem examination To obtain a proper cardiac specimen, the pericardial sack must be opened, the pericardium removed and blood specimen removed by syringe from either the right or left chambers Technique for Specimen Collection Blood-1 25 07/08/2014

Technique for Specimen Collection Blood-2:

Peripheral blood specimens are usually obtained from the femoral vein , although blood from the subclavian vein is also suitable Leg veins are preferred in stead of head and neck-- Due to the anatomical presence of a larger number of valves that resist blood movement from the intestines which reduce redistribution of certain poisons after death It is easier to collect the required volume from the femoral vein than from other peripheral sources Do not massage the muscles of the leg in order to increase specimen volume In some cases, the only available blood material is clot Clot has a strong protection against contamination as a result of poor perfusion of poisons into or out of them Doctors are discouraged from sampling blood from thoracic or abdominal cavity* Technique for Specimen Collection Blood-2 26 07/08/2014

Technique for Specimen Collection Urine-1:

Contains basically metabolites of the poisons. Clinically , urine collection is not invasive and acceptable to many Urinalysis gives an objective evaluation of poisoning and can be useful on an individual level providing complementary information to the overall examination Urine sampling can also provide indicators for studying the evolution of poisoning incidence in different populations , both in a clinical and non-clinical setting Ensure that the urine collected came from the right donor Sodium , chloride, creatinine and pH values done with urine drug screening results to effectively detect resubmitted samples This technique may also have a general application in forensic identification of duplicate samples Technique for Specimen Collection Urine-1 27 07/08/2014

Technique for Specimen Collection Urine-2:

Urine is taken at autopsy directly from the bladder by insertion of a clean hypodermic needle A little more refined skill is required for non-autopsied cases The needle may be inserted directly through the lower abdominal wall, just above the pubic symphysis If possible, up to 100 ml of urine or all available is required If the bladder is empty, aspirate as much urine as possible from both bladder and ureter If no visible urine is available, bladder washing using water (or saline) may be desirable As little as 50  l of undiluted urine is sufficient for some preliminary and essential applications such as chemical spot tests and immunoassay testing Technique for Specimen Collection Urine-2 28 07/08/2014

Technique for Specimen Collection Bile:

Bile is comparable with urine but differs from it because of its relative protein content Presence of free radicals on the walls of gall bladder may be used for morphological evaluation of the organ following exposure to certain types of poisons Gall stone formation may also be related to this Bile is never collected clinically At autopsy, approximately 10 to 20 ml of bile is needed and placed into a properly labelled glass tube The specimen can only be collected at autopsy following the removal of the organ block during the process It is then aspirated from the gall bladder using a clean hypodermic syringe If there is any possibility of contamination, the gall bladder is ligated and cut off from the entire organ This allows bile to be sampled away from the potential source of contamination As a source of a possible liver contamination, bile should be collected prior to the liver specimen collection Technique for Specimen Collection Bile 29 07/08/2014

Technique for Specimen Collection Vitreous Humour-1 :

Diagnostic clinical vitrectomy can be an extremely helpful procedure for establishing the aetiology of ocular inflammation. No single assay is completely sensitive A combination of assays may improve the yield and accuracy of diagnostic vitreous biopsy These advances may aid ophthalmologists in obtaining earlier diagnosis and better outcomes for their patients who may have been chronically exposed to environmental substances Technique for Specimen Collection Vitreous Humour-1 30 07/08/2014

Technique for Specimen Collection Vitreous Humour-2:

With proper technique 2 to 3 ml of fluid can be removed from each eye in an adult , while up to about 1 ml may be removed from a new-born Vitreous humour specimens are obtained by direct aspiration from each eye using a 5 to 10 ml syringe and 20-gaunge needle Vitreous humour can be aspirated from the globe by application of gentle suction Procedure which applies excessive force, like vacuum tubes and heavy suction should be avoided to prevent specimen contamination with retinal fragments and other tissue Vitreous humour specimens obtained from both eyes may be combined in one properly labelled specimen container Technique for Specimen Collection Vitreous Humour-2 31 07/08/2014

Technique for Specimen Collection Gastric Contents:

Quantification of orally ingested poison is one of the determinants in many suicide cases Presence of a large amount of poison in the stomach is certainly not a pointer of the cause of death , as the gastrointestinal tract is an external organ to the body vital organs Because gastric contents are not homogeneous, and because the total volume of gastric fluid is critical in the interpretation of positive findings , the entire contents of the stomach may be required depending on the case history In other cases, only a reasonable sample may be collected. If this is not possible, then the total volume present must be noted and provided with the specimen to the toxicology laboratory If a deadly and poisonous poison like hydogen cyanide is suspected, then a lot more care must be taken to avoid leakage into the laboratory by ligating both ends of the stomach While having the possibility of contamination, the stomach should be opened away from other specimens and tissues, preferably in the fume cupboard if there is history of toxic and volatile gases Technique for Specimen Collection Gastric Contents 32 07/08/2014

Technique for Specimen Collection Tissues:

About a minimum of 50 mg is sufficient for collection Specimen collected is then placed in appropriate and properly labelled airtight container If volatile poisons or inhalants are suspected, quickly collect and seal the specimen in a container as soon as possible after the body has been opened The liver is an organ which is always sampled in every case of poisoning as a matter of principle Sections of the liver least susceptible to contamination should be collected Only liver from deep within the right lobe is recommended for collection for toxicology Technique for Specimen Collection Tissues 33 07/08/2014

Technique for Specimen Collection Labelling:

The first step in the specimen collection process is ensuring that the specimen containers are labelled appropriately Without attention to this detail, all other activities that occur with the specimen are suspect A proper and working sequence should be instituted and incorporated in the S.O.P of the toxicology laboratory In order to ensure the integrity of the sample, the analyst must be working with only one specimen at a time and never two even if the procedures are used is simple The label should contain sufficient, but brief information which is easily readable As a minimum, the label should include the institutional or laboratory case number, name or other identifier, date and time of collection, signature or initials of the toxicologist or whoever collected the specimen Apply tamper-resistant tape with the collector's initials and the collection date should be placed over the specimen lid and container to document specimen integrity Technique for Specimen Collection Labelling 34 07/08/2014


The End 35 07/08/2014

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