O. I. Panasenko, T. O. Samura, O. O. Filatova, A. A. Safonov, T. V. Panasenko, A. S. Gotsulya, R. A. Shcherbina, I. V. Melnik, A. A. Kremzer, V. P. Buryak, V. O. Salionov, S. M. Kulish, N. A. Postol, Yu. V. Timoshik
Zaporozhe State Medical University
GUIDELINES FOR SAMPLE COLLECTION FOR ANALYTICAL TOXICOLOGY

Many analytical toxicology procedures require collection of blood, urine, stomach contents, and “scene residues”, that is material such tablet bottles found at the scene of an incident (table 1). Samples of other appropriate as detailed below, especially when investigating deaths, but may not be required for analysis unless special investigations are required or decomposition is advanced [2, 3]. However, such samples, should be retained (4 – 20 °C) in case they are needed. There are special consideration in sample collection and storage for metal/trace element analysis.

Table 1

Sample requirements for general analytical toxicology

Sample	Notes
Whole blood	10 ml lithium heparin or EDTA tube-use fluoride/oxalate if ethanol suspected; plastic tube of parquet suspected; glass or plastic tube with minimal headspace it carbon monoxide or other volatiles suspected.
Plasma/ serum	5 ml send whole blood if volatiles, metals and some other compounds suspected.
Urine	20-50 ml (plain bottle, no preservative)
Other samples	Vitreous humor (maximum) available, collect separately from bother eyes), bile (2 ml) or liver (5g) can substitute for urine in postmortem work.

Sites in organs such as the brain is recommended if the whole organ is available. For liver use the right lone. The advantages/disadvantages of various specimens are detailed in Table 2. An example of a request form designed to accompany specimens submitted for toxicological investigation has been provided [2]. If poisoning is suspected, a 10 ml blood sample should be taken from adult as soon as possible, for example after admission to hospital.

Table 2

Advantage and disadvantages of different sample types in analytical toxicology

Specimen	Advantage	Disadvantage	Comment
Blood (plasma/ serum)	Detect parent compound. Interpretation of quantitative data	Limo net volume. Low concentrations of basic drug and some other poisons	Interpretation of quantitative result from postmortem blood, may be difficult
Urine	Often large volume. High concentrations of many poisons	Not always available. Quantitative data not often useful	Standard sample for drugs of abuse screening
Saliva/oral fluids	Non-invasive. Qualitative information on exposure to many drugs	Variable sample hence little use for quantitative work. Low concentrations of many analytes.	Different pattern of metabolites to blood or urine for many analytes.
Additional tissues (liver, brain, etc.)	May contain large amounts of poison. If available then large quantity	Interference in analysis. Quantitative data not always easy to interpret	Analysis may ne valuable to help interpret postmortem blood data

In addition, 2 ml of blood should be collected in a fluoride/oxalate tube is ethanol is suspected. Note that tubes of this type for clinical use contain only about 0,1% (w/v) fluoride, whereas about 2% (w/v) fluoride (40 mg sodium fluoride per 2 ml blood) is needed to inhibit fully microbial action in such specimens. Addition of fluoride also helps to protect other labile drugs such as clonazepam, cocaine, and nitrazepam from degradation. If possible the retention of an unpreserved blood sample is advisable. The use of disinfectant swats containing alcohols should be avoided, as should heparin anticoagulant solutions that contain phenolic preservatives. Information recorded on the sample container at the time the sample is collected should include the names (first and family or last name), patient/subject/animal number, the date and time of collection, collection site, and the sample type (including a note of any preservative), and any other appropriate information. The date and time of receipt of all specimens by the laboratory should be recorded and a unique identifying number assigned in each case.

Biological specimens should be stored at 4°C before transport to the laboratory. Exception to this include hair and nail, which are stable at room temperature, and filter-paper adsorbed dried blood, which is a convenient way of storing and transporting blood samples for the analyses if refrigerated transport and storage is not feasible [1]. Dried blood stains and other dried forensic specimens may, of course, be handed similarly. Each specimen bottle should be securely sealed to prevent leakage, and individually packaged in separate plastic bags. Particular attention should be paid to the packaging of sample to be transported be post or courier in order to comply with current health and safety regulations. Sample volumes or amounts smaller than those indicated in Table 2 are often sufficient to complete the analyses required. Submission of very small samples may, however, result in reduced sensitivity and scope of the analyses undertaken, but nevertheless to the laboratory. Any residual specimen should be kept at – 20 °C or below until investigation of the incident has been concluded. In postmortem work, the use of disposable hard plastic. Sterile tubes is recommended. If these are not available then containers with secure closures appropriate to the specimen volumes should be used. Some laboratories provide specimen containers for collecting postmortem blood and urine specimens. It may be important to note if urine was obtained by use of a catheter. Suitable packaging for sending specimens by post may also be supplied. When death has occurred in hospital and poisoning is suspected, any residual ante mortem specimens should be obtained as a matter of urgency from the hospital pathology laboratory and submitted for toxicological analysis in addition to postmortem specimens. Note than the availably of ante- or peri- mortem specimens does not negate the need to collect postmortem specimens. Sampling through tissues containing high concentrations of analyte may lead to contamination of the sample.

Sample integrity is of prime concern if there are medico legal implications as evidence may have to be produced in court. Precautions to ensure sample integrity include: proper sample labeling, use of tamper-proof containers, collections of samples such as hair, nail, and femoral blood before opening the body, and proper accompanying documentation. Samples collected for clinical purposes are often not of “evidential” quality, but such sample may be all that is establish the origin of samples where there has been concern over sample integrity.

In analytical toxicology, plasma or serum is normally used for quantitative assays. However, some poisons such as carbon monoxide, cyanide and may other volatile organic compounds, lead and other heavy, metals, and some drugs, such
as chlortalidone, are found primarily in or associated with erythrocytes and thus haemolyzed whole blood should be used for such measurements. The space above the blood in the tube should be minimized if carbon monoxide, solvents, or other volatiles are suspected. If the samples have been collected and stored correctly, there are usually no significant differences in the concentrations of poisons between plasma and serum. However, if a compound is not present to any extent within erythrocytes then using lysed whole blood will result in approximately a two-fold dilution of the specimen. A heparinized or EDTA whole blood sample will give either whole blood, or plasma as appropriate. The immunosuppressive cyclosporine, sirolimus, and tacrolimus are special cases because redistribution between plasma and erythrocytes begins once the sample has been collected and so the use of haemolyzed whole blood is indicated for the measurement of these compounds. In order to maximize the ratability of measurements performed o postmortem blood, it is recommended that: the interval between death and the postmortem examination is minimized, the bode/samples are stored at 4 °C before the examination/after collection, blood is collected from two distinct peripheral sites, preferably the femoral veins, after tying off the vein proximally to the site of sampling, and a preservative (2 %(w/v) fluoride) is added to a portion of the blood sample/the sample from one vein, and to urine. The exact site of blood sampling should be recorded, as should the time of sampling and time of death of known. If sufficient sample is obtained, this should be divided between unpreserved and preserved tubes, otherwise the entire sample should be preserved unless there is a possibility of poisoning with fluoride or compounds giving rise to fluoride in vivo, such as fluoroacetate. If only heart or cavity blood is available this should be clearly stated. The value of giving as full a clinical, occupational, or circumstantial history as possible, together with a copy of the postmortem report, if available, when submitting samples for analysis cannot be overemphasized. Not only might this help target the analysis to likely poisons, but also the interpretation of any analytical results may be greatly simplified.

Postmortem blood (about 20 ml) for qualitative analysis only should be taken from the heart, inferior vena cava, or another convenient large vessel. The precise sampling site must be recorded on the sample tube. The blood should be free-flowing. Urine is useful for poisons screening as it is often available in large volumes and may contain higher concentrations of drug or other poisons, or metabolites, than blood. The presence of metabolites may sometimes assist identification of a poison if chromatographic techniques are used. A 50 ml specimen from an adult, collected in a sealed, sterile container, is sufficient for most purposes. No preservative should be added. The sample should be obtained as soon as poisoning is suspected, ideally before any drug therapy has been initiated. However, same drugs, such as the tricyclic antidepressants, cause urinary retention, and a very early specimen may contain in significant amount of drug. Conversely, little poison may remain in specimens taken many hours or days after exposure even though the patient may be very ill, for example as in acute paracetamol poisoning

High concentrations of some drugs or metabolites can impart characteristic colours to urine (table 3). Strong smelling poisons such as camphor, ethychlorvynol, and methyl salicylate can sometimes be recognized in urine because they are excreted, in part, unchanged. Acetone may arise from metabolism of 2-propanol chronic therapy with sulfa-drugs such as a sulfonamide may give rise to yellow or green/brown crystals in neutral or alkaline urine.

Table 3

Some possible censes of colored urine

Color	Possible Cause
Yellow / brown	Bilirubin, hemoglobin, myoglobin porphyries, urobilin, enthrone derivatives, fluorescein, mepacrine, methyldopa, quinine
Red / brown	Bilirubin, haemoglobin, myoglobin, porphyrins, urobilin, aminophenazone, furazolidone, furazolium, nitrofurantoin, warfarin
Blue / green	Bill, biliverdin, indicant, acriflavine, amitriptyline, copper salts, indomethacin, nitrofural, phenylsalicylate, triamterene
Black	Blood, homogentisic acid, indicant, porphobilin, cascara, levodopa, pyrogallol, resorcinol, thymol

Characteristic colorless crystals of calcium oxalate may form at neutral pH after ingestion of ethylene glycol, oxalic acid, or water-soluble oxalates. Urine fluorescence may be due to fluorescein added to car antifreeze and possibly to other products to aid leak detection. For postmortem work, if possible, 2·25 ml urine samples should be collected in sterile plastic containers, one with preservative (2%, w/v fluoride). If only a small amount of urine is available, all should be preserved with fluoride in a plain 5 ml plastic or glass tube. Boric acid or thiomersal containers should not be used because of sample contamination with borates and mercury, respectively. Urine specimens collected postmortem are valuable in screening for drugs or poisons, particularly illicit drugs, and are often used for quantitative ethanol analysis to corroborate the results of a blood analysis. Stomach wash-out is rarely performed nowadays in treating acute poisoning. However, if a sample of stomach contents is obtained soon after a poisoning episode, large amounts of poison may be present while metabolites are usually absent. When investigating possible poisoning, it is important to obtain the first sample of any lavage fluid because later samples may be very dilute. A representative portion (about 50 ml) without preservative should be taken for analysis. However, all stomach contents should be retained and the volume noted. If the blood concentration is difficult to interpret, most notably in postmortem work, it can be helpful to measure the amount of poison present in the stomach. Stomach contents are especially useful if poison(s) which are not easy to measure reliably in blood, such as cyanide, have been taken orally. However, great care is needed if cyanide salts or phosphides, for example aluminum phosphide, are thought to have been ingested, particularly on an empty stomach, because highly toxic hydrogen cyanide or phosphine gas may be released due to reaction with stomach acid. Additionally, the presence of these and other volatile materials can lead to cross contamination of other biological specimens unless due precautions are taken. With stomach contents, characteristic colors or smells may indicate a variety of substances. Any other compounds (e.g. ethylchlorvinyl, methyl salicylate, paraldehyde) also have distinctive smells. Examination using a polarizing microscope may reveal the presence of tablet or capsule debris. Starch granules used as “filler” in some tablets and capsules may be identified by microscopy. The local poisons information service or pharmacy will normally have access to publications or other aids to the identification of legitimate and sometimes illicit tablets/capsules by weight, markings, color, shape, an possibly other physical characteristics. Identification of such material by reference to a computerized product database [4] may be possible.

REFERENCES

1. Croes K., McCarthy P. T., Flanagan R. I. Simple and rapid HPLC of quinine, hydroxychloroquine, chloroquine, and desethylchloroquine in serum, whole blood, and filter paper-adsorbed dry blood // J. Anal. Toxicol., 1994. – Vol. 18. – P. 255 – 260.

2. Flanagan R. I. Analytical toxicology guidelines for sample collection postmortem // Toxicol. Rev., 2005. – Vol. 24. – P. 63 – 71.

3. Forrest A. R. W. Obtaining samples at postmortem examination for toxicological and biochemical analysis // J. Clin. Pathol., 1993 – Vol. 46. – P. 292.

4. Hammond M. D. Detection of drugs in blood stains. // Anal. Prac., 1981. – Vol. 18. – P. 299 – 303.