Fundamentals of Analytical Toxicology. Robin Whelpton

SMR, Coucke W, De Baere T, Peters FT. Update of standard practices for new method validation in forensic toxicology. Curr Pharm Des 2017; 23: 5442–54.

2 Sample Collection, Transport, and Storage

      2.1 Introduction

      In analytical toxicology, no matter how complex the equipment and careful the analysis, the results may be rendered worthless if sample collection, transport, and storage have not been performed with the analysis in mind. Thus, it is important to be familiar with the nature and stability of the analyte(s), the nature of the sample matrix, and the circumstances under which the analyses are to be performed. Proper documentation of the history of the sample (origin, mode of collection, transport, storage, chain-of-custody documentation, and the like) is essential.

      The analyte concentration in the specimen is generally assumed to be representative of the concentration in the particular fluid or tissue sampled. Anticoagulated whole blood, plasma (the fluid obtained on centrifugation of anticoagulated blood), or serum (the fluid remaining when blood has clotted) are widely used in clinical work. This is because not only is blood relatively easy to collect, but also a quantitative analysis can give useful information as to the magnitude of exposure and hence the severity of poisoning. Excretions (exhaled air, urine) or secretions (saliva, sweat) are often less useful as regards interpretation of quantitative data, but can be useful in qualitative work.

Variations in bioanalytical measurements may be subject-dependent and reflect normal physiological changes, whilst others may reflect sample collection and handling procedures (Table 2.1). Post-mortem specimens are a special problem because, generally, information on the analyte concentration in blood at the time of death is required. Post-mortem blood concentrations may not accurately reflect peri-mortem blood concentrations for several reasons. Haemolysis is common, for example, whilst haemostasis may lead to changes in the cellular composition of the ‘blood’ being sampled. There is also the possibility of contamination during collection, e.g. with stomach contents, and of leakage of analyte from adjacent tissues into blood. The leakage of intra-cellular potassium into plasma, which begins soon after death, is such an example.

2.2 Clinical samples and sampling

       2.2.1 Health and safety

Biological samples may contain infective agents and must be handled with care, especially if originating from substance misusers, and must always be treated as if they are infective. The major common risks are associated with tuberculosis, hepatitis B and C, and human immunodeficiency virus (HIV). Urine is least likely to be infective. It is thought very likely that following solvent extraction or other robust sample preparation procedure, infective agents will be inactivated, except for variant Creutzfeldt-Jakob Disease (CJD), but in homogenous assays such as immunoassays samples may continue to be infective after incubation, even though diluted. Indeed, incubation may increase the titre of the infective agent.

Table 2.1 Types of variables affecting clinical samples

Variable	Example(s)
(i) Physiological
Age	Markers of bone turnover such as plasma alkaline phosphatase activity are increased in childhood
Sex	Sex hormones
Body weight	Urinary creatinine excretion increases with muscle mass
Recent food intake	Plasma glucose, triglycerides, etc. increase after a normal meal. May delay and/or reduce absorption of some drugs, but increase absorption of others
Diet	Malnutrition or fasting will reduce serum albumin, urea, and phosphate, amongst other parameters
Circadian variation	Some analytes (e.g. cortisol, iron) show diurnal changes in plasma concentration
Menstrual cycle	Plasma concentrations of luteinizing hormone, follicle stimulating hormone, oestradiol, and progesterone vary with the cycle
Seasonal	Vitamin D metabolites higher in summer
Pregnancy	Plasma concentrations of human chorionic gonadotropin, oestradiol, and other biochemicals vary throughout pregnancy
Psychological changes	Venepuncture or hospitalization may increase plasma concentrations of stress-related compounds such as catecholamines, cortisol, and prolactin
Physiological changes	Posture may affect measurements such as plasma aldosterone and albumin. Exercise can change the blood concentrations of compounds such as lactate
Drugs	Drug treatment may alter concentrations of some plasma constituents even in apparently healthy subjects (e.g. trimethoprim increases serum creatinine)
(ii) Sample/analyte variations
Incorrect specimen	Value differences between plasma and serum, venous and arterial blood, random and 24 h urine samples (e.g. potassium is on average higher in serum than in plasma)
Incorrect collection	Contamination with ethanol as a skin disinfectant prior to venepuncture may invalidate ethanol assay; use of 2-propanol or other alcohols may also invalidate ethanol assay if used as an internal standard
Incorrect sample tube	The absence of an appropriate enzyme inhibitor may allow continued enzyme action such as catabolism of glucose or neuropeptides. Failure to acidify urine will decrease urinary catecholamines. Collection into EDTA will decrease plasma calcium. Use of Li heparin tube will invalidate plasma Li assay
Haemolysis	Red cell lysis may lead to changes in plasma constituents, particularly potassium, phosphate, and some enzymes, and may interfere with the analytical method
Cellular contamination	The presence of platelets following incorrect centrifugation will apparently raise plasma serotonin
Incorrect/excessive storage	Some compounds may oxidize even when frozen [e.g. formation of adrenochrome from 5-hydroxyindoleacetic acid (5-HIAA)], or be subject to bacterial degradation (e.g. amino acids in acidified urine). Loss of water when frozen (freeze-drying effect), but may attract water if tube open when contents thawed
Collection during an infusion	Collection near to an infusion site or using a needle used to give an infusion will give misleading concentrations of the compound being infused, or dilute other blood constituents
Drug treatment	Other drugs or metabolites may interfere in the assay

      Staff in regular contact with potentially infective materials must be properly trained in the safe handling