Antemortem Blood

Introduction

Blood is an exceptionally important tissue to the forensic toxicologist, as, in many cases, the concentrations of drugs in antemortem blood samples are interpretable with respect to the effect(s) that the drug may have been exertingon a person at the time of an offense. This article provides an outline of the nature of blood and the use to which it may be put by the toxicologist. The article also offers some thoughts on the considerations that must be taken into account before any meaningful interpretation can be made.

Sample Collection

Antemortem blood samples that are collected and submitted for forensic toxicological analysis usually fall within one of three categories:
1. The administration of a drug or poison to a party with the intent of willfully causing harm (e.g. attempting poisoning).
2. The administration of a drug to a party with the intent of producing incapacitation in them (e.g. sexual assault/date rape and doping).
3. To determine whether the donor of the sample may have been under the influence of a drug at the time an offence was committed (e.g. drunk driving or where a person claims not to have been responsible for his or her actions as a result of being under the influence of a drug).
The presence of a parent or unchanged drug in blood is, in general, often regarded as significant on the basis of the philosophy that if an unchanged drug is circulating in blood, it is potentially available to bind to, and subsequently exert some effect at, its site of action. This is of particular relevance in forensic toxicology where the interest lies with drugs that can influence behavior or exert a harmful or a life-threatening effect.
Blood is a particularly important tissue for the toxicologist because it provides the means whereby drugs, their metabolites and other toxic substances are transported from their point of entry into the body to the site(s) at which they may exert their various effects. Likewise, blood provides the transportation mechanism that assists with the removal of drugs and other substances from the various tissues in the body to their site(s) of elimination.
Since blood provides the transport mechanism that enables drugs and their metabolites to move around the body, the detection of a drug and/or its metabolites in blood may provide the toxicologist with information than can be interpreted with respect to the potential effects that may be exerted on the individual. In every case there are a number of things to consider before attempting to interpret analytical results obtained from an antemortem blood sample:
• the blood sample itself (e.g. whole blood/plasma, preservative);
• type of container used for sample collection and storage;
• analytical method used for detection/quantification;
• time of blood samplingin relation to the incident of interest;
• use of databases;
• pharmacology of the drug(s) – mode of entry into the body;
• actions on the body – druginteractions;
• case history;
• demographic factors;
• disease-related factors;
• extracorporeal factors.
Only after these basic issues have been considered should any attempt be made to interpret analytical results. One thingis certain: in a forensic situation where a livingsubject is concerned, you must get it right the first time. The sample will rarely be perfect or ideal but, unlike in clinical toxicology or therapeutic drugmonitoring, where the patient can be requested to provide a second sample, it is not possible to go back for more at a later time or date. A blood sample that is collected in a forensic investigation is unique to the specific situation/incident that is under investigation.

The Blood Sample

Blood is an exceptionally complex tissue, comprising of red corpuscles (erythrocytes), white cells (leukocytes), proteins and other chemicals suspended in a fluid known as serum. It has been estimated that blood circulates completely around the body in approximately 2-3 min. The total blood volume of the body lies between 52-83 ml kg-1 body weight for an adult male and 50-75 ml kg-1 for an adult female or 2.5-4lm-2 body surface area. The total plasma volume lies between 49 and 59 ml kg-1 body weight or 1.4 and 2.5 lm-2 body surface area. The interstitial fluid volume is approximately three times as great as the plasma volume.
Leukocytes are concerned principally with defense of the body against disease and infection and are not usually of direct interest to the forensic toxicologist, unless they have been measured clinically for the purpose of diagnosis of a patient’s illness. Poisoning by proteinaceous toxins such as ricin may give rise to abnormally high white cell counts and, unless the poisoned victim is beingmonitored, the toxicologist would normally be unaware of the white cell count. The platelets are primarily involved with the clotting mechanism of blood and are not normally considered by the forensic toxicologist.
Erythrocytes are principally associated with the transportation of oxygen to the tissues and carbon dioxide from the tissues to the lungs. Erythrocytes are approximately 7.5 um in diameter and contain no nucleus. The erythrocyte membrane is composed of approximately 40% phospholipids, cholesterol and glycolipids; 50% proteins; and 10% carbohydrates.
Erythrocytes provide the red color to the blood, due to the presence of the complex molecule, hemoglobin, that they contain. Hemoglobin acts as the carrier molecule for gases such as oxygen, carbon dioxide and carbon monoxide. The complex nature of the erythrocytes makes them toxicologically interesting because interactions between toxins and hemoglobin or the red cell membrane can be very significant. Of the plasma proteins, the albumins play an important role as carriers of many drugs around the body.
Blood contains approximately 85% water (w/v) and has a specific gravity of 1.055. The percentage of the volume of the blood that is occupied by the cells is known as the hematocrit, that occupied by plasma is known as the plasmacrit. In males, the normal range of hematocrit values is 42-52%; females 3747%; children 30-43%; and newborn infants 5365%. Consideration of the hematocrit value may be significant in relation to drugs that bind to the ery-throcytes, as subjects havingabnormally low hema-tocrit values would have a low number of cells and subsequently a low hemoglobin concentration (i.e. have a lower drug-binding capacity).
The most frequently encountered blood samples in forensic toxicology are whole blood samples, largely because of the extended time intervals that elapse between sample collection and arrival at the laboratory. Clinical samples normally reach the laboratory within a few hours, at most, from the time of collection, permitting them to be separated before the blood begins to hemolyze. Forensic toxicology blood samples are usually hemolyzed on arrival at the laboratory, havingspent some time in a police station refrigerator before being sent through the postal system and arriving at the laboratory some days after they were collected from the donor. Venous blood is the specimen of choice for forensic antemor-tem samples: it can normally be collected easily through a syringe needle or butterfly cannula from the forearm (cubital fossa) in a reasonable volume without causing too much discomfort to the donor.
There is a valid argument that, because the forensic toxicologist usually has no idea what drug or poison might be present in the sample, whole blood is the most appropriate sample to analyze. The analysis of whole blood facilitates the detection of drugs/poisons that may have associated with a particular blood component or fraction, whereas analysis of plasma only might lead to the analyst missing a substance that has preference for bindingto erythrocytes. The analysis of whole blood is, however, more complex than the analysis of plasma and may require incorporation of a greater number of cleanup steps into the analytical process. Examples of drugs/poisons that have an affinity for erythrocytes are shown in Table 1.

Table 1 Drugs/poisons showing an affinity for erythrocytes

Atropine
Carbamazepine
Carbon monoxide
Chloroquine
Cyanide
Maprotiline
Pethidine in young persons
Salicylic acid
Meprobamate
Mercury
Propranolol

Sample Collection and Storage

The types of containers used for the collection of blood samples are of exceptional importance and can affect the efficiency of analysis and subsequent interpretation of analytical results. It is not always possible for the toxicologist to control the containers used for the submission of samples and all too often the doctor collecting the sample will use whatever sample container is at hand. Many laboratories issue kits for different types of cases, together with detailed instructions about what specimens to collect and how to transport them to the laboratory. Ideally, kits issued for the collection of antemortem blood samples should contain:
• a 10-20 ml syringe, with needle, of large enough volume to collect sufficient sample in one attempt;
• two 5 ml sealed glass septum vials with insert rubber septa containing a fluoride preservative at a concentration of >2%;
• at least one 10 ml vial with a foil-lined cap containing fluoride oxalate preservative.
While clinical specimen vials containing lithium heparin or sodium oxalate do not appear to exert a significant effect on the analysis of common drugs of abuse, clinical tubes containing fluoride and designed for the purpose of collecting blood for glucose measurement are totally unsuitable for the storage of blood requiring alcohol analysis. Not only are the seals/stoppers not sufficiently secure to insure that no loss of the volatile alcohol occurs but the fluoride concentration is not sufficiently high to inhibit microbial activity. Since the toxicologist may occasionally be required to analyze lithium in an antemortem blood specimen, the use of either sodium oxalate, fluoride or tubes containing no preservative are more appropriate for general toxicological analysis than those containing lithium heparin. Caution must also be exercised when selecting swabs for cleaning the skin before blood sampling. Swabs containing any type of alcohol should be avoided lest they interfere with laboratory analysis for volatile substances.
Police surgeons should be encouraged to fill the septum vials so that as little space as possible is left between the specimen and the septum. It may be necessary to use a venting needle in order to achieve this, but many police surgeons are reluctant to use needles because of the risk of needle-stick injuries. However, this is the only satisfactory way to insure that volatile substances, such as alcohol or organic solvents, are not lost. Any remaining blood should be transferred to the screw cap vials. Glass containers are preferred to plastic containers because they offer greater resistance than plastic containers to the loss of volatile substances. Glass containers are also less likely to contain lubricants and plasticizers, which have been known to compete with and displace protein-bound drugs from their binding sites or interfere with analysis. Under no circumstances should plastic containers be used for collecting samples that may require analysis for volatile substances, such as toluene, as significant losses may occur owing to the permeability of plastic to the volatile substance. Care must also be taken not to store blood samples that have been submitted in plastic containers in an environment where contamination by environmental solvents might occur as a result of solvents diffusing into the blood through the walls of the tube.
Blood samples should ideally be stored refrigerated below 4°C while waiting to be analyzed. Blood samples should be analyzed as soon as possible after collection. The time between screening analysis and confirmatory/quantitative analysis should also be kept as short as possible. Knowledge about the stability of most drugs in blood is incomplete. Cocaine and benzoylecgonine are very unstable when stored for even short periods of less than 3 months. Canna-binoid concentrations are also known to fall off significantly, even when stored frozen. One study showed that the stability of morphine fluctuated over a 5 year period in blood samples stored at ambient temperature. The same study demonstrated that methamphetamine, amphetamine and phencycldine (PCP) could all be detected in antemortem blood samples stored at ambient temperature for 5 years, although, on average, the concentrations of each of these drugs decreased over the period of the study.

Analytical Methodology

The German toxicologist, Manfred Moeller, once stated that ‘it would be easier to get forensic toxicologists to use the same toothbrush than for them to use the same analytical method.’ Judging by the numerous analytical methods available for the analysis of drugs in whole blood, one could be forgiven for coming to this conclusion. Almost all laboratories tend to make modifications to published methodology to suit their own purposes. Forensic toxicologists, however, have a requirement placed on them by the courts to be able to interpret their results. In order to do so they must have confidence in the results and be able to demonstrate that if results from different laboratories are used for comparison purposes, they are truly comparable. All analytical methods should be fully characterized so that the analyst knows the specificity, accuracy, precision, limit of detection, limit of quantification, linearity, robustness, reproducibility and the range of concentrations over which the assay performance is valid.
Analytical methodology for forensic purposes is normally a two-stage process comprising an initial screeningtest, to determine whether any substance of interest or significance is present, followed by confirmatory, and possibly quantitative, analysis. The screeningprocess used to determine whether common drugs of abuse may be present usually employs an immunoassay (subject to availability). Immunoassays used for the analysis of whole blood involve either radioisotopes (radioimmunoassay) or microtiter plates, where the drugantibodies are bound to the walls of the microcells at the point of manufacture. For most common drugs of misuse, immunoassays offeringa rapid and relatively inexpensive screening stage, are available.
Although very sensitive, radioimmunoassay necessitates the layingaside of special laboratory areas for the storage and handling of radioisotopes and presents potential problems associated with the disposal of radioactive waste. Microplate assays offer a convenient and cost-effective means of screeningwhole blood samples that is readily automated. The microplate assay utilizes immobilized antibodies bound to the walls of the microplate wells to provide bindingsites for drugs present in the sample. Drug substances present in the blood sample bind to the immobilized antibody duringan initial incubation period. At the end of the incubation period, the blood sample is removed, the cells are washed to remove traces of blood and a further reagent is added; this develops a color reaction that depends upon the presence or absence of substances that have bound to the antibodies. The color intensity can then be read spectro-photometrically.
Drugs for which no immunoassays are available may be screened by gas chromatography (GC), high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) after a preliminary extraction from blood. The advent of ‘bench-top’ mass spectrometry (MS) has made it possible for laboratories to combine the separatingpowers of GC or HPLC with the unique and sensitive identification properties of MS to enable screeningand confirmation to be combined into a single drug identification procedure. The combination of a chroma-tographic separations technique and the ability of MS to yield a molecular fingerprint of most drugs is regarded as providing a high degree of certainty for the positive identification of drugs and/or their metabolites in blood. GC-MS and HPLC-MS also provide an opportunity for quantitative analysis to be undertaken.
Prior to confirmatory analysis by GC-MS or HPLC-MS, the drugs of interest must be isolated from the biological matrix of blood. The complex nature of whole blood compared with plasma means that many methods developed for clinical analysis and therapeutic drugmonitoringare unsuitable for forensic analysis. It is not possible in this short monograph to detail the numerous extraction procedures published for purification of drugs/drug metabolites and the reader is recommended to refer to the specialist toxicological literature.

Interpretation

Interpretation is the ultimate objective of forensic toxicology analyses. Unless some interpretation can be applied to analytical results, there is little point in undertaking analysis. The interpretative process is the most interesting and challenging aspect of forensic toxicology, and that which is most open to challenge and discussion. The most frequently asked questions of the forensic toxicologist are:
• Was a drug/poison present or absent?
• What sort of action could the substance detected have exerted on the donor of the sample?
• If a drug/poison was detected, was it present in sufficient quantity to affect the behavior or well-being of the donor of the sample?
• Could the substance detected have been influencing the donor of the sample at the time of an alleged incident?
Qualitative analysis will usually be sufficient to allow a statement about whether a drug was present above the limit of detection of the analytical method; however, before embarking on expensive quantitative analysis, the toxicologist must consider whether anything meaningful can be gained by it. Drugs affect different people in different ways and can even affect the same person in different ways on different occasions. The concentrations of a number of common drugs in blood and plasma are summarized in Table 2, at what are normally regarded as therapeutic concentrations and at concentrations where some toxic effect may be experienced. Since, however, many commonly misused drugs are not, or have not been, used clinically and have no therapeutic use, there are no ‘therapeutic’ concentrations available that can be substantiated by clinical trials. Most drugs of misuse are taken because they exert a psychotropic effect (i.e. they influence or alter the way a person thinks, behaves, perceives his or her surroundings or reacts to external stimuli). It is these drugs that the forensic toxicologist is requested to analyze and comment upon most frequently.

Table 2 Therapeutic and toxic concentrations of some common drugs of forensic interest in blood and plasma.

Table 2 continued

As most of the therapeutic/toxic concentrations of drugs in blood have been measured and published by clinical laboratories, the data in Table 2 relates primarily to plasma drugconcentrations rather than whole blood. Information concerningthe distribution of drugs between plasma and whole blood is also very limited. Hence, analysts usingthe data in Table 2 should exercise caution in tryingto extrapolate between plasma and whole blood concentrations. Where data have been made available concerning whole blood and plasma drugconcentrations, this has been incorporated in the table. It must be noted that the ranges of results listed under ‘therapeutic’ and ‘toxic’ should be seen as approximate because they have been collated from papers published in the scientific literature where authors will have used different methods of analysis, and there is no definable point below which a drugexerts a therapeutic effect and above which it is toxic.

Information

The extent to which any reasonable attempt at interpretation can be made depends on the information available to the toxicologist, who should always insist on beingprovided with as much information as possible before startinga case and attemptingto make any interpretation. A number of important questions to ask submittingofficers are listed in Table 3. The toxicologist should also strive to keep as up to date as possible with current scientific literature so that the results and case history may be related to the known pharmacology and pharmaco-kinetics of the drugin question. In particular it is important to consider:

Table 3 Minimum information that should be requested to accompany antemortem blood samples requiring toxicological analysis.

1. Name
2. Date of birth
3. Race (e.g. Caucasian / Chinese / Negroid / Other – specify)
4. Sex
5. Body weight
6. Height
7. Build: slim / proportionate / stocky and muscular / stocky and obese /obese
8. Full description of circumstances leading up to arrest
9. Behavior/behavioral characteristics, i.e. speech, stance, appearance (tidy / dishevelled), eye pupils (wide or narrow), shaking, tremors, shivering, face color (ruddy or ashen)
10. Comments of police surgeon
11. Recent medical history – any heart trouble or diabetes?
12. Medications taken within preceding 10 days:prescription over-the-counter medications/preparations cold / flu remedies illegal/illicit drugs
13. Any consumption of alcohol: record all drinks and times, using a 24 h clock, and drinking pattern (obtain evidence in form of cans/glasses if possible)
14. Results of breath alcohol tests if available (copies of printouts should be submitted)
15. Time and description of last meal
16. Occupation: availability / exposure to any occupational toxins or solvents
• mode of entry of the druginto the body;
• distribution of drugwithin the body;
• metabolism of drugwithin the body;
• excretion profile of the drug;
• actions exerted by the drugon the body;
• potential druginteractions;
• whether tolerance may be induced to the effects of the drugand, if so, how this might affect the individual and the subsequent interpretation of results.
Medicinal drugs are normally administered either orally, transdermally or by injection (intravenous or intramuscular). The route by which a drugenters the body can significantly influence the time of onset and intensity of its effect. Since psychotropic drugs exert their psychotropic effects by actingon the central nervous system, the most intense and desirable effects are obtained by deliveringthe active drugin an optimal concentration to the brain as quickly as possible. As cocaine, tetrahydrocannabinol and heroin/morphine undergo extensive first-pass metabolism by the liver when taken orally, they are usually introduced into the body by injection, smoking(heroin, crack cocaine, cannabis) or nasal insufflation (cocaine).
Where the elimination half-life is known, it is sometimes possible to extrapolate backwards from the concentration of drugmeasured in an antemortem blood sample collected at a known time and to estimate what the concentration of drugmight have been at the time of an incident. Any such calculations are based on a number of assumptions, and before any attempt to use such calculations is undertaken, a fully authenticated case history should have been obtained and all appropriate caveats should be included in the statement of interpretation.
Unless robust information is available to justify any interpretation based on a pharmacokinetic back-calculation, the toxicologist should avoid being led into undertaking such a calculation by investigating officers or lawyers. The toxicologist who offers an interpretation that cannot be fully justified is layinghim-or herself open for an uncomfortable and potentially discrediting experience under cross-examination in court.