Panasenko O. I., Samura T. O., Melnik I. V., Parchenko V. V., Kremzer A. A., Gotsulya A. S., Shcherbyna R. O., Safonov A. A.,

Salionov V. O., Postol N. A.

Zaporozhye State Medical University

CLINICAL INTERPRETATION OF ANALYTICAL RESULTATS

Clinical interpretation of analytical results is a complex area. The aim of the analysis is to help understand a clinical or forensic scenario, or to provide evidence for the courts. Detailed knowledge not only of the limitations of the analytical method used, but also of the clinical pharmacology, toxicology and pharmacokinetics of the compound is important.

Patient often respond differently to a given dose of a given compound, especially as regards behavioral effects. Further complicating factors may include the role of pharmacologically active/toxic metabolites [3], and possible toxic effects of drugs on the liver [2].

Postmortem toxicology. The aim of postmortem is usually to establish the role that drugs or other poisons played in the death or events immediately prior to death. In addition it may sometimes be useful to attempt to assess, for example, adherence to prescribed medication or occupational exposure. Bringing together all the information that may be required to provide proper interpretation of analytical findings in individual cases is not easy, especially as investigations tend to be compartmentalized: circumstances: coroner’s officer/emergency services/ accident and emergencies physicians, background medical: GP/ consultant, postmortem examination: pathologist and toxicology: analyst. The importance of individual items may only become clear if and when all the evidence has been assembled.

Analyses postmortem must take into consideration the circumstances under which death occurred, the age of the deceased and the possible presence of other drugs or alcohol. Further potential variables are: the suitability of the analytical investigations employed, the stability of the analyst, the nature of the specimen sent for analysis and the fact that changes may occur postmortem in the composition of body fluids especially blood.

Choice of sample and sample collection site. Blood obtained postmortem is a very variable sample. A degree of haemolysis usual and for this reason whole blood (ideally obtained before opening the body, by needle aspiration from a femoral or other peripheral vein after ligation proximal to the collection sent for analysis and the fact that changes may occur postmortem in the composition of body fluids especially blood.

Choice of sample and sample collection site. Blood obtained postmortem is a very variable sample. A degree of halmolysis usual and for this reason whole blood (ideally obtained before opening the body, by needle aspiration from a femoral or other peripheral vein after ligation proximal to the collection site) is normally analyzed directly.

However, sedimentation of cells and/or clot formation may have occurred before collection of the sample. Nevertheless, whole (i.e. unseparated) blood is commonly used in postmortem toxicology because: it is relatively easy to collect, it is relatively homogenous making it easier to dispense in the laboratory and there are often data on the plasma or serum (or sometimes even whole blood) concentrations of many analyses attained during normal therapy and after acute exposure to provide at least some basis for the interpretation of results.

If the patient was admitted to hospital prior to death, for example, there may be ante- or per mortem samples available for analysis. A compilation of postmortem femoral blood concentration observed in poisoning fatalities is also available to assist in the interpretation of results [4], but simply relying on figures from other cases does not always recognize the possible magnitude of the changes that may take place after death.

Although tissue analysis has become unfashionable to an extent, in part because of a desire to reduce costs, and also because of the comparative lack of information on tissue concentrations could aid in the interpretation of particularly difficult or important cases should not be neglected. Apple [1] drew attention to the use of liver tissue as well as postmortem blood analyses in helping differentiate acute tricycle antidepressant over dosage from chronic exposure to these drugs the relatively high liver drug: metabolite ratio after acute over dosage providing additional interpretative information. Clearly where: the development of tolerance to the acute toxicity of a drug and the possibility of postmortem changes in blood analytic concentrations may make interpretation of the results difficult, the availability of additional information such as the results of tissue analyses could provide further evidence of the nature and magnitude of exposure. However, it should be remembered that site-to-site variation in postmortem drug concentrations have also been found in sample collected from different regions of liver and lung. Hair or nail analysis does, of courses, provide a further option if noncompliance with therapy or illicit drug use in the days or weeks before death us a subject of debate. Measurement of electrolytes such as sodium and potassium has no value postmortem. The presence of acetone in blood and urine may be an indication of ketoacidosis or unrecognized diabetes. The measurement of glycated hemoglobin (HbAlc) is a much more reliable guide to undiagnosed diabetes mellitus as this measurement reflects elevated blood glucose concentrations over a period of time before death. Vitreous humor can sometimes be useful for glucose and lactate measurement, as well as for used and creatinine [5].

Assay calibration. Many assays intended for TDM or clinical toxicology are aimed at plasma or serum, there two fluids often being used interchangeably, and hence are calibrated using analytic-free plasma or serum from an appropriate source. However, after death, intravascular coagulation, cell sedimentation, haemolysis and a variety of other processes may occur, all of which suggest that well-mixed, haemolyzed in an attempt to obtain a representative sample of the contents of the blood vessel. Some analysts use haemolyzed whole blood to prepare calibration solutions, but citrated blood is not usually ideal for this purpose.

An alternative approach is to use assays and calibration standards aimed at TDM analyses, but to dilute whole blood samples 1+4 or 1+9 with the matrix used to prepare the calibration solutions in an attempt to minimized possible matrix effects. Similarly the problems of QC and assay calibration of tissue analyses are often understand. One approach is to use digestion with a proteolytic enzyme such as Subtilisin Carlsberg with subsequent dilution with the matrix used to prepare the calibration solutions, again in an attempt to minimize the possible effect of the matrix on analytic recovery.

Interpretation of analytical results. If a patient either under treatment with, or thought to abuse, a drug or drugs dies, the role of the drug(s) in the death may be questioned. Some issues that may be considered include idiosyncrasy, dose-related toxicity, and accidental acute or chronic over dosage, and deliberate self-poisoning. In such cases the measurement of the concentrations of the drugs in question on a blood specimen obtained postmortem can be important, but the interpretation of the results obtained may not be straightforward [5]. This is in part because blood concentrations of xenobiotics notably lipophilic compounds with relatively large volumes of distribution, may increase after death due to diffusion from surrounding tissue or from the GL tract.

If discovery of a body is delayed the extent of decomposition can make not only sample collection, but also the very difficult. Ensuring that the body is stored at 4 °C prior to the postmortem and that the autopsy is performed as soon as possible after death will minimize the risk of changes in blood analytic concentrations occurring before sampling. Collecting blood by needle aspiration from a peripheral site, ideally after occluding the vein proximally to the site of sampling (see Table 1), may also help. However, such precautions are not always taken, and indeed collection of blood from a central site such as the heart, or even collection of “cavity blood” may occur. Of course, carries the risk of contaminations of the “blood” sample with stomach contents for example, as well as small pieces of tissue(s).
Many poisons are very potent, that in the blood concentrations associated with severe, possibly fatal toxicity are very low, typically mg · L^-1 or even mg · L^-1 and thus even trace contamination of a peripheral blood sample can confound the most careful analytical work. In such instances toxicological analysis can often do
little more than provide evidence of exposure to a particular substance.

Table 1

Factors influencing the likelihood of postmortem change in blood xenobiotics concentrations

Factor	Comment
Site of collection	Central sites (heart, vena cava, or “subclavian” blood) more likely to show changes than peripheral sites. Blood from left ventricle of heart more likely to show change that blood from right ventricle
Time between death and sample collection	A longer elapsed time gives more potential for changes as tissue pH decreases and autolysis proceeds
Position body when found	May result in blood draining from central sites to periphezol sites
Method of sampling	Needle aspiration less likely to result in sample contamination with tissue fluid, for example
Sample preservation	Fluoride needed to help stabilize ordain analyses does not any pre-collection changes
Headspace in sample tube	Volatile analyses will equilibrate between sample and headspace; opening the tube when cold (4°C) will minimize losses
Volume of blood collected	A larger sample volume less likely to be influenced by localized changes in blood composition
Nature of xenobiotics	Lipophilic compounds more likely to show increase than lipophobic compounds.
Presence of xenobiotics in the GL tract	Post-mortem diffusion may alter concentrations in adjacent tissues as well as in blood

There are no currently accepted biochemical markers than can be used to indicate the magnitude of postmortem changes likely to influence drug redistribution into blood. Similarly, there are no well-defined experimental models to study postmortem changes in blood concentrations. As there is a need to study postmortem changes in blood concentrations. As there is a need to obtain blood from control sire such as the vena cava and from peripheral sites such as femoral vein at different times after death, small animals, such as rodents and rabbits, are unsuitable. Pigs have some morphological, physiological and metabolic similarities with man and other primates. They are reported to a attain maturity as regards hepatic drug – metabolizing enzyme activity be 2 months. However, using young pigs it was only possible to obtain small peripheral blood samples up to 48 h postmortem [6]. A human cadaver model has also been used to study postmortem drug distribution. Diffusion of amitriptyline, lithium and paracetamol from the stomach to the base of the left lung and the left lobe of the liver has been demonstrated. Blood concentrations of some drugs with a relatively small volume of distribution may undergo minimal changes after death. However, continued absorption from the gastrointestinal tract may occur after death even with these compounds. A further problem is that some analyses, notably ethanol and other volatile compounds, cocaine, cyanide and insulin, may be lost from, or in the case of ethanol and higher alcohols (propanol, butanol), and produced in postmortem blood.

Enzyme activity continues after death, particularly esterase activity and so esters such as heroin are not often detected in postmortem samples. Anaerobic metabolism of 7-nitrobenzodiazepines (nitrozepam, clonazepam, flunitrazepam) produces the corresponding 7-aminocompounds. The combined effects of postmortem diffusion from tissue and analytic instability can present a confusing picture. Both postmortem changes in blood composition and analytic instability may be exacerbated if the body has been kept at a relatively high temperature before sampling. Clearly the longer the duration of treatment with a lipophilic, centrally acting drug and the higher the dose, the greater the potential for postmortem change as the tissue concentrations are likely to be relatively high.

Finally, there is increasing interest in genetic analysis to aid in the interpretation of postmortem data with the aim of detecting those who could have been predisposed to accumulate a potentially toxics concentration of drug of metabolite.

SUMMARY

All the available evidence must be taken into account when investigating any death or other incident where poisoning is suspected. An overall knowledge of the circumstances, time course, clinical/postmortem observations, and possible poisons involved and their toxicology and metabolism is paramount. Toxicological analysis can provide objective evidence of exposure and of the magnitude of exposure.

REFERENCES

1. Apple F. S. Postmortem tricyclic antidepressant concentrations: Assessing cause of death using parent drug to metabolite ratio / Apple F. S. // J. Anal. Toxicol., 1989. – Vol. 13. – P. 197-198.

2. Burattis S. Drug and the liver: advances in metabolism, toxicity and therapeutics / Burattis S., Lavine Y. E. // Curr. Opin. Pediatr., 2002. – Vol. 14. – P. 601-607.

3. Daple-Scott M. Comparison of drug concentrations in postmortem cardiac and peripheral blood in 320 cases / Degouffe M., Garbutt D., Drost M. A // Can. Sac. Forensic Sci., 1995. – Vol. 28. – P. 113-121.

4. Druid H. Compilation of fatal control concentrations of drugs in postmortem femoral blood / Druid H., Holmgren P. A. // I. Forencic. Sci., 1997. – Vol. 42. – P. 79-87.

5. Drummer O.H. Postmortem toxicology of drugs of abuse / Drummer O. H. // Forensic Sci. Int., 2004. – Vol. 142. – P. 101-113.

6. Flanagan R.I. Effect of postmortem changes on peripheral and central whole blood and tissue clozapine and norclozapine concentration in the domestic pig / Flanagan R. I., Amin A., Seinen W. // Forensic Sci. Int., 2003. – Vol. 132. – P. 9-17.