Under The Microscope: Post-mortem autolysis

It is obvious that the shorter the delay between death and the removal of samples, the better. Though some toxic sub- stances, such as carbon monoxide, form stable compounds in the body, many others (especially volatile substances and some pharmaceutical products) will be broken down by post-mortem autolysis and decomposition. When an autopsy cannot be performed quickly after death, in terms of a few hours, then mortuary refrigeration is the first line of defence to slow up putrefactive and autolytic processes. If delay is foreseen, usually because of administrative problems in obtaining consent or authority for autopsy, it may be possible to obtain a sample of blood through the body surface, such as puncturing the femoral vein by needle and syringe. The blood can then be kept in optimal conditions, with preservative where needed, and perhaps with the serum or plasma separated from the cells to avoid haemolysis. Similarly, urine could be drawn off by catheter or even suprapubic puncture, unless strict regulations forbid this as anticipating autopsy permission.

In some jurisdictions, authority for autopsy may be particularly hard to obtain, from religious, financial or administrative reluctance. Where poisoning is suspected, permission may be granted only for external examination and sampling; here venous blood, urine and perhaps vitreous humour may have to suffice for all investigations.

When samples are submitted to the toxicologist, they should be accompanied by the best possible information relevant to the case. It is both counterproductive and an unprofessional discourtesy merely to record the personal details of the deceased and list the samples, with a terse demand such as 'Any poisons?' on the request form. Such a demand could legitimately be refused by the toxicologist, as it is quite inad- equate information upon which he can be expected to function effectively and safely. The following information should be supplied and where necessary, supplemented by direct discussion either in person or by telephone:

-          The personal details of the deceased, including age, sex arid where thought relevant, the occupation ,(especially if in agriculture or industry)

-          Brief details of symptoms, if any, and length of illness.

-          The post-mortem interval before samples were obtained, and the actual date and time of sampling.

-          The name, address and telephone number of the pathologist.

-          A list of all samples provided, with an indication of the sampling site for each.

-          The nature of any preservative in each of the samples.

-          If there has been a delay in submitting or transporting the samples, a note of the condition under which they have been stored (for example, refrigeration or deep-freeze)

Any special risk associated with the samples must be communicated to the laboratory. The most obvious are infective conditions, especially hepatitis B or C virus or HIV infection in the deceased, though other diseases such as tuberculosis, tetanus, anthrax, gas gangrene or any other bacterial or viral condition must also be specifically reported. In relation to hepatitis and HIV, even if these are not definitely confirmed, the toxicology laboratory must be told if the deceased was in a high-risk group, such as a drug addict or a homosexual. In many areas, including Britain, some toxicology laboratories will not accept samples from such high-risk groups until a blood sample has been screened for hepatitis B and HIV antibodies; if positive, they may decline to carry out the analysis or perform it only under strictly controlled conditions. Similar warnings must be given to the analyst if there is any possibility of certain harmful substances, such as radioactive isotopes or certain war gases, being present in the samples.

When a death has criminal aspects, such as a murder or manslaughter, then the usual strict precautions must be taken for continuity of wild rice. Each container m1.w be carefully labelled and preferably countersigned by the pathologist. Some jurisdictions will require actual seals on the containers themselves or the package into which they are placed for transport, or both. Accompanying signed 'exhibit labels' with serial numbers corresponding exactly with numbers on the jars may be required. The containers must be given by the pathologist to a named person, usually the 'exhibits officer' of the police investigating team (or to forensic scientists if they attend the autopsy). The police officer must hand the samples personally to a member of the laboratory staff and a record of this chain of evidence must be kept, so that there can be no criticism levelled at anyone when the matter comes to court, raising doubts about the correct identity of the sample.

In warm conditions, fermentation by yeasts and other alcohol-producing flora can produce appreciable quantities of alcohol: it has been reported that as much as 150 mg/100 ml have been generated within 24 hours after death. Once the blood or urine has been withdrawn from the body, however, further changes can be arrested by preservatives. In most cases the delay before analysis is relatively short, being measured in days - often with refrigerated storage - so the long-term experiments mentioned here do not apply. When the ambient temperature is high, much more caution needs to be used. It would seem that for general use, a concentration of 10 mg/ml of sodium or potassium fluor- ide is satisfactory. Fluoride should also be added to urine and vitreous humour if alcohol estimations are required. Cocaine and its metabolites are also labile in vitro, and fluoride should be added to samples submitted for analysis for this drug. Cyanide may be formed in considerable quantities in plain blood samples, which are of little use for cyanide estimation. Fluoride should be added to such specimens, as well as for carbon monoxide (carboxyhaemoglobin) if the analysis is to be delayed. In all analyses for pharmaceutical drugs, two samples of blood should be submitted, one plain in large volume of at least 25 ml and another smaller sample in fluoride.

When insulin assay is required on a blood sample, special precautions should be taken. Haemolysis of the red cells releases enzymes which will reduce the [1]S-S bonds in insulin and destroy its immunoreactivity, so the sample should be centrifuged as soon as it is obtained to separate the serum. A heparinized sample should also be taken for glycosylated haemoglobin estimation and fructosamine assay. Vitreous humour, blood and urine samples should be placed in fluoride and sent for glucose estimation, as well as tissue samples from around any putative injection site, together with a control tissue sample from a site elsewhere on the body where insulin is unlikely to have been recently injected.

It is now obvious that, in the past, serious errors were made in toxicological analyses from lack of care or consideration concerning the source of body fluid samples. Significant variation can be found in the concentration of many sub- opposed to venous blood is used, as tissues may take up the compound from the arterial supply, the concentration then being lower in the venous return. Similarly, portal blood may have a substantially higher concentration of a sub- stance that is being absorbed from the intestine, before it is extracted by passage through the liver. After death, most variation is caused by uneven destruction by enzymatic and microbiological activity - and by diffusion from sites of higher concentration. The barriers formed by living cell membranes break down after death and small molecules in particular may move easily through the tissues into vascular channels.

Post-mortem levels of many substances are unreliable because of this diffusion effect, making the interpretation of physiological components, such as sodium, potassium, calcium, glucose, urea and many others extremely difficult, if not impossible. Applying these facts to toxic compounds, the concentrations may vary considerably according to the sampling site. As an illustration, [2]the Moorgate Tube disaster in London showed that blood-alcohol levels may vary widely between different sampling points. The driver of an under- ground train was killed, along with a number of passengers, but his body could not be recovered from the warm environment for several days. As it was obviously vital to know if drink had contributed to the accident, four samples were taken from various sites in the body. On analysis, a fourfold variation from between 20 and 80 mg/ 100 ml was obtained, presumably because of variable rates of putrefactive alcohol production, as there was no evidence to show chat the driver had imbibed alcohol before death. Most research in respect of sampling variation has been directed at alcohol. One point of dispute, which is relevant even when post-mortem changes are minimal, is whether alcohol in the stomach can diffuse after death to neighbouring organs and produce a falsely elevated alcohol concentration in blood at those sites.

The relevance is clear, in that, if a person drinks alcohol immediately before death, there will be insufficient time for it to be absorbed. Thus ethanol remaining in the stomach after death cannot have contributed to his ante-mortem blood-alcohol level, and hence his cerebral function and consequent behaviour. If however, analysis of a sample of blood taken from the heart cavities is contaminated by post-mortem diffusion from the adjacent stomach, then a falsely high reading will be obtained, which may have serious legal implications, if the error is not appreciated.

After a delay of 48 hours, an autopsy was carried out and drug concentrations measured in samples taken from various sites. Whilst femoral vein blood remained clear of these substances, samples from vessels in the thorax showed up to 130mg/100ml of alcohol with a mean of 58 in a pulmonary vein - and up to 1934 mg/l of paracetamol (mean 969), both obviously spurious due to post-mortem diffusion. This situation could have occurred from agonal regurgitation of stomach contents with recently swallowed drugs back into the air passages and shows the dangers of inappropriate sampling sites, with the femoral the place of choice.

 

For the reasons given above, peripheral vein blood should be used whenever possible, avoiding heart blood as a potential source of error for alcohol and presumably other easily diffusible substances.

 

Acknowledgements:

www.aived.nl    AIVD – @Erik Akerboom ©

www.politie.nl  Politiekorpschef  @Janny Knol©

www.politie.nl WEB Politie - @Henk van Essen©

 

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^{In chemistry, a disulfide (or disulphide in British English) is a compound containing a R−S−S−R′ functional group or the S2−
 anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In inorganic chemistry, the anion appears in a few rare minerals, but the functional group has tremendous importance in biochemistry. Disulfide bridges formed between thiol groups in two cysteine residues are an important component of the tertiary and quaternary structure of proteins. Two kinds of disulfides are recognized, symmetric and unsymmetric. Symmetrical disulfides are compounds of the formula RSSR. Most disulfides encountered in organo sulfur chemistry are symmetrical disulfides. Unsymmetrical disulfides (also called heterodisulfides or mixed disulfides) are compounds of the formula RSSR'. Unsymmetrical disulfide are less common in organic chemistry, but many disulfides in nature are unsymmetrical. Illustrative of a symmetric disulfide is cystine.}

 

[2] The Moorgate tube crash occurred on 28 February 1975 at 8:46 am on the London Underground's Northern City Line; 43 people died and 74 were injured after a train failed to stop at the line's southern terminus, Moorgate station, and crashed into its end wall. It is considered the worst peacetime accident on the London Underground. No fault was found with the train, and the inquiry by the Department of the Environment concluded that the accident was caused by the actions of Leslie Newson, the 56-year-old driver.

The crash forced the first carriage into the roof of the tunnel at the front and back, but the middle remained on the trackbed; the 16-metre-long (52 ft) coach was crushed to 6.1 metres (20 ft). The second carriage was concertinaed at the front as it collided with the first, and the third rode over the rear of the second. The brakes were not applied and the dead man's handle was still depressed when the train crashed. It took 13 hours to remove the injured, many of whom had to be cut free from the wreckage. With no services running into the adjoining platform to produce the piston effect pushing air into the station, ventilation was poor and temperatures in the tunnel rose to over 49 °C (120 °F). It took a further four days to extract the last body, that of Newson; his cab, normally 91 centimetres (3 ft) deep, had been crushed to 15 centimetres (6 in).

The post-mortem on Newson showed no medical reason to explain the crash. A cause has never been established, and theories include suicide, that he may have been distracted, or that he was affected by conditions such as transient global amnesia or akinesis with mutism. The subsequent inquest established that Newson had also inexplicably overshot platforms on the same route on two other occasions earlier in the week of the accident. Tests showed that Newson had a blood alcohol level of 80 mg/100 ml—the level at which one can be prosecuted for drink-driving—though the alcohol may have been produced by the natural decomposition process over four days at a high temperature.

In the aftermath of the crash, London Underground introduced a safety system that automatically stops a train when it is travelling too fast. This became known informally as Moorgate protection. Northern City Line services into Moorgate ended in October 1975 and British Rail services started in August 1976. After a long campaign by relatives of the dead, two memorials were unveiled near the station, one in July 2013 and one in February 2014.