Forensic Science, 8 (1976) 33 - 36 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

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INVESTIGATION OF SUDDEN DEATHS FROM MYOCARDIAL ISCHAEMIA*

BERNARD KNIGHT

Institute of Pathology, Royal Infirmary, Cardiff (Gt. Britain)

The material to be investigated comes from the routine forensic pathology service of the Welsh National School of Medicine, part of the University of Wales. The autopsy load is approximately 1200 cases per annum, dealt with by 2 full-time forensic pathologists, who act as part of the Medical School Pathology Department, there being no seperate medicolegal department. About two-thirds of the work load comes from the city of Cardiff and the remainder consists of coal-miners from neighbouring districts, where the autopsy is to evaluate the degree of pneumoconiosis, if any.

As in all routine forensic services, approximately 70% of the work load consists of sudden natural death, mostly of cardiac origin. The following routine is carried out in the investigation of this latter group.

1 If the autopsy reveals an obvious well-established myocardial infarct, especially if there are secondary complications such as rupture into the pericardium, no further investigation is thought necessary, except for academic interest.

2 Where there is coronary artery disease, either occlusive or severely stenotic atheroma or thrombosis etc., but no obvious infarct is visible in the muscle, histological investigations are carried out. Similarly, in cases of death where there is left ventricular hypertrophy from hypertension or aortic valve disease, histology is also carried out.

3 Formerly, enzyme-staining of macroscopic dimensions was attempted, using the TTC (triphenyl-tetrazolium chloride) technique. However, this proved so unreliable, by giving many false negative results, that it has been abandoned.

4 Routine formalin-fixed, paraffin-embedded blocks are taken from 4 areas of the heart, normally cut to a distinctive shape which identifies the area of the heart from which they are taken. These are processed in the usual way and both routine stains performed as well as retaining some spare sec- tions for other techniques.

5 Haematoxylin-eosin stain is first evaluated, and an attempt to detect ischaemic-anoxic lesions is made by evaluation of increased eosinophilia, corrugation of fibres, cellular oedema, and in later stages, aggregation of the

*Paper presented at the First Meeting of the International Study Group of Natural Unexpected Death (ISNUD), Stockholm, December 11-13, 1975.

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contractile material within the fibres. It has been noted that loss of stria- tions, loss of muscle nuclei and inflammatory cell infiltration are most unreliable as guides to early infarction.

6 PAS (periodic-acid Schiff’s stain) has been used in the past, since a diastase-resistant muco-protein appears in the ceil in the form of a purplish- blue material at about the time when haematoxylin-eosin changes are definite. This however is no advance in detecting early infarction.

7 PTAH (phosphotungstic acid haematoxylin) (Mallory’s) stain has also been used, which gives an excellent demarcation of the infarcted areas, but is of no advantage in detecting early infarction.

8 Fluorescent techniques using acridine orange have recently been used and appear to show considerable promise. If a paraffin section is brought to water or a frozen section is used, immersion in a 1:lOOO aqueous solution of acridine orange for 3 or 4 seconds will cause normal myocardium to fluoresce golden-brown under ultraviolet light. Any anoxic or ischaemic damage to the cell will change this secondary emission to green and this change appears to be an early and sensitive index of cell damage. The techni- que is very rapid and simple and on frozen sections can be performed within minutes of autopsy. The reaction must be performed within a certain range of pH but this does not appear to be as critical as described in various original publications.

9 Micro-enzyme staining has been performed for some years and has been found to be very reliable. Though the literature quotes SDH (succinic dehy- drogenase) and LDH (lactic dehydrogenase) as useful tests, it is MDH (malate dehydrogenase) that is the author’s stain of choice. This appears to be sensitive enough to show early infarction within approximately 4 hours after onset and is also resistant enough to post-mortem autolysis to be usuable in refrigerated bodies up to at least 4 or 5 days after death. The technique is a standard one as described by Pearce and others and the preparations are permanent. The technique appears to be reliable and reproducible, and correlates well with methods of detecting early infarction.

10 Recently the haematoxylin basic fuchsin picric acid technique (HBFP) has been investigated again, it having been tried several years ago and discarded as unreliable. Although the results obtained are now somewhat equivocal, it is felt that the method has considerable promise but may show too many false positives in its present form. This is being further evaluated and it is hoped to collaborate with other workers in trying to assess its reliability.

The above techniques are mainly applicable to the ischaemic-anoxic damage caused by focal infarction following blockage of a coronary vessel. However, during this investigation, it was noted that hypertrophied left ventricles, in the absence of coronary artery disease also showed extensive changes using these techniques. This was further investigated, using hearts where there was no coronary artery disease but where evidence of left ventricular hypertrophy from either hypertension or aortic stenosis was present.

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REFERENCES

1 G. Farrer-Brown, Br. Heart J., 30 (1968) 527. 2 B. H. Knight, The post-mortem diagnosis of early myocardial infarction, M.D. Thesis,

University of Wales, (1966). 3 B. H. Knight, Med. Sci. Law, 5 (1966) 31. 4 B. H. Knight, J. Forensic Med., 14 (1967) 101. 5 B. H. Knight, in F. E. Camps (Ed.), Gradwohl’s Legal Medicine, 2nd. edn., Wright,

Bristol, 1968, p. 251. 6 B. H. Knight, in F. E. Camps (Ed.), Recent Advances in Forensic Pathology, Church-

chill, London, 1969, p. 20. 7 B. H. Knight, in C. Wecht (Ed.), Legal Medicine Annual, Appleton-Century-Crofts,

New York, 1971, p. 69. 8 A. J. E. Pearce, Histochemistry: Theoretical and Applied, 3rd edn., Vol. 2, Appendix

21, Churchill Livingstone, Edinburgh and London, 1972, p. 1342. 9 J. T. Lie, K. E. Holley, W. R. Kampa and J. L. Titus, New histochemical method for

morphologic diagnosis of early stages of myocardial ischaemia, Mayo Clin. Proc., (1971) 319.

10 V. B. Sahai and Bernard Knight, The post-mortem detection of early myocardial infarction by a simple fluorescent method, Med. Sci. Law, 16 (1976) 17.

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It was found that a very striking lesion was almost constantly present where death was attributable to the left ventricular failure. This took the form of an extensive loss of enzyme activity in the inner part of the ventricular wall. This sometimes extended right up to within a few cells of the epicardium, which were always spared. In less advanced cases, the lesion was restricted to the inner third of the ventricular wall, though here again the layer of cells immediately deep to the endocardium were spared. Using malate dehydrogenase stains, the inner third of the ventricular wall frequent- ly showed gross enzyme abnormalities and this was correlated with work done by micro-anatomists on the vascular supply of the left ventricular wall. At this point, reference should be made to the original publication, but in summary, it can be said that the sub-epicardial zone tends to be supplied by arteries with many branching terminations which offer a profuse and concentrated blood supply. A second set of vessels passes straight through the ventricular wall without division to supply the papillary muscles etc., while another group tends to run circumferentially parallel with the epicardium, giving branches both inwards and outwards.

Farrer-Brown (1968) found the termination of these vessels to be of 2 types. In the subendocardial zone, the endings continued in the general line of the main artery and the area of myocardium supplied by these vessels was small. This was in contrast to the other pattern in the middle of the myo- car-dial wall, where the arterial branches turn more at an angle to the main artery and then divide to supply a much wider area of myocardium than was supplied by the terminal branch in the subendocardial zone.

These anatomical differences in the various layers of the left vetricular wall suggest that the vessels in the mid zone each have to supply a larger volume of myocardial wall than in other layers. This might well explain the greater sensitivity of this zone to hypoxic changes, as indicated by reduced enzyme staining. In both aortic stenosis and hypertensive heart disease, even in the absence of coronary stenosis, the enlarged left ventricular muscle mass requires more blood to supply its greater mass, but eventually might outgrow the available supply and thus the middle zones of the ventricular wall would become the most vulnerable to anoxia.

Returning to the general problem of protecting anoxic-ischaemic damage to the myocardium, from whatever cause, it seems essential that a battery of techniques be used and an overall survey be made of the spectrum of available tests, before deciding whether or not the muscle is damaged. To date, the most useful techniques appear to be enzyme staining and fluores- cence with acridine orange, though experience is necessary in their inter- pretation. They certainly produce results at an earlier stage than the classical methods of haematoxylin-eosin, PAS and PTAH staining. The potential value of HBFP is doubtful at the moment, but it appears to hold consider- able promise if reliability and false positive results can be standardized. It is hoped that co-operation between interested workers in this field will improve the reliability of all these tests in the near future.