hypoxic damage

Forensic Science International, 45 (19901 135- 141 Elsevier Scientific Publishers Ireland Ltd.

FURTHER EVALUATION ING IN THE DIAGNOSIS HYPOXIC DAMAGE

STEPHEN

LEADBEATTER”,

OF IMMUNOCYTOCHEMICAL STAINOF EARLY MYOCARDIAL ISCHAEMICl

HELEN M. WAWMANb and BHARAT JASANP

of Forensic Pathology, IS.2 and bDepartment of Pathology, Park, Cardiff CFL 4XN (U.K.1

“Sub-Department

Institute

University

of Pathology, of Wales

Cardiff Royal Infirmary, CF2 College of Medicine, Heath

(Received August 8th, 19891 (Accepted September 22nd, 19891

Summary The pattern of immunocytochemical staining with antibodies to caeruloplasmin, myosin, myoglobin and C-reactive protein seen in myocardium taken from deaths with macroscopic evidence of myocardial infarction and/or significant coronary artery atherosclerosis and from deaths with neither of these lesions has been correlated with H&E, PTAH and HBFP staining of myocardium and circumstances of each death indicative of antemortem hypoxia and/or ischaemia. Loss of staining with these antibodies correlated well with fuchsinorrhagia and both techniques are more sensitive than H&E and PTAH staining in the detection of early ischaemic/hypoxic damage to myocardium. However, their sensitivity is such that they appear to detect agonal changes and, therefore, cannot be used for specific diagnosis of early myocardial infarction. Key words: Myocardium;

Ischaemiclhypoxic

damage; Immunoperoxidase

staining

Introduction

The results of immunocytochemical staining with several antibodies in selected cases of ischaemiclhypoxic damage to myocardium showed that the patterns of staining seen with antibodies to caeruloplasmin, myosin, myoglobin and C-reactive protein (CRP) were consistently meaningful [l]. The object of the present study was to apply these antibodies to myocardium sampled from a relatively large population of deaths and compare the patterns of staining with those seen with haematoxylin and eosin (H&E), phosphotungstic acid haematoxylin (PTAH) and haematoxylin basic fuchsin picric acid (HBFP). Correlation of these patterns with gross post-mortem findings and the history of the circumstances of the deaths was attempted to assess the overall specificity and sensitivity of immunocytochemical staining in the diagnosis of early ischaemiclhypoxic damage to myocardium. 0379-0738/90/$03.50

0 1990 Elsevier Scientific Publishers Printed and Published in Ireland

Ireland Ltd.

136

Materials and Methods

Deaths included in the study were placed in one of three populations: Population 1 - macroscopic evidence of myocardial infarction. Population 2 - No macroscopic evidence of myocardial infarction but significant coronary artery atherosclerosis (obstruction of more than 50% of the diameter of the lumen of at least one major epicardial coronary artery, as judged by the naked eye). Population 3 - Neither macroscopic evidence of myocardial infarction nor significant coronary artery atherosclerosis. Details of these populations are given in Table 1. The selection of tissue blocks at post-mortem and staining techniques have been detailed previously [l]. Briefly, serial sections of myocardium were stained respectively with H&E, PTAH, HBFP [2] and the immunoperoxidase DNP hapten sandwich technique [3] using antibodies to caeruloplasmin, myosin, myoglobin and CRP. The patterns of staining obtained with each technique were correlated by all three authors, a concensus being reached. The history of the circumstances of, and the gross post-mortem findings in, each death in Populations 2 and 3 were examined for evidence of external cardiac massage or other blunt chest injury and circumstances or lesions associated with hypoxia or ischaemia. Results

The patterns of staining seen in myocardium taken from Population 1 are summarized in Table 2 and reflect those previously described [l]. In all 15 cases the immediately subendocardial myocytes provided a reliable ‘in-built control’ consistently showing no abnormality with H&E, PTAH and HBFP stains and showing strong positive staining with all four antibodies. One of the cases showed only granulation tissue repair: of the remaining 14 cases showing earlier stages of infarction fuchsinorrhagic myocardium did not stain with the antibodies. Three cases showed no histological abnormality TABLE 1 DETAILS OF POPULATION OF CASES Population

Postmortem

details

Macroscopic infarction Coronary artery atherosclerosis only Coronary artery atherosclerosis and additional hypoxiclischaemic factor Neither macroscopic infarction nor coronary artery atherosclerosis

No.

Age range (years)

Post-mortem interval lhl

18 15 11

39-83 37-100 37-81

12- 120 16- 108 3-65

33

8 weeks-84

3-90

Macroscopic infarction

STAINING PATTERNS

TABLE 2

(15)

Loss of staining with antibodies

Loss of staining with antibodies

Fuchsinorrhagia Fuchsinorrhagia (2) No fuchsinorrhagia (1)

I

No loss of staining with antibodies

No fuchsinorrhagia -Fuchsinorrhagia/ no fuchsinorrhagia

Repair (1) Necrosis and polymorphs (5) Hypereosinophilia (6) PTAH clumping contraction bands No change (3)

(1)

(1)

(13)

DNP-hapten

HBFP

1

H&E, PTAH

SEEN IN POPULATION

138

with H&E and PTAH staining and in one of these cases the myocardium did not show fuchsinorrhagia and did not stain with the antibodies. No histological abnormality was seen on preliminary scanning 01 myocardium stained with H&E and PTAH taken from cases in Populations 2 and 3. Tables 3 and 4 show the correlation of the HBFP and immunocytochemical staining patterns with the histories and post-mortem findings in Populations 2 and 3, respectively. In 19 of 26 cases in Population 2 fuchsinorrhagic myocardium did not stain with the antibodies. Myocardium in 6 of the remaining 7 cases showed strong positive staining with the antibodies and did not show fuchsinorrhagia. Of the 33 cases in Population 3, 15 cases showed fuchsinorrhagic myocardium which did not stain with the antibodies and 15 cases contained myocardium which showed strong positive staining with the antibodies and did not show fuchsinorrhagia: only 3 cases showed discordant results. The circumstances of death were suggestive of ante-mortem ischaemia or hypoxia in 9 of 15 cases with fuchsinorrhagic myocardium which did not stain with the antibodies. In several of the cases in Populations 2 and 3 where the myocardium showed fuchsinorrhagia and no staining with the antibodies high power scrutiny of those sections stained with H&E and PTAH revealed contraction band necrosis [4].

TABLE 3 CORRELATION OF PATTERNS POPULATION 2 History and post mortem findings

Coronary artery atherosclerosis only Coronary artery atherosclerosis, external cardiac massage Coronary artery atherosclerosis, other pathological findings or circumstances associated with hypoxia or ischaemia e.g. cardiac hypertrophy, pneumonia, chronic obstructive airways, disease As above, with external cardiac massage

OF STAINING

AND CIRCUMSTANCES

OF DEATH IN

Staining patterns FuchsinorrhagziJ loss of staining with antibodies

No fnchsinonhagti no Eoss of staining with antibodies

FuchsinorrhagW no loss of staining with antibodies

7

2

1

5

-

-

4

3

-

3

1

-

139 TABLE 4 CORRELATION OF PATTERNS POPULATION 3

History

Suggestive of antemortem hypoxia only Suggestive of antemortem hypoxia, external cardiac massage Chest injury only Chest injury, external cardiac massage External cardiac massage only No evidence of any of above factors

OF STAINING WITH CIRCUMSTANCES OF DEATH IN

Staining patterns Fuchsinorrhagid loss of staining with antibodies

No fuchsinorrhagd no loss of staining with antibodies

Fuchsinorrhagiul no loss of staining with antibodies

4

-

2

5

-

1

2 1

2 2

-

1

9

-

2

2

-

Analysis of data from Populations 2 and 3 shows that correlation of HBFP and immunocytochemical staining patterns for the detection of early ischaemiclhypoxic damage to myocardium has a sensitivity of 82% and a specificity of 71%. Discussion

The staining patterns in myocardium taken from macroscopically evident infarctions reflect those previously reported [l]: loss of staining with antibodies correlating with fuchsinorrhagia, this pattern being seen in two out of three cases where H&E and PTAH staining showed no abnormality. Good correlation of HBFP and immunocytochemical staining, again as previously reported [l], was seen in 69 of 74 cases scrutinized, 47 showing loss of staining with antibodies in fuchsinorrhagic myocardium, the obverse pattern, positive staining with antibodies in myocardium which did not show fuchsinorrhagia, pertaining in the other 22 cases. Five ‘rogue’ results may reflect the technical capriciousness of the HBFP technique [5], even in the hands of an experienced operator. Similar fuchsinorrhagia and loss of staining with antibodies was found in 19 of the 26 cases in Population 2, where there was adequate pathological reason to infer ischaemic and/or hypoxic damage to myocardium. None of these cases showed any histological abnormality on low-power scanning of myocardium stained with either H&E or PTAH; high-power examination of those foci showing fuchsinorrhagia and loss of staining with antibodies revealed, in several cases, contraction band necrosis, but this change, visible

140

more easily with PTAH than H&E, was never as extensive as the areas of fuchsinorrhagia or loss of staining with antibodies. Failure to sample areas of myocardial ischaemic/hypoxic damage may explain the finding, in 6 cases, of the obverse pattern of positive staining with antibodies in myocardium which showed no fuchsinorrhagia. Inferences are drawn less easily from the results obtained with Population 3. Myocardium taken from 15 of 33 cases showed fuchsinorrhagia and loss of staining with antibodies, the obverse pattern being seen in an identical number of cases. In nine of the former cases the circumstances of death were suggestive of ante-mortem ischaemia or hypoxia, these circumstances including intra-operative haemorrhage, inhalation of smoke, mechanical asphyxia or convulsions. In the absence of continuous. physiological monitoring in an intensive care environment objective measurement of the degree and duration of ischaemia and hypoxia is impossible: assumptions are made from the history, which may not be reliable [6], and morbid anatomical findings, whose correlation with functional deficit may be poor. Blunt cardiac trauma might explain the fuchsinorrhagia and loss of staining with antibodies seen in four cases, but a history of similar blunt injury to the chest was present in 13 of 15 cases showing the obverse pattern (although none of these latter showed contraction band necrosis on H&E and PTAH staining). It might be argued, therefore, that: 61 subdivision of Population 3 in Table 4 is arbitrary; (ii) more extensive sampling of myocardium would reveal foci of fuchsinorrhagia and loss of staining with antibodies in all cases in Population 3; (iii) fuchsinorrhagia and loss of staining with antibodies reflects only agonal change. None of these arguments can be refuted. It is not unreasonable, however, to postulate that ischaemia and/or hypoxia are integral components of the agonal (or terminal1 period, the duration of which can rarely, if ever, be determined with accuracy and precision in human postmortem material. We conclude that loss of staining with antibodies to caeruloplasmin, myosin, myoglobin and C-reactive protein is a valid indicator of myocardial damage correlating well with HBFP staining, that both techniques are more sensitive than H&E and PTAH staining, but their sensitivity is such that, like fluorescence microscopy [7&i], they detect immediately-terminal or agonal changes, regardless of cause of death. These methods, therefore, cannot be used alone for the specific diagnosis of myocardial infarction as a cause of sudden death. Acknowledgements

This work was supported by the Welsh Scheme for the Development of Health and Social Research. Research Grant: JR 116 864 E 042. JR 116 864 E 110 and JR 116 864 E 955.

141

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myocardial

infarc-

of sudden

cardiac