The value of succinate dehydrogenase stain in the post-mortem diagnosis of early acute myocardial infarction

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Iv. consecutive medicolegal material comp~rising 79: pesetas, the].formazan i test revealed, recent, myocardial infarction in 16 cases (20~k]L Ol,~ly] 41 of t h e s e c~e*:(25%) " were demonstrated by maeroscb~iC examinatiOni ~:~y mea.~~ cif mlci~i,ose0pie.:exam[na~ion,. :' the 4 infarctions recognized macroseopical|y a n d 5; ocherca.~@s WhiFfs.Wet @demonstrated by the formazan t e s t c o u ! d he confirmed. If the formazan =best i.~'.employe d a~an...indeper~daat demoustration of m3,ocardiet infezctioa, a total of 16 cases chulfi:.:be demon-. strated (as mentioned above), or .four times as.m~m3 a~ .at.macr0~eoPic examim~tion'at autc,psy. •

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. • .... " -i:. : ::!. :.:~-:..:.::."i:i:~:: Routine pose-me,tern examination : o f ' the. :heart .::is.::insu.ff6~i~nt::::i"o. identify an early myocardial infarction. TO: approach thiadiagn6sis.a m a c r o : h~st0ehemiea[-enzyme- study :ofthe myoea~diu m ~m~ .be pe~rf6rmed , ( f o r m ~ a n test)., . ' ' .: : ': : " ":" ".:i : ".: . ...... In consecutive.medicolegaI autopsy.material; t h e present.au%h0m tried to determine how Often early anoxic Changes: of:.:the rh:~0caxdium:egcape •medicolegal detection if ~he formazan testAs not used.i :: ::: . ' " Mater~l ~ d methods .

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Du~Ar~g a period of tw~ raon~hs consecutive malarial was :selected ~,or

the Zo~nazan test at the Un.[ve~ity .Institu.te of Forensfc Medicine, Cope nhagen. 'The to'~A number, oZ a~t0ps!e~ done. during this. period was 7,O .... S, ~"~f l~hese 79 were considered suitable; 44 w e r e excluded.becau.se.the Pre-se~ 72-hour .limit had been e.xeeede5 (cadaverosis).. O~ '~he 7 9 ¢~es, 47 We:re males, and 32 femaIes. Figure !.shows t h e age distribution ~ d sex ra~i~: The mor~,esofdeath: .lad been ~ tol~ows-: ~.atural deat,]~,:42=.cases;:accidenf.~, 23 c~es; suicide, 12 cases; homici. ~e', i case; ~had.in 1 .case the m o d e o.~ death ~,,vasunkno~wa (Table ! ) . :. . : " . ' : • ..... ,; ..At autopsy ÷'~neimmedia:~e!i~ .v~ible i{~f~aCt~ou changes ~ d : localhz~ifl .~brotic lesions w e r e reco:'ded. Thereafter,: a formaz~a~ t e s t was d o n e o n a .

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Fig. 1. Age distributic)n and sex ratio in the consecutive material. TABLE

Distribution of me~d,esof death and {'or~mazan-negativecases in the consecutive mater~al. Modes of death

Male

Female

Mate form~zan aegative

Female formazan negative

A~u~e heart failure

18 12 12 5 0 0 47

7 5 11 7 1 1 32

5 2 3 1 0 0 11

I' ]L 8 0 0 0 .5

Other natural death Accident Suicide Homicide Uuknown Total:

cross-section o f the m y o e a r d i u m representing the t r a n s i t i o n f r o m the :middle t o the d i s t i l third ot! the ventricles. The theoret~ca[ basis fbr the histoehemistry o f . t h e f o r m a z a n . t e s t has been described by Nach!~s and S h n i t k a [1] and by B r o d y et at. [2].. I n t a c t my'ocardium contains substrate and a d e h y d r o g e n a s e able to reduce the yellow ~etrazoLium salt Nitzo-BT t o dark-blue f o r m a z a n . A n infarcted area o f t h e my ocardium has been irrevemibly deprived o f d e h y d r o g e n a s e activity [3 • 5] and in the f o m m z a n test it will be stained faintly blue or yellowish b r o w n because it d,~e~.J n o t reduce NiCro-BT. Such m y o c a r d i a l areas will stand ou~ in sh~.~p ~ontrast ~o the dark bluish-pm]ole i n t a c t ;~,yocardium still capable o f ~educ[ng Lhe Nitro-BT. T h e formazan ~est was carried o u t ~ fellows. T h e myocardial slice was carefu~y ~u~d quicld]~ cle~med o f b l o o d a n d t h e n placed f o r 3G m ~ n i n an i n c u b a t o r at 37 °C in ~: so!u~:ion pre-heated t~ the same t e m p e r a t u r e and cen,~sting of 10-9 mg Ni~ro-BT in 2 0 0 mI o f 0 . t M S4.:rensen p h o s p h a t e buffer (pH 7.4). Care was t a k e n t o c o v e r ~he m y o c a r d i a l slices w i t h . a b u n d a n t Nitro-BT solution which was freshly m a d e each time. T o ensure,, a sufficient c o n c e n t r a t i o n o f substrate (paying regm'd to:autolysis.) 2 a m i o f . 0 ; ! M

s0dium suc cinate. Were added; After:.incubatign the ra!~ocardiurh: ,was .~sess.ed . a n d . the.:infareti0ns traced'. on' a graph(see: rab!e. 2 ) . H e a r ~ s ~ h o ~ g . . s i ~ . c ; f ~iinfarcti0n:sare .~designatecl if o r m ~ aii-fl~gatlve.::in"this-paper.: The:: depth :anfl:;~e : -; surface :extent :.'hi~-the.i infa~tion z,..were.-eva~iiated.,- -ai!id::they., were. classified. : ia'~0, the° following type~:" :(1) total:"~d local.:~transmural":.iufarctions;::/.e;. :in:. f,~zction,~:extending. from the: endocardium ~o.~the pe!.Scardium;' (2). ~t.~l 5u d. local subend0thelial- :infarctions, i.e.:invOtvemer~:cf-atmost:the.endocardi~ taalf of the. mY0Cardiumi. (3) combined tr, msi~firal.and"subendocardial .infarc-. tions; and (4) isolated ififarcti0ns .of..the papillary.miiscles., o n l y anntiln.r infarctions were interpreted as tot.al. :.." . .:" : :/:. .: . . ::.i...~':: : ~!:: "'... : "... In the present study" the effect of cadaver0sis on clehydJ~genase activity was not te.c;ted, as i~,.had been :demonstrated Pre~o~sly that ~he col0ur re:a:ction in the fo/~nazan. ~est does not weaken h i ihe. fi~.,t 72 hours afterdeath, fthebc
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In 64 o f t h e 79 fonnazan-teste:d Cases :the"c()ro~a~ arteries :exhibited arteriosclerotic changes. In. 25 case~ these changes '~vere./mild.and :in..17 moderate,, whereas. 22.,cases sl/owed. ,~evere,:...stenos~rig ,cor0nary sclerosis." Freshthro:mbi were demonstrated i~ 9.m~,]e~ .and:2 females, all..~Vith sev,ere coronarychange~. HYpei~rop~ay of .th,e:hear~ !was four~d:]n 10 cases. Acc~)rding' to the autopsy findings the cause o f death was.assumed 'tb be aC(i'~e' heart,,-- failu.~e in 2 5 cases.: In 4 autopsiesi:fresh, myocm
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.226 •TABLE ,2: 'Clinical and pathologlcM ,data No.

Age and

Autopsy No~.

"Possiblesbc~:k I" .pa~hogenesis

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D 88175 ' 6 3 M

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D192175

83 F

Contusion of the brain

Contuslol~ of the brain

Accident

4

D104/75

84 F

Contusion o f [he brain

Contu~ion of the bra~n

Accident

5

DI08175

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C o n t u s i o n and laceration o f the brain

Contusion and lee,>. tartan of the brain

Accident

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D17t1/75

74 M

Bil!at. fract, o f ~st - 12th ribs

Accident

7

D119/75

69 M

1Vhmopurtt~ent ~,~nchiolitis

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Acute heart failure

Natural

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D145175 [ 5 5 F

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Aliment. asgiration to resp. tract

Accident

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Emollit:.on o f the brain

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D146175

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11

D148175

73 M

12

F t~9175

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90To Ist, 2rid, and 3rd degree burns

Accident

F 74/75

61 M

Acute heart failure

Natured

Acute heart lahore

Natura~

A c u ~ heart fait~re

Natural

Poisoning

Suicide

Emollition o f the brM n

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15

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El 8~9175

'70 M

LonT:l~ti~g Lt nc:oD ~c]ol.t~|e~

• Graphlc presentation o£ ~he for~azan-tested myoeardinl ring ~iew,~d from above w~th the antetier wall turning downwards. Fo~:nazan-nega'~ive a~oa5 black.

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MicroscOpic examinatkm revealedinfarction changes: in ,-9of.the la form;_:;an-negative'hear~% ill.8 ma][es ,and"I, female.,All ~i.macroscopical~.y identified infarctions were Confirmed by miCrOSCopic examinati0m On!,y 2 of the8 cases with infarcti~)nand diagnosed Shockwer~ demonsLzated.,micrcscopically, whereas the h~t.3 microseopiea~Iy dem0ns~rated infercfions were found i.a C~ses without diaguosed shock(Table 4 ) . . " " . "..... : ....." At the si~es of the macroseopieatly der~oustrated infazetion~ there was acute inflammatory reactiou. A p a r t f~'om, tha~, neui~ophilic ~anutoeytes were observed ,only in Cases 7 and 1!. ..... . , Comparison of infm'ctLon size before and after~he.formazan test in the 4 macroscopicadly identified cases revealed ~,T~eement in 2 (Cases g. mad 15), whereas in the other 2 (Cases 1 and 13) ~.e f o r m ~ a n 'h;sL showed a considerably greater extent of the infarction.

D!iscussion

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!nfa~ction chahges h~ the heart do not sh0w.ind:ubi~able macroscop,ic. te:;~ons until 24 laou~s after cbronar: occ!us!on,.butmicroseopica~ly theY a~e demonstrable much earlier, a t times as early as 6 hou~s.:after a n o c c l u s i o n . :

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228 '[7, 8], cases ~th no rnacroscop,i.cch~mgesloften escape detedtionbeeau~e.0f •lack .of.suspicion, but even, when~.the history or the l?atho!ogieal findings indicate an :infarction, the patho]0gist...often~lacks micr0sc0picconfinnation because-the topography of theinfarction is unknown.. The formazan.test, which demonstrates reduced de,hydrogenase activity in infarctedtissue,: renders possible an earlierm~.croscopic detection and secures cdrrect reirnov~l Of tissue for microscopiic examina:~ion. " • " " .... . . : Nachlas and Sb,n:itka, Brodey e t a l . , and Ramkissoon C0uld':"demonstmte reduced dehydrogenase activity in human ma~eri.al by t]he Nitro-BT method only if the patients had exhibil~.ed clinical symptoms for 6 - . 8 hours before death [1, 2, 9]. In anot,her t w o ;~eries the latent period is stated to be 5 hours and 1.5 hours, respectively [10, 11]. In a hospital seMes Andersen and Fischer Hansen investigated how often a fresh infarction escaped detection at autopsy [6]. On formazan testing of 100 consecutive hospit~d au'topsies they demonstrated 40 fresh myocardial infarctions. In 15 otF these cases thorpe had been no suspicion of fresh myocardial infarction at tbe macroscopic examination. In 7 cases the formazan test showed infarctions of a greater extent than assumed at the macroscopic assessment. All the unexpected cases of infarctio~ were confirmed by microscopic examination..Andersen and Fischer Hansen found a latent period of 8 hours before definite changes were demonstrable by the formazan test, Thus, false-positive formazan reactions, i.e. lacldng demonstration of reduced debydrogenase activity, ought to be possible only within this period. There were no false-negative fora~a~an reactions. The material was predominated by elderly patients having mainly severe coronary sclerosis, but coronary thrombosis was found in only 5 cases. A n u m b e r of the patients had been in shock for a varying length of time before death:~ a condition believed to have contributed to the occurrence of subendocardial infarction. Unlike the hospital series, the forensic serins comprises many cases of sudden, unexpected, na~:ural death in which the c~mse is often sta~;ed as acute failure of a heart having coronary sclerosis without fresh infarction changes or other heart ,disease, apart from dispersed fibrosis. In addition, the medicolegal au$opsies comprise m a n y cases in which the cause-of death must be ascribed to the group of violence, accidents: suicide, or homic.ide, :arid a demonstration of enzyme depletion in the myocardium, assessed by the formazan test, may entail important perspectives in forensic 1nedicine and in ":nsurance. in the present, material 12 cases (15%) exhibited with the formazau test a ![resh myocardial infz~rct~on of which t]he macroscopic assessmep.~ at autopsy had given no indication. '~'he mode of death had been classified in 5 c,f the cases as natural, whereas i.n 6 cases it was accident and in 1 suicide. Three of the patients who had been killed in accidents (Cases 3 , 4 , ,.~md 5 in Table 2) had severe injmT of the brain. They had been unconsciou~:~ and in shock, and death had occurred from 26 hours to 1 i days a f t e r t h e accident. '~he case of suicide (Ce.se 16) was due to the intake of a fatal dose of barbiturate (aprobarbi~one), The duration of .unconsciousness was unkr;own

.229 ' in .thi s. case,., but" it' could, not :.have ex< ,~.eded 7...hours:. Ir~..these.. 4: cases the . infarctions we~..subendocardial or.else:iuvolved.only;apapillary.musele~and" none Could b e confirmed:by fight microscopy, -~ - In.one.ca~e of. an accident (Case 6)~ a.74-year-ol[d man.had been:killed instantly, in. a. traffic: accident...The, formazan, test. Sh0wed.a local,, subendo, cordial . posterior-waU: infareti6n Which"was Confirmed: bY ligb.t..mibrosc0py.. This man .had been on the whole in good health, zmd in particular he had not had any symptoms or. signs of heart disease. His. coronary arte~.es, were normal at their origin and in their coume,.and, showed. :merety~ moderate arteriosclerosis..In the anterior intervent~cuta~ branch there was moderat2 ste~.osis. No thromboses .were.present and no localized fibrotic le~ions. The heart was n o t hypertrophic. After the autopsy the cause of death Was stated to be the severe injuries sustained, in pa~icular the severe injuries to the chest and I u n ~ with consequent][y hnpaired respirat.h~n, tn this case, then, the formazan test gave occasion for reflecthms concerning reconsideration of the cause as welt as the mode of death. . Case 10 was a 71-year-old man who" suffered a traffic accident. This man had severe coronary sclerosis and loc~dized fibrosis in the posterior wall of the left ven~Lricle. He had not been in shock nor was unconscious: There was an open ,Eracture of the left lower ~eg .and scattered bruises. Cardiac arrest occurred 26 hours after the accident.. The for.mazantest s h o w e d a total subendocardial }nfarction which was confirmed by microscopic examination. The cause of death was established, even at the autopsy, as acute cardiac failure. . .. The last case (Case 1 2 ) w a s a 73-year-old womm, w h ° ' h a d previously .... threatened to commit suicide. She w a s i n the' habit o~l~smoldng in bed. and had been found dead in her bed ~dth severe ..bums."At autopsY" severe coronary sclerosis was demonstrated, 'the cause of death was assumed t b b e the bums, :possible combined with c ~ b o n monoxide poisoning. Analysis o f the blood showed only 17% carbon monoxide. saturation; b u t an.almost fatal concentration of aproba~bitone. The mode Of. '.death was thereafter assumed to be suicide, By the fonnazan ~es~;a locM subendocardial infarction was found in the posterior wall of the tef~ ventricle. This infarction was Conrimmed by microscopic examination.

Conclusion Demonstration of an inf~xction not observed macroscopical!y ~.ay acquire legal import~tee hnL the final assessment of:fl~e mode o f deatl~ in cases where it has been assessed primarilyas violent. The fo.rm~an test will be of greatest significance in ,certain cases of sudden death related ~a accidents or assumed suicides where demons~mti0n of a zecent infarction m a y resuh~, in revision of the mode of death with insurance~tnd/or compensation consequences for t h e parties involved.. . " i "• : : :. .

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::: : ::i:::;,::Despite the: time limitations of: the f o n n a z a n test: (7 2 : h 0 u r s ) ; we : h a v e :: ' : f o u n d i t t o b e :Oi:practical v ~ u c ; i and rapid : ~ l d easy::::~b:administer. ]~he:~x~! : :: ~:t e n s i v e / n u m b e r 0f: ::unexpeCted :ihfarc~ibns dem0n~trateci:i::~9: tiie: f 0 m ~ !: ::t~i~t:in:: especially: a m o n g : : u n n a m r a l m o d e s o f death,: appears:::to justi[fy the:use ,0f t h e f o r m a z a n t e s t i n meldic01egal r h a t e r ~ a l s , : at:i:least:-~hen:,, t h e c~nse a n d ] 0 r m o d e 0 f d e a t h i s n0ti:e~ident :at :autopsy:

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Acknowledgement This s t u d y was carried o u t w i t h the technical assistance o f Sys J $ r g e n s e n and Aase Lindballe.

References 1 N.M. Nachlas and T, K. Shnitka, Amer. J. PathoI., 42 (1963) 379 - 396. "2 G. L. Brody, W: A. I~eLding~R. M. ]~eldiag and ~. A. Fetdma~l, Arch. Pathol.~ 84 (ltJC7) 3 1 2 - 317. 3 M. W. Hamolsky and N, O. Kapian, Circulation, 23 (1961) 102 - 110. 4 P. Roegsegger, J. Nydich,. A. Freimann and J. S. LaDue, Circ. Res., 7 (I959) 4 - 10. 5 P. E. Strandjord, K. E, Thomas and L. P, WhD~e,d. Ciin: Invest., 38 (1959) 2~11 -

2118. 6 J. A. Andersen and B. F. Hansen, Ugeskr. Laeg., 134 (1972) 1716 - 1720. 7 G, Fine, A. R. Mordes and J. R. Scerpe|la, Arch. Pathol., 82 (1966) 4 - 8. 8 W. E. Gould (ed.), Pathology of The Heart, Charles C. Thomas, Springfield, Ill., 1950, ~p. 5~9 - 606. 9 R. A. Ramkissoon, J. C!in. Pathcl., 19 ~(1966) ~79 - 481. 10 E. Knight, J. Forensic Med., 14 (1967) 101 - 107. 11 A. R. Morales and G. Fine, Arch. Pathol., 32 (196G) 9 - 14.