THE NEW ARMENIAN MEDICAL JOURNAL

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1 THE NEW ARMENIAN MEDICAL JOURNAL Vol.9 (2015), Nо 1 p THE USE OF LUMINESCENCE MICROSCOPY FOR DIAGNOSIS OF SUDDEN CARDIAC DEATH DUE TO ISCHEMIC HEART DISEASE IN THE FORENSIC PRACTICE Khchtryn P.S. 2,3*, Zilfyn A.V. 1, Bishryn M.S. 2,, Avgyn S.A. 1 1 Scientific-Reserch Center, Yerevn Stte Medicl University, Yerevn, Armeni 2 Repulicn Scientific-Prcticl Center of Forensic Medicine, Ministry of Helth, Yerevn, Armeni 3 Deprtment of Pthologicl Antomy nd Clinicl Morphology, Yerevn Stte Medicl University, Yerevn, Armeni Received 8/29/2013; ccepted for printing 02/28/2015 Astrct Sudden crdic deth remins mjor helth issue ccounting for significnt percentge of ll crdic deths. The mjority of deths investigted y forensic pthologists is certified s nturl. The use of pproprite microscopy in these cses will ssist the pthologist in identifying the disese process nd ny other underlying medicl conditions tht my exist. Microscopy in dily expert prctice requires the use of hemtoxylin nd eosin, which re mndtory for ech forensic exmintion. Histologicl smpling of the myocrdium my revel n cute infrct or crdiosclerosis, ut sometimes there were no significnt chnges while using routine hemtoxylin-eosin stining. In these cses it is importnt to use different techniques. Over the yers histologicl techniques use distinguished method of luminescent microscopy y cridine ornge stining to ssess the morphofunctionl condition of the myocrdium. We exmined three groups of decesed from sudden deth: the first group included 15 cses of lunt hed trum without dmging of orgns in thorcic cvity, the second group with 15 cses of sudden coronry deth without mcroscopic chnges in the hert. The finl third group included 15 cses of myocrdil infrction with the presence of necrotic chnges visile y nked eye. Tinctoril fetures of crdiomyocytes in the processing of microscope slides in ll three groups /deth from csul trum without dmged myocrdium, nd sudden crdic deth cses in the presence or sence of mcroscopiclly visile chnges/ treted with cridine ornge show green fluorescence loclized within structurlly intct cells, green nd yellow in crdiomyocytes t the erly stges of dystrophic processes, ornge-red in the muscle cells in dvnced dystrophic processes in the myocrdium. Thus, the method of myocrdil smples stining with cridine ornge including susequent luminescence microscopy exmintion is very informtive in determining the severity of the dmge of crdiomyocytes nd the spreding pttern of regionl pthologicl process. Keywords: sudden crdic deth, histologicl exmintion, cridine ornge, luminescent microscopy. Address for Correspondence: *Deprtment of Pthologicl Antomy nd Clinicl Morphology, Yerevn Stte Medicl University fter M. Hertsi, 2 Koryun Street, 0025, Yerevn, Armeni Tel.: ( ) E-mil: prik82@gmil.com Introduction Sudden crdic deth is currently descried s nturl, unexpected deth occurring within one hour of the onset of finl symptoms. The term sudden deth hs een used in literture for over 250 yers, ut no single definition hs een identified yet. Sudden deth is ssumed to e either instntneous or the one tht occurs during severl minutes, 1 or 6 or up to 24 hours fter the onset of continul symptoms of mortl disese. However, such wys of determining it re often useless ecuse sudden deth is unwitnessed in out 40% cses, for exmple when it occurs during sleep. Most often sudden deths hve crdiovsculr cuse when this time lpse is short [de l Grndmison GL, 2006]. According to the World Helth Orgniztion (WHO) every yer n estimted 20 million people 83

2 die of crdiovsculr diseses glolly, prticulrly more thn hlf from different forms of ischemic hert disese [WHO, 2012]. According to the Ministry of Helth of Repulic Armeni more thn 6000 people die from CVD, including 55% from hert ttck. Specific feture of sudden deth due to ischemic hert disese is the deth of 30-40% of victims in the first 10 minutes, nd pproximtely the sme in the next 2 hours. Two thirds of lethl outcomes occur t the prehospitl stge, even in the presence of well-dministered emergency medicl cre. [Kirichenko AA,2002; Chugh SS et l., 2008; Bokeri LA, Revishvili AS, 2011]. Dignosis of ischemic hert disese in forensic utopsies prctice is hindered y the lck of medicl documenttion with clinicl records. However, dignosis of chronic forms of ischemic hert disese usully does not present gret difficulties, since t the time of post-mortem exmintion it is lredy possile to revel significnt pthologicl chnges of the hert [Milroy CM, 2012; Chng J et l., 2013]. The gretest difficulties rise in the dignosis of cute forms of ischemic hert disese, especilly during sudden deth of reltively young people in the presence of seeming well-eing. It is known tht the focus of necrosis in ptients with cute myocrdil infrction is formed only y the end of the first dy fter the onset of ischemic ttck [Boyko YA, 2000; Goldstein JA et l., 2000; Bsso C et l., 2008]. In these situtions, when sudden deth occurs t very erly stge of myocrdil infrction, the myocrdil chnges re nonspecific nd cnnot esily e detected y trditionl mcroscopic exmintion, or histologicl stining. The medicl literture contins informtion out the possiility of using luminescence method in postmortem dignosis of erly ischemic dmge of myocrdil tissue. Evlution of structurl chnges in res of myocrdil ischemi ws evluted y the uthors sed on the spontneous luminescence of the myocrdil tissue, i.e. without the use of fluorescent proes. Menwhile, over the yers histologicl techniques use distinguished method of luminescent microscopy y cridine ornge (AO) stining to ssess the morphofunctionl condition of the myocrdium [Rnji M et l., 2009; Gohel H et l., 2012]. Possiilities of luminescent microscopy using usul stining of smples with hemtoxylin-eosin nd those emedded in polystyrene were studied [Khromov АМ, Vliullin DN, 2001]. Fluorescent stining should e used when ville in ll forensic cses of sudden deth y hert ttck to ensure its highly prole determintion, which is otherwise not visile grossly nd on routine histopthology exmintion y hemtoxylin nd eosin stins. However, this very informtive method hs not found proper ppliction in specilized forensic lortories. Unvilility of the UV fluorescent microscope nd the old technique of the procedure with cryostt sections might ecome limiting fctor. The fct tht the study cn e conducted on prffin emedded sections elimintes this hurdle, s the tissue locks cn e miled to such referrl centers, if the equipment is not ville loclly. Mterils nd Methods Myocrdil smples tken from 45 ded odies of persons ged were exmined to revel the possiilities of luminescence microscopy method in the dignosis of sudden crdic deth from ischemic hert disese in forensic prctice. In ll cses there were no comoridities nd no trces of lcohol or drugs in the lood or other fluids of the ody. The mteril ws divided into three groups: the first group included 15 cses of lunt hed trum without dmged orgns in thorcic cvity. In second group 15 cses of sudden coronry deth without mcroscopic chnges in the hert. The finl third group included 15 cses of myocrdil infrction with the presence of necrotic chnges visile y nked eye. In order to determine the severity of the structurl chnges in the myocrdium in ll groups studied, we hve used cridine ornge stining method with susequent exmintion of slides under luminescence microscope y Bertlnffy Bikis [Pirs E, 1960]. This method is very informtive ecuse it llows in ech cse (even on the sme slide) to determine foci of structurlly intct crdiomyocytes, re of myocrdium with pprent dmge, including the evlution of the prevlence of regionl pthologicl process nd profundity of dystrophic chnges in prenchym nd stroml structures. Luminescence method of dyeing with AO is sed on the dynmic of trnsformtion of the crdiomyocytes tinctoril properties 84

3 depending on the prevlence nd severity of regionl disese process in wide vriety of crdiovsculr diseses. For stining procedure fresh 1% AO solution ws prepred (ph 7.2). Prffin emedded sections were treted with AO solution for 3-5 seconds nd repetedly rinsed in phosphte uffer for minutes chnging the uffer solution 2-3 times to wsh wy non-specific deposits of AO. The slides were immeditely viewed under UV fluorescent microscope ("Boeco", Germny). The entire gmm of tinctoril properties ws oserved most prominently in the longitudinl sections of the myocrdium. Thus, structurlly intct crdiomyocytes during the process of stining with AO gin intense green color. The nucleus, nucleoli nd cytoplsm of crdiomyocytes, including intrcellulr longitudinlly oriented myofirils re lso endowed with intense green fluorescence. Initil stges of dystrophy in myocrdil tissue, s rule, mnifested mrked decrese in the green fluorescence, which is replced with the yellowornge fluorescence in compound components of crdiomyocytes. During deeper destructive injury of myocrdil structures (dyscomplextion of djcent srcomeres, foci of myofirils destruction, myocytolysis, kryopyknosis nd kryolysis) res of myocrdium with prevlence of ctolic processes egin to cquire new tinctoril properties. Thus, s result of depolymeriztion nd decy of nucleus, nucleolus nd structurl components of cytoplsm ll of the forementioned intrcellulr components of crdiomyocytes stin in ornge, ornge-red nd red colors, depending on the severity of dmge. Results In the first group cytongiorchitectonics of the myocrdium ws intct in ll prts. Longitudinlly nd trnsversely oriented srcomeres mnifested loose orienttion to ech other, resulting from moderte edem of the interstitium. Crdiomyocytes showed modertely compct distriution of myofirils in the cytoplsm nd the presence of intensively sophilic ovl shped nuclei. Cpillries were clerly seen in intermusculr connective tissue long longitudinlly oriented crdiomyocytes. In generl, the structure of the orgniztion of the myocrdium in persons who died from n ccidentl injury (I study group) did not differ from tht of typicl myocrdium of prcticlly helthy popultion of the sme ge. However, in some res there were signs of focl ctivtion of firolstic processes (Fig. 1). Figure 1. Structurl chnges in the myocrdium of study group I. H&E о. 20, оc. 10. Cytongiorchitectonics of the myocrdium is intct. Moderte swelling of the strom. In the second study group the cytongiorchitectonics of myocrdium ws chnged. Everywhere there were signs of dyscomplextion of myofirils, pyknosis or, on the contrry, swelling of nucleus, lumpy destruction (Fig. 2а). Microcircultory disturnces with swelling of endotheliocytes nd perivsculr edem were detected in the res with dystrophic chnges of crdiomyocytes. Erythrodipedesis indicted high degree of vsculr permeility. In this condition, some res showed signs of ctivtion of the firolstic cells developing looseness in connective tissue, which ws reveled in the foci of dystrophy nd destruction of crdiomyocytes. Microvessels wlls (venules, cpillries, rterioles) were in the stte of firosis, loclized especilly in the intrmusculr connective tissue (Fig. 2). The polymorphism of structurl chnges in myocrdium tht we hve found indictes tht regionl pthologicl process hd prolonged progressive nture. Thus, reltively fresh res with prevlence of ltertive processes were found due to ctivtion of focl reprtive-prolifertive rections (firosis nd sclerosis in intrmusculr connective tissue, including wlls of vessels). During the exmintion of the slides prepred from the smples of the third group under the light 85

4 Figure 2. Structurl chnges in the myocrdium of study group II. hemotoxylin-eosin, о. 40, оc. 10. ) Diffuse chnging of cytongiorchitectonics of myocrdium. Signs of lumpy destruction of crdiomyocytes in the presence of modertely edemtous strom nd focl ctivtion of firolstic processes; ) Pronounced firolstic processes in the wlls of djcent microvessels in the presence of prolifertion of endothelil nd smooth muscle cells of rterioles. Signs of focl dyscomplextion nd crdiomyocytes decy, ctivtion of firolstic cells. microscope the structure of myocrdium ws norml in some res. In these res crdiomyocytes hd а strict liner orienttion. Intermusculr rteriolr nd venulr microvessels were lso detected very clerly. In most cses exmined in this study group were found micronecrotic foci of myocrdium. In these res the oundry etween djcent groups of srcomeres ws not contoured. They were presented y structureless (lumpy or even homogenous) eosinophilic msses quite often showing the signs of kryorrhexis (hemtoxylin odies) (Fig. 3а). Exudtive processes previled in the perifocl re, presented y signs of incresed vsculr permeility (plsmorrhgi, perivsculr swelling, erythrodipedesis, nd moderte swelling in connective tissue) (Fig. 3). In this condition, signs of ctivtion of firolstic processes were detected in some res. As result, res of firosis were found t the plce of previous locliztion of crdiomyocytes, sclerotic processes were reveled in the thickness of some vessel s wlls, including cpillries. The results of our fluorescence-microscopic nlysis hve demonstrted tht trnsversely oriented muscle cells with intense green luminescence of their constituent components were oserved in the myocrdil smples of the first study group throughout the entire myocrdil res (Fig. 4а). Besides crdiomyocytes, stroml cells endothe- Figure 3. Structurl chnges in the myocrdium of study group III. hemotoxylin-eosin, о. 40, оc. 10. ) Signs of focl droplet-lumpy decy nd crdiomyocytolysis of djcent srcomeres with the presence of hemtoxylin odies ; ) Destruction re s perifocl zone. Pronounced diltion nd hyperemi of intermusculr microvessels. 86

5 Figure 4. Fluorescent-microscopic chrcteristics of first study group. Stining with cridil-ornge. ) Longitudinlly oriented crdiomyocytes endow green fluorescence of their cytoplsm о. 10, оc. 7; ) Chnging of tinctoril fetures of muscle cells on the ckground of green glow of longitudinlly oriented crdiomyocytes. A focus of yellow nd ornge fluorescence long some srcomeres ws reveled о.40 оc. 10. liocytes, smooth muscle cells of vsculr wll, firolsts, nd dventitil cells lso displyed intense green fluorescence. Green glow ws replced y yellow-green only in few visul fields on very limited re over the djcent crdiomyocytes, (Fig. 4). In the second study group, AO stining of slides reveled mosic pttern. Groups of srcomeres with green nd green-yellow fluorescence lternted with djcent srcomeres of crdiomyocytes whose cytoplsm nd nucler mteril displyed intense red-ornge or red fluorescence (Fig. 5, ). It is noteworthy tht the ornge-red fluorescent foci loclized in the crdiomyocytes were oserved not only in the res of their decy, ut lso in those where integrity of the muscle cells ws reltively intct. In the third study stining of the slides with AO reveled reltively severe chnges, strictly loclized in the foci of micronecrosis nd in the perifocl re. Thus, res of necrotic myocrdil tissue hd n intense homogeneous ornge-red fluorescence (Fig. 6а). In some res free of infrction zones, in the presence of diffuse green fluorescence in most groups of the srcomeres, only in some res the srcomeres chnged their tinctoril properties, i.e. the glow gve wy to ornge-red (Fig. 6). Figure 5. Fluorescent-microscopic chrcteristic of II study group. Stining with cridil-ornge.. о.10 оc. 7. ) Diffuse intense nd moderte green fluorescence of crdiomyocytes cytoplsm; ) Focl green fluorescence of muscle cells lternting with res in which crdiomyocytes hve yellow-ornge fluorescence. 87

6 Figure 6. Fluorescent-microscopic chrcteristics of study group III. Stining withcridil-ornge.. ) Focl orngered glow in the field of dystrophy nd crdiomyocytes decy о.40, оc. 10. Minly yellow-ornge fluorescence, including few cells with green glow in the cytoplsm in the perifocl re of djcent srcomeres. ) In res remote from necrosis mosic pttern is oserved: fields with green glow of the cytoplsm of crdiomyocytes re interspersed with ptches of yellow fluorescence of djcent srcomeres о. 10, оc. 10. Discussion Thnks to the reserch findings, in our cse, tht is, in ll the three study groups, the method of myocrdil smples stining with AO followed y luminescence microscopy exmintion ws very informtive in determining the severity of the dmge of crdiomyocytes nd the spreding pttern of the regionl pthologicl process. Tinctoril fetures of crdiomyocytes in the tretment of microscope slides with AO in ll the three groups were mnifested s green fluorescence loclized within structurlly intct cells: green nd yellow in crdiomyocytes in the erly stges of dystrophic processes, ornge-red in the muscle cells, in fr dvnced dystrophic processes in the myocrdium. The fluorescent method we used AO stining of slides lso in conjunction with other informtive morphologicl methods, llows for differentil dignosis of pthologicl processes occurring in the myocrdium in the groups we studied. The method is sed on the unique tinctoril properties of AO, which mkes it possile to determine the severity of the regionl pthologicl process sed on identifile trnsformtion of crdiomyocytes color rnge of fluorescence from green to ornge-red. REFERENCES 1. Bsso C, Burke M, Fornes P, et l. One ehlf of the Assocition for Europen Crdiovsculr Pthology. Guidelines for utopsy investigtion of sudden crdic deth. Virchows Arch. 2008;452: Bokeri LА, Revishvili АS. [Sudden crdic deth] [Pulished in Russin]. Moscow: GEO- TAR-Medi p. 3. Boyko YA. [Anlysis of cses of sudden crdic deth due to different forms of ischemic hert disese] [Pulished in Russin]. Modern questions of Forensic medicine nd prctice. Novosiirsk: 2000;5: Chng J, Nir V, Luk A, Butny J. Pthology of myocrdil infrction. Dignostic Histopthol- ogy. 2013;19(1): Chugh SS, Reinier K, Teodorescu C, et l. Epidemiology of sudden crdic deth: clinicl nd reserch implictions. Prog Crdiovsc. Dis. 2008;51(3): De l Grndmison GL. Is there progress in the utopsy dignosis of sudden unexpected deth in dults? Forensic science Int. 2006;156: Gohel H, Desi N, Tripthi S, Gupt B. Postmortem study of hert in cses of sudden crdic deth using cridine ornge fluorescence nd hemtoxylin nd eosin stin. Indin Journl of Medicine nd Helth cre. 2011;1(5):

7 8. Goldstein JA, Demetrion D, Grines CL, et l. Multiple complex coronry plques in ptients with cute myocrdil infrction. N Engl J Med. 2000;343(13): Khromov АМ, Vliullin DM. [Luminescent method ppliction in histologicl Forensic medicine prctice] [Pulished in Russin]. 2001;1-1(01): Kirichenko АА. [Angin pectoris nd cute coronry syndrome] [Pulished in Russin]. Interntionl medicl Journl. 2002;2: Milroy ChM. Coronry rtery disese, sudden deth nd implictions for forensic pthology prctice. Mini-Symposium: Crdiovsculr pthology-ii. Dignostic Histopthology. 2012; 19(1); Pirs E. [Histochemistry] [Pulished in Russin]. Foreign Literture Pulishing House Moscow: p. 13. Rnji M, Mtsur M, Leshnower BG., et l. Quntifying cute myocrdil injury using rtiometric fluorometry. IEEE Trns Biomed Eng. 2009;56(5): World Helth Orgniztion. Mortlity nd glol helth estimtes Retrieved from: disese/en/ 89