detection ofmyocardial infarction immunohistological fixed, … · detection ofmyocardial...

4 JClin Pathol 1996;49:34-37

Detection of myocardial infarction byimmunohistological staining for C9 on formalinfixed, paraffin wax embedded sections

J P Doran, A J Howie, J N Townend, R S Bonser

AbstractAims-To evaluate an immunohistologicalstain for complement component C9 asa method of detecting early myocardialinfarction and to compare this with (1)an enzyme histochemical method and (2)conventional histological staining.Methods-(1) Eight hearts taken at nec-ropsy were stained using the nitrobluetetrazolium/phenazine methosulphatemethod and an immunohistological stainfor C9. (2) Twenty five hearts from casesof suspected or confirmed myocardial in-farction and 25 from cases without con-ventional evidence of infarction werestained for C9 and by haematoxylin andeosin.Results-(I) The histochemical methodindicated myocardial necrosis in fivehearts and the C9 method in seven, all ofwhich had clinical evidence ofmyocardialdamage or a reason for it. The histo-chemical method required fresh myo-cardium, was difficult to use and wasdifficult to interpret. (2) Of 25 hearts withsuspected or confirmed infarction, 24 werestained by the C9 method. Staining withhaematoxylin and eosin showed infarctionin 16 of these, all with infarcts at least24 hours old; the other eight had clinicalevidence of infarction less than 24 hoursold. Tne heart not stained by C9 was froma patient who, on review, had no evidenceofinfarction. Ofthe 25 control hearts, nonehad infarction on staining with haema-toxylin and eosin, but three were stainedby the C9 method. These three were frompatients with septicaemia or anotherreason for myocardial damage.Conclusions-The immunohistologicalmethod for C9 is a simple, reliable andsensitive method for the detection of earlymyocardial necrosis that could be usedon formalin fixed, paraffin wax embeddednecropsy material. This had advantagesover a histochemical method and con-ventional staining with haematoxylin andeosin.(Jr Clin Pathol 1996;49:34-37)

Keywords: C9, myocardial infarction.

Recent myocardial infarction can be impossibleto detect either by direct examination at nec-ropsy or using conventional stains on histo-logical sections. Histochemical techniques can

show early infarction, but these are difficult tointerpret and rarely used. A method of de-tection of infarction that could be used onformalin fixed, paraffin wax embedded sectionswould be of great value.Complement component C9, part of the

C5b-9 membrane attack complex of com-plement, is a reliable marker of complementdeposition and can be detected immuno-histologically on formalin fixed, paraffin waxembedded sections of renal biopsy specimens.1This membrane attack complex has also beenfound in necrotic skeletal muscle fibres2 andin infarcted myocardium.3 We wished to seewhether the immunohistological stain for C9could be used as a routine method for dem-onstrating recent myocardial infarction, bycomparing it with an established histochemicaltechnique, the nitroblue tetrazolium/phenazinemethosulphate reaction,4 and against con-ventional staining with haematoxylin and eosin.

MethodsHISTOCHEMICAL STUDYTransverse slices, about 1-2 cm thick, weretaken from the left ventricle of eight patientsat necropsy (table 1).

Nitroblue tetrazoliumlphenazine methosulphatemethod'The slices of myocardium were rinsed andstored at 4°C in Sorensen's phosphate buffer(0 1 M, pH7 4) containing 0 1 M ascorbicacid. Fresh slices were cut just before staining.These were incubated in Sorensen's buffer con-taining nitroblue tetrazolium, 50 mg/i00 ml,and phenazine methosulphate, 2 5 mg/100 ml,for 15 minutes at 37°C. The slices were rinsed,immersed in 10% formol saline, and pho-tographed.

Immunohistological method for C9After fixation for at least 18 hours, histologicalblocks were cut from the myocardial slices,using the photographs as guides to the sites ofblocks. Blocks were embedded in paraffin waxon a Shandon Hypercenter with a routine pro-cessing schedule. Sections were cut at 5 ptm,dried on glass slides at 60°C for 30 minutes,rehydrated, and immersed in 1% hydrogenperoxide in methanol to block endogenous per-oxidase. Sheep antiserum to C9 (The BindingSite, Birmingham, UK) was used at a dilutionof 1 in 500 in 0-15 M phosphate buffered saline

University ofBirmingham,Birmingham:Department ofCardiovascularMedicineJ P DoranJ N Townend

Department ofPathologyA J Howie

Department ofCardiothoracicSurgeryR S Bonser

Correspondence to:Dr A J Howie,Department of Pathology,The Medical School,Birmingham B 15 2TT.Accepted for publication5 September 1995

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Tablel Comparison of staining of myocardium with haematoxylin and eosin with the histochemical technique and immunohistology for C9

Nitroblue tetrazoliunilPatient Haematoxylin phenazine nmethosulphate(sex/age in years) Clinical details and eosin staining C9 inlmunohistological staining

F/33 Died 24 hours after heart One group of contraction Patchy pink/red Extensive patchy staining oftransplant band necrosis single cells and groups of cells

M/44 Died two months after heart Severe acute rejection with Patchy pink/red Extensive patchy staining oftransplant patchy necrosis single cells and groups of cells

F/50 Died one hour after collapse No infarction identified Dark blue Nonefrom dysrhythmia:no clinical evidence ofmyocardial infarct:coronary arteries patent

F/55 Died from septicaemic shock in No infarction identified Dark blue A few scattered cellsliver failure

M/62 Died seven days after Infarcted area White area Heavily stained areamyocardial infarct

M/68 Died two days after myocardial Infarcted area White area Heavily stained areainfarct

M/73 Died 24 hours after myocardial No infarction identified Patchy pink/red A few groups of cellsinfarct

MJ83 Died six hours after myocardial No infarction identified Dark blue Scattered single cellsinfarct

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Figure 1 Slice of left ventricle from a 62 year old manstained by the nitroblue tetrazoliumlphenazinemethosulphate method seven days after a myocardialinfarct. Non-infarcted myocardium appears dark andinfarcted myocardium appears white. The junction betweenthe dark and white areas is irregular, producing a mottledpattern at the edges of the main white area.

and applied to sections for 60 minutes at roomtemperature on a Shandon Sequenza im-munostaining centre. After washing, per-oxidase conjugated donkey antiserum to sheepIgG at a dilution of 1 in 100 was applied for45 minutes. After another wash, peroxidasewas detected with hydrogen peroxide and tetra-amino-biphenyl hydrochloride (diamino-benzidine), and sections were counterstainedwith haemalum, dehydrated and mounted.

STUDY OF CONVENTIONAL STAININGNecropsy records were reviewed and 25 con-secutive cases with clinically suspected or con-firmed myocardial infarction were identified.Another 25 cases without clinical or con-ventional pathological evidence of myocardialinfarction were also identified. Sections offormalin fixed, paraffin wax embedded myo-cardium, stained with haematoxylin and eosin,were inspected. The immunohistologicalmethod for C9 was applied to sections cut fromthe same blocks.

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Figure 2 Histological section taken from the heart in fig1 at the junction of macroscopically dark and white areas,stained by the immunohistological method for C9.Infarcted myocardial cells appear dark.

ResultsHISTOCHEMICAL STUDYMyocardium without obvious infarctionstained dark blue with the nitroblue tetra-zolium/phenazine methosulphate method(table 1). Unstained areas, virtually white, wereseen in two hearts with well established in-farction (fig 1). Intermediate staining withpatches of pink to red coloration was seen inthree hearts.

Intracellular deposition of C9 was detectedimmunohistologically in the areas of infarctionas defined by the enzymatic method, with

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Doran, Howie, Townend, Bonser

Table 2 Comparison of staining of myocardium with haematoxylin and eosin (H andE) with immunohistology for C9 in 50 hearts

25 hearts without25 hearts with suspectedl obvious myocardialconfirmed myocardial infarction infarction

H and E showed infarct; 16 0C9 positiveH and E showed infarct; 0 0C9 negativeH and E did not show 8 3infarction; C9 positiveH and E did not show 1 22infarction; C9 negative

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Figure 3 Immunohistological staining for C9 on a sectionof myocardium fr-om a 64 year old man five days after amyocardial infarct. Subendocardial myocardium is viableand unstained, with immunostaining at the edge of a wellestablished infarct.

greater intensity of staining at the edges ofinfarcts (fig 2). There was patchy staining withC9 in the hearts that had pink/red histo-chemical staining. Scattered single cells stainedwith C9 in two hearts that had dark bluehistochemical staining. C9 was not deposited inmuscle cells in one heart which had no clinical,conventional pathological or histochemicalevidence of infarction. C9 was detected in ar-terial walls in viable myocardium and was auseful internal positive control in all hearts.

STUDY OF CONVENTIONAL STAINING

Comparison of conventional staining with im-munohistology for C9 is presented in table 2.The eight hearts in the group of suspected orconfirmed myocardial infarction stained by C9but with no conventional evidence of infarctionwere all from patients with clinical evidence ofinfarction less than 24 hours old, whereas the16 identified by staining with haematoxylin andeosin and also stained with C9 had infarctsfrom 24 hours to 20 days old (fig 3). The single

Figure 4 Immunohistological staining for C9 on a sectionof myocardium from a 57 year old man who died ofperitonitis. Two myocardial cells contain C9 and appeardark.

heart in this group that seemed to be normalon staining with both haematoxylin and eosinand C9 was from an 81 year old man who was

thought to have had a myocardial infarct but onreview of clinical records had unstable anginawithout electrocardiographic evidence of in-farction. Three hearts in the control group wereC9 positive. These were from a 43 year oldman with septicaemic shock caused by an intra-abdominal abscess, a 57 year old man withperitonitis caused by a perforated gastric ulcer,and a 78 year old woman with Henoch-Schoenlein purpura. In these three cases a fewsingle myocardial cells scattered throughoutthe sections stained positively with C9 (fig 4).

DiscussionImmunohistological staining for C9 was betterthan the nitroblue tetrazolium/phenazine metho-sulphate method for detecting myocardial in-farction. The C9 method had the advantagethat formalin fixed, paraffin wax embeddedmaterial could be used. Unlike the technicaldifficulties of the histochemical method, therewas no need for special storage or treatment ofsamples from hearts. The histochemicalmethod was unreliable on an infarct under 24hours old, whereas the C9 method stainedthis infarct. The histochemical method is a

macroscopic test whereas the C9 method ismicroscopic and permitted much finer res-

olution, detecting staining of single cells. TheC9 method was so sensitive that it detectedscattered single myocardial cell necrosis in threeof the 25 hearts without other evidence ofmyocardial damage, but with an adequate clin-

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Detection of myocardial infarction

ical explanation for such damage. This findingwas similar to that in the 55 year old womanwith septicaemic shock in the histochemicalstudy (table 1). The C9 method was superiorto conventional staining for detecting infarctsless than 24 hours old. Hearts without evidenceof infarction were not stained by the C9method.

Activation of the complement system anddeposited C5b-9 complex is known to ac-cumulate in infarcted myocardium, both inhumans35 and experimental animals.67 Com-plement deposition in infarcted areas occursafter a few hours, two to four in the rat7 and fiveto six in the rabbit,6 except when reperfusionoccurs, when complement deposition is rapid.6This observation shows that circulating bloodis necessary for the activation and depositionof complement,8 and accords with our findingthat C9 deposition was most noticeable at theedges of well established infarcts.The immunohistological stain for C9 has

many potential uses such as in retrospectivestudies of hearts to detect unsuspected myo-

cardial infarction and in the study of diffusenon-regional necrosis of individual myocardialcells.We are grateful to the British Heart Foundation for a grant tosupport this work, and to Mrs M C Williams for technical help.

1 Howie AJ, Gregory J, Thompson RA, Adkins MA, Niblett AJ.Technical improvements in the immunoperoxidase study ofrenal biopsy specimens. J Clin Pathol 1990;43:257-9.

2 Engel AG, Biesecker G. Complement activation in musclefiber necrosis: demonstration of the membrane attack com-plex of complement in necrotic fibers. Ann Neurol 1982;12:289-96.

3 Schafer H, Mathey D, Hugo F, Bhakdi S. Deposition of theterminal C5b-9 complement complex in infarcted areas ofhuman myocardium. .7 Immunol 1986;137:1945-9.

4 Flint EJ. Phenazine methosulphate and the nitroblue tetra-zolium macroreaction for recent myocardial infarction. .7Clin Pathol 1984;37:477-8.

5 Hugo F, Hamdoch T, Mathey D, Schafer H, Bhakdi S.Quantitative measurement of SC5b-9 and C5b-9(m) ininfarcted areas of human myocardium. Clin Exp Immunol1990;81:132-6.

6 Mathey D, Schofer J, Schafer HJ, Hamdoch T, JoachimHC, Ritgen A, et al. Early accumulation of the terminalcomplement-complex in the ischaemic myocardium afterreperfusion. Eur Heart .7 1994;15:418-23.

7 Vakeva A, Morgan BP, Tikkanen F, Helin K, Laurila P, MeriS. Time course of complement activation and inhibitorexpression after ischaemic injury of rat myocardium. AmJ Pathol 1994;144:1357-68.

8 Kilgore KS, Friedrichs GS, Homeister JW, Lucchesi BR. Thecomplement system in myocardial ischaemia/reperfusioninjury. Cardiovasc Res 1994;28:437-44.

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