Abstract
The analysis of tissues and cells first obtained at forensic autopsy can present particular technical problems. While all established conventional histological staining methods are used, the immunohistochemical visualization of defined antigens has also made progress in forensic histopathology. Tissue that has frequently already undergone autolytic changes or putrefaction can make both conventional and immunohistochemical staining challenging, if not impossible. It is precisely for this reason that the mastery of staining techniques is essential. In the field of immunohistochemistry, the choice of fixative and the duration of fixation play a role, as does the option to pretreat tissues for antigen demasking, which often involves varying incubation times with primary and secondary antibodies. Other histopathological methods are occasionally used, such as in situ hybridization or apoptotic cell detection using the TUNEL technique. The actual practice of microscopy evaluation requires knowledge of possible distortions and artifacts but also depends on selecting the correct stain or the primary antibodies that are appropriate for the immunohistochemical question at hand. Experience in microscopy can significantly reduce problems such as interobserver variability. It is also important not to underestimate the importance of converting microscopic findings into a written expert appraisal. The qualification and microscopic quantification of defined cell types raises additional issues that, in many cases, can only be answered when one also has good knowledge of the relevant literature and its critical review. Highly specialized microscopy investigations, such as confocal laser scanning microscopy and electron microscopy, are used more rarely. However, histopathological findings must always be classified within the overall context of a “case,” taking other findings and information into consideration.
References
- Ananian V, Tozzo P, Ponzano E, Nitti D, Rodriguez D, Caenazzo L (2010) Tumoural specimens for forensic purposes: comparison of genetic alterations in frozen and formalin-fixed paraffin-embedded tissues. Int J Legal Med 125(3):327–332CrossRefPubMedGoogle Scholar
- Bankfalvi A, Navabi H, Bier B, Böcker W, Jasani B, Schmid W (1994a) Wet autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry. J Pathol 174:223–228CrossRefPubMedGoogle Scholar
- Bankfalvi A, Riehemann K, Öfner D, Checci R, Morgan JM, Piffko J, Böcker W, Jasani B, Schmid KW (1994b) Feuchtes Autoklavieren. Pathologe 15:345–349CrossRefPubMedGoogle Scholar
- Ben-Ezra J, Johnson DA, Rossi J (1991) Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction. J Histochem Cytochem 39:351–354CrossRefPubMedGoogle Scholar
- Biro C, Kovac P, Palkovic M, El-Hassoun O, Caplovicova M, Novotny J, Jakubovsky J (2010) Potentialities of scanning electron microscopy and EDX analysis in bullet wounds. Rom J Leg Med 18:225–230CrossRefGoogle Scholar
- Bratzke H, Schröter A (1995) Immunhistochemie in der Rechtsmedizin. Hänsel-Hohenhausen, EgelsbachGoogle Scholar
- Brinkmann B, Fechner G, Püschel K (1984) Identification of mechanical asphyxiation in cases of attempted masking of the homicide. Forensic Sci Int 26:235–245CrossRefPubMedGoogle Scholar
- Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol 8:515–520PubMedGoogle Scholar
- Cardinetti B, Ciampini C, D’Onofrio C, Orlando G, Gravina L, Ferrari F, Di Tullio D, Torresi L (2004) X-ray mapping technique: a preliminary study in discriminating gunshot residue particles from aggregates of environmental occupational origin. Forensic Sci Int 143:1–19CrossRefPubMedGoogle Scholar
- Chiquet-Ehrisman R, Chiquet M (2003) Tenascins: regulation and putative functions during pathological stress. J Pathol 200:488–499CrossRefGoogle Scholar
- Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, McDonald S, Pulford AF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immuno complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP-complex). J Histochem Cytochem 32:219–229CrossRefPubMedGoogle Scholar
- Cuevas EC, Bateman AC, Wilkins BS, Johnson PA, Williams JH, Lee AHS, Jones DB, Wright DH (1994) Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. J Clin Pathol 47:448–452CrossRefPubMedPubMedCentralGoogle Scholar
- Dettmeyer R (2016) Postmortem diagnostics of a (post-)viral myocarditis in putative sudden infant death syndrome. Case report for avoidance of errors in the diagnostics. Rechtsmedizin 26:514–519CrossRefGoogle Scholar
- Dettmeyer R, Baasner A, Schlamann M, Padosch SA, Haag C, Kandolf R, Madea B (2004a) Role of virus-induced myocardial affections in sudden infant death syndrome (SIDS): a prospective post-mortem study. Pediatr Res 55:947–952CrossRefPubMedGoogle Scholar
- Dettmeyer R, Baasner A, Schlamann M, Padosch SA, Haag C, Kandolf R, Madea B (2004b) Role of virus-induced myocardial affections in sudden infant death syndrome: a prospective postmortem study. Pediatr Res 55:1–5CrossRefGoogle Scholar
- Dettmeyer R, Stiel M, Madea B (2006) Heat shock protein 60 (cHSP60) as a marker for chronic infection with Chlamydia pneumoniae in atherosclerosis—investigation of atherosclerotic coronary arteries by immunocytochemistry. Forensic Sci Med Pathol 2:173–178PubMedGoogle Scholar
- Dettmeyer R, Sperhake JP, Müller J, Madea B (2007) Cytomegalovirus-induced pneumonia and myocarditis in 3 cases of suspected sudden infant death syndrome (SIDS): diagnosis by immunohistochemical techniques, in-situ-hybridisation and molecular pathologic investigations. Forensic Sci Int 174:229–233CrossRefPubMedGoogle Scholar
- Dettmeyer R, Baasner A, Haag C, Bruch S, Schlamann M (2009a) Immunohistochemical and molecular pathological diagnosis of myocarditis in cases of suspected sudden infant death syndrome (SIDS)—a multicenter study. Legal Med 11:S124–S127CrossRefPubMedGoogle Scholar
- Dettmeyer R, Bruch S, Haag C (2009b) Letter to the editor on “myocardial inflammation, cellular death, and viral detection in sudden infant death caused by SIDS, suffocation, or myocarditis”. Pediatr Res 66:714CrossRefPubMedGoogle Scholar
- Dettmeyer R, Friedrich K, Schmidt P, Madea B (2009c) Heroin-associated myocardial damages—conventional and immunohistochemical investigations. Forensic Sci Int 187:42–46CrossRefPubMedGoogle Scholar
- Dokhan DB, Kovatich AJ, Miettinen M (1993) Nonenzymatic antigen retrieval in immunohistochemistry—comparison between different antigen retrieval modalities and proteolytic digestion. Appl Immunohistochem 1:149–155Google Scholar
- Dreßler J, Bachmann L, Koch R, Müller E (1998) The detection of P-selectin in paraffin embedded sections by wet autoclave technique. J Cell Pathol 3:139–143Google Scholar
- Dubrovin IA, Dubrovina IA (2003) The influence of a shot distance on a profile of a wound canal in flat bones. Sud Med Ekspert 46:11–13PubMedGoogle Scholar
- Edston E, Gröntoft L, Johnsson J (2002) TUNEL: a useful screening method in sudden cardiac death. Int J Legal Med 116:22–26CrossRefPubMedGoogle Scholar
- Erel O, Gun B, Tataroğlu C, Dirlik M (2014) Evaluation of apoptosis in myocardial injury in cases of medicolegal death. Rom J Leg Med 22:267–274CrossRefGoogle Scholar
- Fechner G, Petkovits T, Brinkmann B (1990) Ultrastructural pathology of mechanical skeletal muscle damage. Z Rechtsmed 103:291–299CrossRefPubMedGoogle Scholar
- Feiden W (1995) Einführung in die Immunhistochemie. In: Bratzke H, Schröter A (eds) Immunhistochemie in der Rechtsmedizin. Hänsel-Hohenhausen, Egelsbach, pp 7–13Google Scholar
- Fracasso T, Heinrich M, Hohoff C, Brinkmann B, Pfeiffer H (2009) Ultrasound-accelerated formalin fixation improves the preservation of nucleic acids extraction in histological sections. Int J Legal Med 123:521–525CrossRefPubMedGoogle Scholar
- Frick R (1981) Stenosierende Koronarsklerose und Färbung des Myokards nach Lie et al. 1971 (Fuchsinorrhagie). Pathologe 2:246–254Google Scholar
- Gaaloul I, Riabi S, Evans M, Hunter T, Huber S, Aouni M (2016) Postmortem diagnosis of infectious heart diseases: a mystifying cause of sudden infant death. Forensic Sci Int 262:166–172CrossRefPubMedPubMedCentralGoogle Scholar
- Gavrieli Y et al (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501CrossRefPubMedGoogle Scholar
- Gown AM, de Wever N, Battifora H (1993) Microwave-based antigenic unmasking—a revolutionary new technique for routine immunohistochemistry. Appl Immunohistochem 1:256–266Google Scholar
- Grasl-Kaup B et al (1995) In situ detection of fragmented DNA (TUNEL-assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21:1465–1468CrossRefGoogle Scholar
- Hasday JD, Sing IS (2000) Fever and the heat shock response: distinct, partially overlapping processes. Cell Stress Chaperones 5:471–480CrossRefPubMedPubMedCentralGoogle Scholar
- Hausmann R, Bock H, Biermann T, Betz P (2004) Influence of lung fixation technique on the state of alveolar expansion—a histomorphometrical study. Legal Med 6:61–65CrossRefPubMedGoogle Scholar
- Havel J (2003) Energy-dispersive X-ray fluorescence spectrometry—a forensic chemistry method for determination of shooting distance. Soud Lek 48:57–60PubMedGoogle Scholar
- Havel J, Zelenka K (2003) Energy dispersive x-ray fluorescence spectrometry—a forensic chemistry method for detection of bullet metal residue in gunshot wounds. Soud Lek 48:22–27PubMedGoogle Scholar
- Holgate CS, Jackson P, Pollard K, Lunny D, Bird CC (1986) Effect of fixation on T and B lymphocyte surface membrane antigen demonstration in paraffin processed tissue. J Pathol 149:293–300CrossRefPubMedGoogle Scholar
- Hsu SM, Raine L, Fanger H (1981a) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577–580CrossRefPubMedPubMedCentralGoogle Scholar
- Hsu SM, Raine L, Fanger H (1981b) A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin-complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75:734–739CrossRefPubMedGoogle Scholar
- Imam SA (1995) Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissue for immunostaining. Anticancer Res 15:1153–1158PubMedGoogle Scholar
- Javid B et al (2007) Structure and function: heat shock proteins and adaptive immunity. J Immunol 179:2035–2040CrossRefPubMedGoogle Scholar
- Kage S, Kudo K, Kaizoji A, Ryumoto J, Ikeda H, Ikeda N (2001) A simple method for detection of gunshot residue particles from hands, hair, face, and clothing using scanning electron microscopy/wavelength dispersive X-ray (SEM/WDX). J Forensic Sci 46:830–834CrossRefPubMedGoogle Scholar
- Kardasewitsch B (1952) Eine Methode zur Beseitigung der Formalinsedimente (Paraform) aus mikroskopischen Praeparaten. Z Wiss Mikrosk 42:322–324Google Scholar
- Kinoshita H, Nishiguchi M, Ouchi H, Minami T, Kubota A, Utsumi T, Sakamoto N, Kashiwagi N, Shinomiya K, Tsuboi H, Hishida S (2004) The application of a variable-pressure scanning electron microscope with energy dispersive X-ray microanalyser to the diagnosis of electrocution: a case report. Legal Med 6:55–60CrossRefPubMedGoogle Scholar
- Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibodies of predefined specificity. Nature 256:495–497CrossRefPubMedGoogle Scholar
- Kok LP, Boon ME (1992) Microwave cookbook for microscopists. Art and science of visualization, 3rd edn. Coulomb Press, LeidenGoogle Scholar
- Krous HF, Ferandos C, Masoumi H, Arnold J, Haas EA, Stanley C, Crossfeld PD (2009) Myocardial inflammation, cellular death, and viral detection in sudden infant death, caused by SIDS, suffocation or myocarditis. Pediatr Res 66:17–21CrossRefPubMedGoogle Scholar
- Kuhn H, Krugmann J (1995) Einfluß von Formalinfixierung und Fixationsdauer auf die DNA-Amplifizierung von verschiedenen Paraffin-eingebetteten Geweben. Verh Dtsch Ges Pathol 79:600Google Scholar
- Kwok S, Higuchi R (1989) Avoiding false positives with PCR. Nature 339:237–238CrossRefPubMedPubMedCentralGoogle Scholar
- Labat-Moleur F et al (1998) TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement. J Histochem Cytochem 46:327–334CrossRefPubMedGoogle Scholar
- Leong ASY (1996) Microwaves in diagnostic immunohistochemistry. Eur J Morphol 34:381–383CrossRefPubMedGoogle Scholar
- Leong ASY, Milios J (1993) An assessment of the efficacy of the microwave antigen-retrieval procedure on a range of tissue antigens. Appl Immunohistochem 1:267–274Google Scholar
- Lie JT, Holley KE, Kampa WR, Titus LL (1971) New histochemical method for morphologic diagnosis of early stages of myocardial ischemia. Mayo Clin Proc 46:319–327PubMedGoogle Scholar
- Login GR, Schnitt SJ, Dvorak AM (1987) Methods in laboratory investigation—rapid microwave fixation of human tissues for light microscopic immunoperoxidase identification of diagnostically useful antigens. Lab Investig 57:585–591PubMedGoogle Scholar
- Lozinski GM, Davis GG, Krous HF, Billmann GF, Shimizu H, Burns JC (1994) Adenovirus myocarditis: retrospective diagnosis by gene amplification from formalin-fixed, paraffin-embedded tissues. Hum Pathol 25:831–834CrossRefPubMedGoogle Scholar
- Lucitti JL, Dickinson ME (2006) Moving toward the light: using new technology to answer old questions. Pediatr Res 60:1–5CrossRefPubMedPubMedCentralGoogle Scholar
- Mason JT, O’Leary TJ (1991) Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 39:225–229CrossRefPubMedGoogle Scholar
- Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orscheschek K, Moubayed P, Feller AC (1995a) ImmunoMax—a maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. Lab Investig 73:149–156PubMedGoogle Scholar
- Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orscheschek K, Moubayed P, Feller AC (1995b) Methods in laboratory investigation ImmunoMax. Lab Investig 73:149–156PubMedGoogle Scholar
- Meyer R, Niedobitek F, Wenzelides K (1996) Erfahrungen mit der Formalinersatzlösung NoTox. Pathologe 17:130–132CrossRefPubMedGoogle Scholar
- Miething F, Hering S, Hanschke B, Dressler J (2006) Effect of fixation to the degradation of nuclear and mitochondrial DNA in different tissues. J Histochem Cytochem 54:371–374CrossRefPubMedGoogle Scholar
- Moloney WC, McPherson K, Fliegelman L (1960) Esterase activity in leucocytes demonstrated by the napthol-ASD-chloracetate substrate. J Histochem Cytochem 8:200CrossRefPubMedGoogle Scholar
- Nakatone M, Matoba R, Ogura Y, Tun Z, Iwasa M, Maeno Y, Koyama H, Nakamura Y, Inoue H (2002) Detection of cardiomyocyte apoptosis in forensic autopsy cases. Int J Legal Med 116:17–21CrossRefGoogle Scholar
- Nikolaou P, Papoutsis I, Dona A, Spiliopoulou C, Athanaselis S (2013) Toxicological analysis of formalin-fixed or embalmed tissues: a review. Forensic Sci Int 233:312–319CrossRefPubMedGoogle Scholar
- Noll S, Schaub-Kuhnen S (2000) In: Höfler H, Müller KM (eds) Praxis der Immunhistochemie. Urban and Fischer, MünchenGoogle Scholar
- Nwariaku FE, Mileski WJ, Lightfoot E, Sikes PJ, Lipsky PE (1995) Alterations in leukocyte adhesion molecule expression after burn injury. J Trauma 39:285–288CrossRefPubMedGoogle Scholar
- Ortmann C, Brinkmann B (1997) The expression of P-selectin in inflammatory and non-inflammatory lung tissue. Int J Legal Med 110:15–158CrossRefGoogle Scholar
- Paulin D, Li Z (2004) Desmin: a major intermediate filament protein essential for the structural integrity and function of muscle. Exp Cell Res 301:1–7CrossRefPubMedGoogle Scholar
- Pelstring RJ, Allred DC, Esther RJ, Lampkin SR, Banks PM (1991) Differential antigen preservation during tissue autolysis. Hum Pathol 22:237–241CrossRefPubMedGoogle Scholar
- Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, Ascani S, Santini D, Piccaluga PP, Leone O, Damiani S, Ercolessi C, Sandri F, Pieri F, Leoncini L, Falini B (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183:116–123CrossRefPubMedGoogle Scholar
- Sabattini E, Bisgard K, Ascani S, Poggi S, Piccioli M, Ceccarelli C, Pieri F, Fraternali-Orcioni G, Pileri SA (1998) The ENVision system: a new immunohistochemical method for diagnosis and research: critical comparison with the APAAP, ChemMate, CSA, LABC and SABC techniques. J Clin Pathol 51:506–511CrossRefPubMedPubMedCentralGoogle Scholar
- Schiller PI, Puchta U, Ogilvie AJL, Graf A, Kind P, Sander CA (1998) In-situ-PCR und PCR-in-situ-Hybridisierung am Paraffingewebe. Pathologe 19:313–317CrossRefPubMedGoogle Scholar
- Seidl S, Burmeister R, Hausmann R, Betz P, Lederer T (2005) Contact-free isolation of sperm and epithelial cells by laser microdissection and pressure catapulting. Forensic Sci Med Pathol 1:153–157CrossRefPubMedGoogle Scholar
- Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748CrossRefPubMedGoogle Scholar
- Shi SR, Chaiwun B, Young L, Imam A, Cote RJ, Taylor CR (1994) Antigen retrieval using pH 3.5 glycine-HCI buffer or urea solution for immunohistochemical localization of Ki-67. Biotech Histochem 69:213–215CrossRefPubMedGoogle Scholar
- Shi SR, Imam A, Young L, Cote RJ, Taylor CR (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43:193–201CrossRefPubMedGoogle Scholar
- Shi SR, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem 45:327–343CrossRefPubMedGoogle Scholar
- Taylor CR, Shi SR, Cote RJ (1996) Antigen retrieval for immunohistochemistry. Status and need for greater standardization. Appl Immunohistochem 4:144–166Google Scholar
- Torre C, Mattutino G, Vasino V, Robino C (2002) Brake linings: a source of non-GSR particles containing lead, barium, and antimony. J Forensic Sci 47:494–504PubMedGoogle Scholar
- Trani N, Bonetti LR, Gualandri G, Barbolini G (2008) Immediate anaphylactic death following antibiotics injection: splenic eosinophilia easily revealed by pagoda red stain. Forensic Sci Int 181:21–25CrossRefPubMedGoogle Scholar
- Turillazzi E, Karch SB, Neri M, Pomara C, Riezzo I, Fineschi V (2007) Confocal laser scanning microscopy. Using new technology to answer old questions in forensic investigations. Int J Legal Med 122:173–177CrossRefPubMedGoogle Scholar
- Uno AT, Morita S, Furukawa S, Nishi K, Hitosugi M (2016) Determination of both fetus’and mother’s blood type from an autopsy case immersed in formalin for over 50 years. J Forensic Sci 61:1131–1134CrossRefPubMedGoogle Scholar
- Vandewoestyne M, van Hoofstat D, van Nieuwerburgh F, Deforce D (2009) Automatic detection of spermatozoa for laser capture microdissection. Int J Legal Med 123:169–175CrossRefPubMedGoogle Scholar
- von Wasielewski R, Werner M, Nolte M, Wilkens L, Georgii A (1994) Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies. Histochemistry 102:165–172CrossRefGoogle Scholar
- Werner M, Wasieleweski VR, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253–260CrossRefPubMedGoogle Scholar
- Wiegand P, Domhöver J, Brinkmann B (1996) DNA-degradation in formalin-fixiertem Gewebe. Pathologe 17:451–454CrossRefPubMedGoogle Scholar
- Williamson SLH, Steward M, Milton I, Parr A, Piggott NH, Krajewski AS, Angus B, Horne CW (1998) Technical advance—new monoclonal antibodies to the T cell antigens CD4 and CD8—production and characterization in formalin-fixed paraffin-embedded tissue. Am J Pathol 152:1421–1426PubMedPubMedCentralGoogle Scholar
- Wyss A, Lasczkowski G (2008) Vitality and age of conjunctival petechiae: the expression of P-selectin. Forensic Sci Int 178:30–33CrossRefPubMedGoogle Scholar