HIPPOKRATIA 2015, 19, 2: 164-171 ORIGINAL ARTICLE The importance of hemosiderin deposition in the infant brain: an autopsy study Türkmen İnanır N1, Eren F2, Akgöz S3, Eren B2, Çetin S4, Gündoğmuş UN5, Çomunoğlu N6, Çomunoğlu C7 1Forensic Medicine Department, Uludağ University Medical Faculty, Council of Forensic Medicine of Turkey, Bursa Morgue Department, Bursa 2Bursa Morgue Department, Council of Forensic Medicine of Turkey, Bursa 3Biostatistics Department, Çanakkale Onsekizmart University Medical Faculty, Çanakkale 4Kayseri Morgue Department, Council of Forensic Medicine of Turkey, Kayseri 5Forensic Medicine Institute, Istanbul University, Council of Forensic Medicine of Turkey, Istanbul 6Pathology Department, Istanbul University Cerrahpaşa Medical Faculty, Istanbul 7Pathology Department, Acıbadem University Medical Faculty, Istanbul Turkey Abstract Background/aim: Iron is an essential element involved in many metabolic processes. Presence and accumulation of iron in various body systems can result in different outcomes. Its accumulation in the central nervous system (CNS) cannot be detected routinely by application of hematoxylin-eosin staining. Detection of the presence of hemosiderin in the brain and cerebellum by application of Perls’ dye is of importance in cases of infant deaths. Material and Methods: In this study, brain and cerebellar specimens obtained from 52 eligible infants (aged 0-1 years) autopsied in our institute between the years 2010 and 2013, independent of the cause of death, were analyzed in order to detect possible presence of hemosiderin. Perls’ dye was used to detect histopathological staining intensity and distribu- tion of hemosiderin in the brain and cerebellum. Results: Cases did not differ significantly as for the patients’ age and gender (p =0.473), type of the culprit trauma (p =0.414), death/crime scene (p =0.587), and diagnosis groups (p =0.550). In this autopsy study blue colored hemosiderin granulations, stained with Perls’ dye were detected in the brain (n: 39, 75%), and cerebellum (n: 35, 67.3%). A weakly negative, but significant correlation was detected between the postmortem interval and intensity values of cerebel- lar hemosiderin (Spearman’s correlation coefficient: -0.381, p =0.024). A statistically significant difference was found between the distribution scores of cerebral hemosiderin in cases with and without trauma history (p =0.03). Median cerebral hemosiderin distribution scores were 2.5 and 2, respectively. Conclusions: The detection of a correlation between the presence of cerebral and cerebellar hemosiderin, and post- mortem interval in the age group of 0-1 years, should be interpreted as an important finding in the analysis of cerebral iron. The presence of hemosiderin in the CNS may be a significant finding in the elucidation of infant deaths and this procedure should be carried out on a routine basis.Hippokratia 2015; 19 (2):164-171. Keywords: Brain, hemosiderin, infant, autopsy Corresponding authors: Bülent Eren, MD, Associate Professor, Council of Forensic Medicine of Turkey, Bursa Morgue Department, 16010, Bursa, Turkey, tel: +902242220347, fax: +0902242255170, e-mail: [email protected] Ü.Naci Gündoğmuş, MD, Forensic Medicine Specialist, Associate Professor, Istanbul University Forensic Medicine Institute, Fatih/Istanbul, 34303, Turkey, tel: +902124143000, fax: +902125880011, e-mail: [email protected] Introduction stress and vice versa. Also, it has been stated that iron load Although processes related to iron metabolism and its is important in the etiopathogenesis of neurovegetative accumulation in the body have been the subject of dif- pathologies6. Sudden fetal death and neonatal death can ferent investigations, we have limited knowledge about occur respectively in fetuses and neonates with increased these processes, especially in children1-5. The number of cerebral tissue hemosiderin levels who were previously available publications about the accumulation of iron in exposed to oxidative stress3. Iron is an essential microele- the central nervous system is still limited3,4,6. It has been ment for cerebral metabolism; iron also plays critically indicated that cerebral iron load increases with oxidative important roles in many metabolic processes including HIPPOKRATIA 2015, 19, 2 165 numerous enzymatic activities. Besides, it acts as a co- ing throughout the section score). The staining intensity factor for cytochromes required in the energy production was recorded using a semiquantitative scoring system (0: and is involved in neurotransmitter synthesis and many no staining, 1: weak, small punctate staining, 2: accumu- distinct physiological processes1,4,7,8. Excessive amounts lations with greater staining intensity, 3: strong, dark ob- of iron are encountered in cerebral, and cerebellar tissues served staining, 4: very strong, the darkest observed stain- in Parkinson’s disease, hemosiderosis, various neurode- ing). Postmortem interval periods were determined for all generative hereditary diseases, and Friedreich ataxia3,9,10. cases. The results obtained were subsequently analyzed Accumulation of iron in various cerebral structures is regarding autopsy findings, sociodemographic charac- generally detected with the aging process, including spe- teristics (age, gender), and parameters such as causes of cialized cerebral regions like basal ganglia1,6,9,11,12. death, postmortem interval, signs of trauma, and toxico- Owing to the critical role of iron in metabolism, the logical findings. In statistical analyses, diagnoses were inability to be detected on histopathological examina- grouped as ‘absence’ or ‘presence’ (of hemosiderin etc) tion of hematoxylin-eosin (H&E) stained slides, and the and evaluted accordingly. potential significance of cerebral/cerebellar hemosiderin detection in the clarification of infant deaths we design Statistical analysis this study. We aimed to detect histochemically the accu- Statistical analysis was performed using IBM SPSS mulation of hemosiderin, in cerebral and cerebellar tis- (Armonk, NY, USA) version 19.0 for Windows. Con- sues from autopsied infants (0-1 year of age), using Perls’ tinuous variables were expressed as mean and standard stain and also to discuss the results in comparison with deviation (SD) or median and range, while categorical those of other studies. variables as frequencies and percentages. For continu- ous variables, after controlling for the assumption of Material and methods normality, non-parametric tests as Mann-Whitney U and In this study were included 52 infants (0-1 year of Kruskal-Wallis, were used in comparisons. For compari- age) who were among the 5,334 cases been autopsied sons of the distribution of categorical variables the Pear- from 2010 until 2013 in Bursa morgue department of the son’s chi-square and Fisher’s Exact test were employed. council of forensic medicine of Turkey, for whom we For correlations between measurements the Kendall’s tau had complete data regarding clinical status, death/crime b and Spearman’s correlation coefficients were calculat- scene, and postmortem interval. Cerebral and cerebellar ed. p <0.05 was considered statistically significant. tissue specimens of these cases amenable to staining with Perls’ dye were analyzed within 24 hours after their death. Results Without taking into consideration the causes of death, all In total 52 cases were examined. At autopsy, no gross cerebral and cerebellar tissue specimens obtained were congenital anomaly was detected on external examina- immersed for at least five days in 10% formaldehyde so- tion. Toxicological examinations could not detect the lution, so as to analyze the blocks. After completion of presence of any illicit substance in the blood, urine sam- the follow-up procedures, paraffin blocks were cut into ples, and visceral organ specimens that were analyzed 5-micron sections and stained with H&E before their mi- during systemic analyses. croscopic examination. Descriptive data of all cases are presented in Table To detect hemosiderin deposition, histochemical 1. Age of death in these cases ranged between one and staining with Prussian blue was used. In this technique, 350 days (mean: 79.56, SD: 84.98, median: 60 days). No Perls’ dye was applied to produce ferric ferrocyanate and significant difference was detected between age and gen- also fixate nonheme ferric ions (Fe3+) which necessitated der (p =0.473), history of trauma (p =0.414), death/crime the combined use of 2% hydrochloric acid (100 ml) and scene (p =0.587), and diagnoses groups (p =0.550). 2% potassium ferrocyanide solutions. Independently from Blue-coloured hemosiderin granulations that dis- the cause of their death, we utilised the method previous- closed nonheme iron (Fe3+), after staining with Prussian ly used to detect hemosiderin deposits in the lungs, liver, blue, were detected in the brain and cerebellum in 75% and spleen in pediatric cases2,5, in order to determine the (n: 39) and 67.3% (n: 35) of cases, respectively. Median hemosiderin distribution scores of the brain and cerebel- hemosiderin distribution score in the brain specimens lum. However, intensity scores were evaluated according (Figures 1 a,c,d) of male and female infants was two to the intensity of staining. Specimens of the brain and points (mean ± SD: 1.59 ± 1.25 and 1.80 ± 1.26, respec- cerebellum were examined for hemosiderin distribution tively). Median intensity score (Figures 2 a,c,d) was one and intensity scores under magnification (x 10) using an point in male