Sudden infant death syndrome (SIDS) is the sudden and unexpected death in a child under the age of one year, which remains unexplained after a scene investigation, complete autopsy, and review of the history/medical records. SIDS is the most common cause of death in a child between the ages of one month and one year. Although we are aware of certain risk factors associated with SIDS, the etiology remains unknown. When one considers SIDS as a cause of death, other conditions such as metabolic diseases, toxicologic deaths, and electrolyte disturbances must be ruled out. Often these victims have a negative autopsy or subtle, nonspecific findings. A metabolic screening currently used on pediatric autopsies is the NeoGen screen (NeoGen Screening, Pittsburgh, PA 412-341-8658). The screening methods used include tandem mass spectrometry, immunoassays, and confirmatory molecular analyses. Fresh blood is dropped onto a filter paper card provided by NeoGen. From the dried blood spots, the screen is performed. Numerous metabolic diseases can be screened for including Medium Chain Acyl CoA Dehydrogenase deficiency (MCAD). Metabolic testing is also important in determining diseases that mimic child abuse such as Shaken Baby Syndrome (SBS). One of these metabolic diseases is glutaric aciduria type I, an autosomal recessive aminoacidopathy . This disease can result in retinal and subdural hemorrhages similar to those of the SBS and thus be mistaken for inflicted trauma. By performing metabolic screening, the pathologist not only clears the caretakers of homicide but also diagnoses a hereditary disease, information very important to the family.

Toxicology should be performed on all pediatric victims, especially those of unexplained deaths. Accidental pediatric poisonings have decreased over the recent years with updated safety standards in packaging and an increased public awareness. However, pediatric poisonings do occur with a bimodal age distribution, half of deaths occurring in the toddler years and the second peak in the adolescent years. Both accidental and intentional poisonings must be considered and excluded before assigning the cause and manner of death in the pediatric victim. Toxicologic deaths usually present as unexpected deaths with no significant findings at autopsy. Urine can be used for a drug screen. Peripheral blood should be drawn and placed into a gray top tube containing sodium citrate or sodium fluoride (preservative). Drugs including alcohol have been detected in both accidental and homicidal pediatric deaths; suicidal intentional ingestions are seen in the adolescent age group. The vitreous may also be used for alcohol detection, levels remaining constant and correlating well with premortem blood levels.

Vitreous electrolytes can be analyzed to detect disturbances especially related to diabetes mellitus/diabetic ketoacidosis (DKA), renal failure, and dehydration. Using a needle and syringe, vitreous (2-3 ml) is slowly drawn from the globe and placed into a sterile red top tube. Postmortem levels of sodium and chloride and urea nitrogen reflect premortem values. Potassium rises after death with the increasing postmortem interval. Due to gycolysis, vitreous glucose normally falls after death in a non-diabetic, so a low glucose is insignificant. However, the detection of an elevated glucose (usually over 200 mg/dL) and ketones in the vitreous is important when considering diabetes mellitus and DKA. Hypochloremia may be seen in cases of vomiting. An elevated urea nitrogen without an increase in sodium and chloride may be seen in uremia. Dehydration can be reflected by an increase in sodium, chloride, and urea nitrogen and is important in cases of neglect. However, dehydration is also seen in viral gastroenteritis, such as Rotavirus. Rotavirus gastroenteritis can be very dangerous in children. Four methods have been studied over the past several years for detecting rotavirus in fecal samples: latex agglutination, electron microscopy, immunofluorescence, and ELISA. Simple, rapid, and cost-effective manual tests have recently been perfected. Two studies, Dot-ELISA and Rapid Latex, have proven sensitive and specific in detecting rotaviral gastroenteritis. Not only can we detect dehydration via vitreous electrolytes, but we can define the viral etiology.

Immunology techniques are also at the forefront via immunohistochemistry and its application in cases of alleged non-accidental head trauma such as SBS. The latest in histopathology uses the beta-amyloid precursor protein (BAPP) to detect diffuse axonal injury in brain (white matter) and optic nerve sections. The acceleration-deceleration action of the shaking stretches and “shears” the axons, resulting in damage to the cytoskeleton and disruption of the axoplasm and axonal transport. Axonal retraction balls, or swellings, result as certain proteins including BAPP accumulate in the axon at or near the site of injury. After 12 to 24 hours, these swellings can be seen with routine hematoxylin and eosin staining and silver impregnation techniques but are often difficult to recognize. Immunohistochemistry allows for easier and earlier detection of the axonal injury as early as 2 hours after injury. By applying the BAPP antibody to these tissue sections, axonal injury can be detected under light microscopy, supporting the diagnosis diffuse axonal injury secondary to head trauma and a non-natural manner of death.

No discussion on current studies in pediatric forensic pathology would be complete without including molecular pathology and cytogenetics. Whether diagnosing fatal complications of chromosomal abnormalities or confirming sexual assault, DNA and chromosomes are on the cutting edge of technology and legal evidence. As previously mentioned, NeoGen metabolic screening applies various methods to detect diseases in postmortem blood samples. The same filter card can be analyzed for cystic fibrosis mutations as well as additional molecular diagnostic studies. Due to the severe malabsorption that may occur with cystic fibrosis, children may present with starvation and malnutrition. These children are also susceptible to dehydration. A positive CF mutation, such as the common cystic fibrosis mutation Delta F508, could explain such physical findings in a child and rule out neglect. DNA analysis has also recently been applied to the diagnosis of cardiac abnormalities, in particular the prolonged QT syndrome (PQTS). The disease PQTS is highly lethal if untreated and often results in sudden death in children and teenagers. Four genes responsible for PQTS have been identified on chromosomes 3,7,11,and 21. These genes all encode for an ion channel and thus ionic control of cardiac rhythm. By using DNA technology, we can study penetrance (the probability for an individual with an affected genotype to manifest the disease clinically) and family pedigrees. We can also properly assign a cause of death to these children who would have previously been classified as SIDS or undetermined.

Cytogenetics with karyotyping is used to diagnose chromosomal abnormalities in children. The specimens of choice are skin and Achilles tendon; kidney, liver, spleen, and bone marrow may also be sampled. The specimen should be collected within 24 hours after death, best if within 6 hours, and transported in sterile Hank’s solution, which is either at room temperature or chilled. Cytogenetic studies are especially important in sudden perinatal deaths, including stillbirths, and infant deaths when an abnormality is clinically subtle or not yet manifested by outward signs and symptoms. It has also been discovered that many children with chromosomal abnormalities are more prone to electrolyte disturbances and dehydration mimicking physical neglect. Thus, cytogenetic studies aid in alleviating suspicions and in adequately diagnosing the underlying disease resulting in death. Another method used in cytogenetics is fluorescence in situ hybridization, or FISH. Recently FISH has been applied to forensic pathology in cases of sexual assault. FISH uses non-radioactive fluorescent probes directed at a specific chromosome or chromosomal region of interphase cells. When analyzing cervicovaginal smears from sexual assault victims, these sensitive and specific probes can be directed at the X and Y chromosomes to allow for identification of male and female cells on the smear. Using a fluorescent microscope, the cells on the smear are easily identified as male or female. The XY cells originate from the penis, hand, or mouth of the male assailant and can support an allegation of sexual contact.

The pediatric forensic autopsy has benefited greatly from the recent advances in ancillary studies of the pathology laboratories. From improved chemical and toxicological analyses to the arena of DNA and cytogenetics, these new advances are changing the quality and utility of the autopsy. Although not available at or applicable to all institutions and cases respectfully, the forensic community needs to be aware of available ancillary tests and their applications. By continuing to research and perfect such ancillary laboratory procedures, we will eventually be able to answer more of those once impossible questions related to the pediatric forensic autopsy.

Suggested References

Gleckman AM, Evans RJ, Bell MD, Smith TW. Optic nerve damage in shaken baby syndrome. Arch Pathol Lab Med 2000;124:251-256.

Gleckman AM, Bell MD, Evans RJ, Smith TW. Diffuse axonal injury in infants with nonaccidental craniocerebral trauma. Arch Pathol Lab Med 1999;123:146-151.

Coe JI. Postmortem chemistry update: Emphasis on forensic application. Am J Forensic Med Pathol 1993;14(2):91-117.

Collins KA, Rao PN, Hayworth-Hodge R, Schnell S, Tap MP, Lanyz PE, Geisinger KR, Pettenati MJ. Identification of sperm and non-sperm male cells in cervicovaginal smears using fluorescence in situ hybridization: Applications in alleged sexual assault cases. J Forensic Sci 1994;39(6):1347-1355.

Schwartz PJ. Long QT Syndrome. Cardiology Special Edition: Pacing and Electrophysiology. 1999, p. 43-47.

Kohno H, Akihara S, Nishio O, Ushijima H. Development of a simple and rapid latex test for rotavirus in stool samples. Pediatr Int 2000;42(4):395-400.

Anand T, Raju TA, Vishnu C, Rao LV, Sharma G. Development of Dot-ELISA for the detection of human rotavirus antigen and comparison with RNA-PAGE. Lett Appl Microbiol 2001;32(3):176-180.