Forensic evidence in fatal milk-aspiration

We reported the case of a three-month-old female infant found deceased after being transported in a traditional African baby carrier, with an otherwise unremarkable clinical history. At autopsy, no significant gross abnormalities were found, except for a slight increase in the weight and consistency of the lungs and the detection of whitish, milky material in the lumen of the trachea, esophageal lumen and stomach. Histological examination of the lungs (i.e., hematoxylin and eosin staining) revealed bilateral, widespread, weakly eosinophilic, often brownish granular, amorphous material mixed with birefringent translucent globules within bronchial and bronchiolar lumens, as well as alveolar spaces. This was associated with intralveolar histiocytes displaying granular cytoplasm, occasional small lymphocytic aggregates, and focal endoalveolar hemorrhages. Material with the same characteristics was also detected in the stomach. These histological findings, with evidence of the same material in both stomach and lungs, suggested milk aspiration, likely resulting from regurgitation of gastric contents, as the cause of death. The gross and microscopic findings align with previous reports [8, 9, 12, 18] describing milk aspiration as a cause of acute respiratory failure due to mechanical airway obstruction and potential chemical irritation from gastric contents.

Post-mortem diagnostic challenges

Post-mortem diagnosis of fatal milk aspiration and its causal inference in determining the cause of death pose a significant challenge for the forensic pathologist. Indeed, conventional forensic examinations alone may yield negative or inconclusive results. At gross examination, fatal milk aspiration may show the presence of white milk-like liquid material in the nostrils, mouth, trachea, esophagus and stomach [8, 12]. Other non-specific findings may include hyperemia and oedema of the tracheal mucosa [12] and pleural petechiae [9]. The histopathological analysis often shows amorphous material, finely granular eosinophilic material or weakly basophilic particles in the alveoli and bronchioles [5, 8, 12]. This material may be associated with inflammatory cells [12], suggesting a response to the aspiration. However, the absence of significant inflammation might indicate a rapid or sudden death [8]. Clumps of bacteria may also be histologically detected [5]. The presence of lipid-laden alveolar macrophages may indicate the inhalation of lipid-containing substances, aligning with the presence of aspirated milk in suspected cases [9, 12]. In addition, distinguishing milk from proteinaceous edematous fluid, fibrin, and other airway debris becomes increasingly challenging over time due to bacterial and chemical modification [5]. In our case, the extensive distribution of the material throughout all lung fields classified the aspiration as severe [5]. Hence, additional post-mortem diagnostic tests were performed, supplementing standard histopathological analyses, to investigate the nature of the material found.

Milk type detection through immunohistochemical and ultrastructural analysis

The mother’s refusal to disclose the type of milk used to feed the infant posed an additional diagnostic challenge, requiring the use of immunohistochemical markers able to identify both feeding options, i.e. human breast milk and formula milk. Immunohistochemical analysis represents a reliable diagnostic tool in post-mortem tissues [19] to detect fatal milk aspiration and distinguishing between formula milk and human breast milk. Beta-lactoglobulin, a major whey protein found in cow’s milk, serves as a specific marker for formula milk since it is absent in human breast milk [20, 21]. Immunostaining for beta-lactoglobulin produces a distinct reaction, making it a reliable indicator of formula milk aspiration [9]. Conversely, alpha-lactalbumin, present in both human and cow’s milk, reacts with both types of milk proteins, leading to cross-reactivity and potential nonspecific background staining, thus limiting its use for differentiating between the two types of milk. In our case, immunohistochemical analysis provided crucial information about the nature of the aspirated material, revealing positive results for beta-lactoglobulin and inconclusive results for alpha-lactalbumin. The positivity for beta-lactoglobulin in both gastric and pulmonary samples confirmed the ingestion and subsequent aspiration of formula milk. The detection of translucent hyaline globules and fibrillary fragments, presumably of vegetable origin, suggested possible contamination or additives in the formula, such as milk thickeners. This finding raises concerns about the quality and safety of the formula used. To test the hypothesis that the birefringent hyaline globules represent additives for infant formula, acting as milk thickeners such as starch, we performed an electron microscopy study on lung tissue. In support of our hypothesis, these globules exhibited a reticular appearance at the ultrastructural level, characteristic of various organic compounds, including carbohydrate polysaccharides such as starch.

Distinguishing ante-mortem from post-mortem milk aspiration

The main diagnostic challenge was determining whether milk aspiration occurred ante-mortem or post-mortem. Microscopic detection of milk not only in the proximal airways but, most importantly, in the distal airspaces and across multiple examined lung fields, along with the presence of reactive macrophages, strongly suggested ante-mortem aspiration as the primary cause of death. A potential role of respiratory infection in the cause of death could not be excluded, as the post-mortem nasopharyngeal swab tested positive for rhinovirus/enterovirus. Notably, at 15 days of age, the infant was hospitalized for dyspnea and persistent cough. At that time, she was treated with oxygen and discharged with a clinical diagnosis of suspected bronchiolitis. It can be hypothesized that bronchiolitis may be related to an episode of gastric content aspiration secondary to gastroesophageal reflux. Furthermore, gastroesophageal reflux, central apnea, and laryngospasm are common in infants with respiratory infections, potentially exacerbating the risk of aspiration [22]. Focusing on the circumstances surrounding the death, it remains uncertain whether the bus experienced significant vibrations or shaking during the journey. However, it should be considered that physiological gastroesophageal reflux peaks around 2–3 months of age [23] due to the immaturity of the critical components of the anti-reflux barrier (e.g., lower esophageal sphincter and crural diaphragm) [24]. Thus, it can’t be ruled out that even minimal movements during the bus journey may have contributed to reflux-related complications, potentially acting as a predisposing factor for milk aspiration. Moreover, the use of the soft back carrier for infant transport raised additional concerns [15]. It is noteworthy that the US Consumer Product Safety Commission (CPSC) warns about suffocation risks for infants, especially those under 4 months, when using infant sling carriers [25, 26]. However, to the best of our knowledge, there is no robust scientific evidence directly linking soft carriers to asphyxiation in infants. Overall, our comprehensive forensic investigation, including additional non-routine examinations, allowed us to state that the infant’s death was caused by acute respiratory failure due to the aspiration of formula milk into the airways and alveolar spaces, following gastric content regurgitation. The death due to respiratory failure is supported by the extensive presence of material in lung samples, especially at the endoalveolar level. This evidence suggests that the infant was alive and breathing independently, rather than passive inhalation occurring. This evidence exonerated the mother from criminal liability, as the aspiration was determined to be an ante-mortem event caused by regurgitation rather than the result of improper feeding, external compression, or suffocation by the soft carrier or the adult’s body.