A Standardized Four-Domain Operational Workflow for Postmortem Research in Initially Unexplained Infant Deaths

1. Introduction

Sudden unexpected infant death (SUID) is an investigative presentation rather than a single etiologic category. Sudden infant death syndrome (SIDS) is a classification applied when death remains unexplained following the required investigation [1]. The triple-risk model provides a conceptual rationale for investigating physiological vulnerability during critical developmental periods [2], but does not establish a validated measurement panel for individual deaths. Routine medicolegal autopsies generally do not systematically assess hepatic CYP450 protein abundance, inflammatory markers, or oxidative stress markers as potential indicators of physiological vulnerability. Consequently, infants receiving the same final death classification may have different underlying biological findings that remain uncharacterized.

The published five-domain Metabolic Vulnerability Index (MVI) framework [3] provides the conceptual foundation for a narrower, four-domain operational workflow. The selected domains address biologically interconnected processes: Domain 1 assesses a protein-abundance correlate of hepatic CYP450-mediated metabolic capacity through measurement of age-referenced CYP3A4, CYP2C19, and CYP2D6 protein abundance in qualified frozen liver [4]; Domains 2 and 3 assess inflammation-associated processes through hepatic C-reactive protein (CRP) immunoreactivity [5] and oxidative stress through 4-hydroxy-2-nonenal (4-HNE) protein-adduct immunoreactivity [6], respectively, using immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE) liver tissue; and Domain 4 addresses documented xenobiotic exposure through contextual interpretation of finalized routine forensic toxicology findings. The four complementary domain measurements are assigned provisional Tier 0–3 classifications, which are subsequently converted to binary indicators (0 for Tier 0 and 1 for Tiers 1–3), generating 16 distinct four-bit patterns representing candidate combinations of physiological vulnerability-related findings. These patterns are descriptive research classifications, not independent demonstrations of metabolic impairment or established physiological mechanisms.

Empirical support for hepatic immunohistochemical marker selection. Published findings support the use of tissue-based immunohistochemistry to characterize selected inflammatory and lipid-peroxidation-associated processes. In 100 sudden unexpected infant death cases, Pryce et al. evaluated hepatic CRP immunoreactivity across investigative categories, including infection-related and unexplained deaths [5]. In a series of 314 forensic autopsies, Quan et al. described diffuse, periportal, and focal hepatic CRP staining patterns associated with circumstances of death and survival interval [7]. Podszun et al. characterized the analytical performance of a hepatic 4-HNE protein-adduct immunohistochemistry method and demonstrated measurable differences in human liver tissue [6]. These investigations substantiate the selection of CRP and 4-HNE as biologically relevant hepatic tissue markers, but do not establish sensitivity for all responses, reliability under the proposed infant postmortem preservation conditions, or validity of the provisional MVI percentage-based tiers.

Biological rationale for selecting interconnected but narrower markers. Cytochrome P450 enzymes participate in Phase I biotransformation of numerous exogenous and endogenous substrates, while their expression and catalytic activity may be influenced by developmental maturation, inflammatory signaling, and cellular redox conditions [8,9,10,11]. Inflammatory responses may downregulate certain CYP enzymes, potentially reducing substrate-specific metabolic capacity. Depending on the CYP isoform, substrate, and physiological context, CYP-dependent reactions may also generate reactive oxygen species through imperfect catalytic coupling, potentially contributing to lipid peroxidation and oxidative stress; conversely, oxidative stress may alter CYP expression or function [12]. These relationships provide a biological rationale for examining hepatic CYP abundance, CRP immunoreactivity, and 4-HNE protein adducts together while considering documented xenobiotic exposures. Nevertheless, these interactions are complex, potentially bidirectional, and dependent on developmental stage and physiological conditions. Co-occurrence of abnormal findings does not establish a causal sequence or demonstrate that inflammation or oxidative stress has impaired CYP-mediated biotransformation.

Direct quantification of hepatic CYP protein abundance provides information about expressed enzyme levels that cannot be fully inferred from genotype alone. In human liver tissue, CYP2D6 protein abundance has been shown to explain substantial interindividual variability in CYP2D6-mediated activity beyond that captured by genotype-based activity scores [13]. However, protein abundance is not equivalent to catalytic activity or individual drug clearance, particularly during infancy when enzyme ontogeny changes rapidly. The selection of protein abundance rather than catalytic activity is particularly important in postmortem research because CYP450 catalytic function may deteriorate substantially after death even when measurable enzyme protein remains. Quantitative protein abundance therefore provides a potentially more suitable postmortem endpoint, subject to specimen-preservation requirements and analytical qualification [14,15]. Similarly, hepatic CRP immunoreactivity reflects an acute-phase-associated tissue signal rather than circulating cytokine concentrations or systemic inflammatory status, while 4-HNE protein-adduct immunoreactivity provides a lipid-peroxidation-associated measurement rather than a comprehensive assessment of mitochondrial energetics or oxidative injury. Routine toxicology identifies analytes detected within the scope and sensitivity of the investigative panel, not total xenobiotic burden or all possible exposures. These limitations reinforce the importance of treating the four domains as complementary, potentially interrelated research observations requiring future calibration and validation.

Operational adaptation of the original MVI framework. The principal methodological change is to prioritize specimens that forensic pathologists can reasonably obtain during routine infant autopsies without materially delaying examination or release. The original five-domain MVI framework included a neurochemical/autonomic integrity domain investigating potential abnormalities involving tryptophan hydroxylase 2 (TPH2), serotonin (5-hydroxytryptamine; 5-HT), and related brainstem pathways involved in respiratory and autonomic regulation. Obtaining appropriately oriented medullary and pontine specimens for these investigations presents substantial practical challenges. Fresh infant brain tissue is delicate, and reliable identification and sampling of specific brainstem structures may require specialized anatomical expertise, preservation procedures, and neuropathological interpretation. Although such specimens have been obtained successfully in specialized research settings, their consistent acquisition cannot be presumed feasible across routine medicolegal offices. Consequently, the neurochemical/autonomic integrity domain is excluded from the operational four-domain workflow, without implying that the associated neurochemical and respiratory-autonomic regulatory vulnerabilities lack biological importance.

A second modification eliminates the requirement for additional femoral venous blood for research-based inflammatory measurements. Because available blood volume may be limited in infants and must remain available for medicolegal priorities, particularly forensic toxicology, the revised workflow substitutes hepatic CRP immunoreactivity for the original blood-based inflammatory assessment and hepatic 4-HNE protein-adduct immunoreactivity for the broader redox assessment. Both measurements can be performed on FFPE liver tissue, while CYP450 protein abundance is assessed in qualified frozen liver and toxicologic exposure is evaluated using finalized routine forensic findings. This methodological paper specifies a standardized, specimen-adaptive workflow that distinguishes specimen acquisition and qualification, laboratory acceptance, analytical evaluability, interpretive scorability, and missing evidence. Its immediate purpose is to establish a framework for evaluating whether the proposed measurements can be obtained reproducibly and efficiently within routine medicolegal practice. Subsequent investigations would calibrate and validate age-appropriate CYP450 abundance measurements and hepatic CRP and 4-HNE assessments, refine provisional classification thresholds, and explore whether reproducible candidate mechanistic vulnerability patterns demonstrate concordance with clinical, developmental, environmental, and toxicologic contextual variables. Such findings could inform further hypothesis-generating studies and eventually provide additional investigative context for otherwise unexplained infant deaths. No cohort has yet been evaluated under this specification, and no diagnostic-performance estimate, established physiological impairment, or individual causal attribution is advanced.

2. Methods

2.1. Study Design and Operational Framework

The four-domain operational Metabolic Vulnerability Index (MVI) is a proposed research-use methodology for initially unexplained sudden deaths among infants younger than one year undergoing medicolegal investigation. It is not a validated physiological vulnerability scale or diagnostic instrument. The framework is designed for prospective evaluation of specimen-acquisition feasibility, laboratory measurement reproducibility, and integration with routine medicolegal procedures.

The framework incorporates four biologically interconnected domains: hepatic CYP450 protein abundance (Domain 1), hepatic CRP immunoreactivity (Domain 2), hepatic 4-HNE protein-adduct immunoreactivity (Domain 3), and finalized routine forensic toxicology findings (Domain 4). Domain 1 employs investigator-selected, literature-informed, provisional age-specific minimum-abundance thresholds for CYP3A4, CYP2C19, and CYP2D6. Domains 2 and 3 use investigator-proposed immunohistochemical scoring conventions based on the percentage of evaluable hepatocytes exhibiting qualifying CRP or 4-HNE staining. Domain 4 applies prespecified interpretive criteria to documented toxicological findings.

Each scorable domain receives a provisional Tier 0–3 classification according to domain-specific rules. Tier 0 indicates that the applicable operational reference or negative criterion is satisfied, except for the separately flagged first-week developmental classification in Domain 1. Tiers 1–3 represent progressively greater departures from the selected CYP450 abundance minima, increasing percentages of qualifying CRP or 4-HNE staining, or increasing categories of toxicological findings, as applicable. These tiers are descriptive research classifications rather than validated measures of physiological impairment or vulnerability severity. Original laboratory measurements, staining percentages, toxicology findings, and supporting assessment data are retained independently of their assigned tiers, preserving the underlying evidence for subsequent analyses of distributions, variability, interdomain associations, and empirical refinement of classification thresholds. The proposed minima, staining conventions, and interpretive boundaries require prospective assessment and subsequent empirical calibration and biological validation before their physiological significance can be established.

The workflow may be implemented by authorized coroner or medical examiner offices in collaboration with qualified laboratories. Participating institutions must establish eligibility criteria, family authorization procedures, specimen-handling responsibilities, laboratory arrangements, and prospective evaluation criteria before implementation. Routine medicolegal investigations, diagnostic requirements, and body-release priorities take precedence over research activities.

2.2. Medicolegal Priority and Authorization

The responsible coroner or medical examiner retains authority over the investigation, diagnostic specimens, routine toxicology, certified cause and manner of death, and release of the body. Research collection is limited to material that can be obtained without compromising forensic requirements.

Participating institutions must establish appropriate research authorization and family-permission procedures, designate qualified collection personnel and forensic-pathology oversight, and document responsibilities for specimen custody, laboratory evaluation, data security, and referral of potentially explanatory findings. Research collection must not delay required medicolegal procedures.

2.3. Specimen Collection, Handling, and Qualification

The operational workflow utilizes two forms of hepatic tissue and existing routine forensic toxicology findings. An adequate formalin-fixed, paraffin-embedded (FFPE) liver block or suitable unstained sections are required for C-reactive protein (CRP) and 4-hydroxy-2-nonenal (4-HNE) immunohistochemistry. Qualified frozen liver is required for age-applicable CYP450 protein-abundance measurements after the first postnatal week. Both hepatic immunohistochemical assessments require adequate tissue morphology and independently acceptable staining.

Frozen liver specimens should be stored at −80 °C when required by the performing laboratory and transported under validated conditions, ordinarily using dry ice. Collection records should document relevant postmortem intervals, tissue removal and preservation times, storage conditions, transfers, and receipt condition. Laboratory qualification must establish acceptable specimen amounts, fixation and preservation requirements, handling procedures, and analytical quality-control criteria.

Specimen acquisition, laboratory acceptance, analytical evaluability, and interpretive scorability are assessed and documented separately. A specimen may be successfully collected but subsequently rejected, or produce an analytically valid measurement that cannot be interpreted under the applicable classification rules. Domains lacking sufficient qualifying evidence receive a not-scorable (NS) designation, with the specific reason documented. Missing or unevaluable evidence is not assigned Tier 0 or interpreted as a normal finding. Valid measurements and supporting laboratory observations are retained even when incomplete evidence prevents assignment of a domain tier or generation of a complete four-domain profile.

2.4. Domain 1: Hepatic CYP450 Protein Abundance

Domain 1 assesses a protein-abundance correlate of hepatic CYP450-mediated metabolic capacity through age-referenced measurements of CYP3A4, CYP2C19, and CYP2D6 in qualified frozen liver. These enzymes participate in the metabolism of both exogenous and endogenous substrates. Protein abundance provides information about expressed enzyme levels but does not directly establish catalytic activity, metabolic clearance, or physiological impairment.

Specimen sampling and duplicate analysis. A liver specimen of approximately 2 g, or another mass specified by the performing laboratory, will be collected, preferably from deep within the right hepatic lobe, consistent with published recommendations for standardized postmortem liver sampling [16,17]. All planned CYP450 protein-abundance assays will be performed in duplicate using two independently prepared aliquots from the same specimen. For each age-applicable isoform (CYP3A4, CYP2C19, and CYP2D6), the mean of two quality-control-acceptable measurements satisfying prespecified agreement criteria will determine the provisional Tier 0–3 classification. Any single-valid-measurement exceptions or unresolved discrepancies will be handled according to laboratory-qualified adjudication rules. Both original measurements, associated quality-control results, and final classifications will be retained to permit subsequent evaluation of analytical reproducibility and measurement variability.

Quantification is proposed using qualified targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS). Measurements are normalized to analytically comparable adult-liver reference preparations and expressed as a percentage of adult-reference abundance. Laboratory qualification must also evaluate CYP450 protein stability and measurement reproducibility across relevant postmortem intervals, preservation conditions, and specimen-handling procedures; the existing human and animal evidence does not establish infant-specific stability for every selected isoform [14,15].

For each isoform i and measurement j, normalized abundance, A, is calculated as:

A_ij = 100 × C_ij / R_ij

where C_ij is the measured infant protein abundance, and R_ij is the corresponding adult-reference abundance. Measurements must use comparable protein preparations and normalization procedures.

For classification, the prespecified summary should be the mean of the two quality-control-acceptable normalized replicate measurements for each required isoform, provided they satisfy laboratory-qualified agreement limits. Adult-reference material, isoform-specific denominator selection, normalization basis, batch comparability, lower quantification limits, and treatment of nonquantifiable measurements must be documented before applying the provisional tiers. A reference denominator must not be changed post hoc to obtain a preferred classification.

Developmental studies support age-dependent interpretation of hepatic CYP450 expression [4,18,19]. However, the age-specific minima in Table 1 are provisional, literature-informed operational estimates rather than validated physiological reference limits.

The proposed age-specific minima were selected heuristically from published evidence of isoform-specific CYP maturation, including quantitative pediatric hepatic CYP abundance reported by Subash and Prasad [4], developmental changes in hepatic CYP2D6 expression reported by Stevens et al. [18], and developmental CYP2C19 expression reported by Koukouritaki et al. [19]. These studies support age-dependent interpretation, but their specimens, analytical endpoints, age groupings, and interindividual variability do not independently establish the exact percentages or boundaries in Table 1. Koukouritaki et al. also documented substantial early-life heterogeneity in CYP2C9 expression [19]; CYP2C9 is not included in the operational assay panel, and those findings are cited as evidence of developmental variability rather than as numerical minima for the selected isoforms. The listed age bands and minima are therefore investigator-selected, literature-informed operational conventions for exploratory classification. Future studies should characterize continuous age-specific distributions, calibrate the provisional minima empirically, and evaluate sensitivity to reasonable alternative thresholds.

For infants aged 0–7 completed postnatal days, CYP isoform tiers are not assigned because reliable developmental classification thresholds have not been established. Domain 1 receives a separately flagged developmental Tier 0 for purposes of the proposed classification system; this designation does not represent a measured normal result. At ages 8–28 days, CYP3A4 and CYP2D6 are assessed. Beginning on day 29, CYP2C19 is included.

For analysis and reporting, cases assigned a first-week developmental D1 Tier 0 will be identified as a separate stratum. Although the operational scheme permits a complete four-domain score and binary pattern when the other domains are scorable, these profiles have a structurally unavailable nonzero D1 assignment and are not directly comparable with profiles having assay-derived D1 tiers. Future studies should report overall profile completion separately from completion based on assay-derived D1 measurements and conduct sensitivity analyses excluding first-week developmental profiles from pooled score or archetype-frequency comparisons.

For each required isoform, the normalized abundance A is compared with its age-applicable minimum M in Table 1:

• Tier 0: A ≥ M 
• Tier 1: 0.80M ≤ A < M 
• Tier 2: 0.70M ≤ A < 0.80M 
• Tier 3: A < 0.70M 

These provisional thresholds classify the extent to which measured protein abundance falls below the selected age-applicable minimum; they do not establish degrees of functional metabolic impairment.

The 0.80M and 0.70M subdivisions are investigator-selected operational boundaries, not demonstrated transitions in catalytic capacity or biological injury. Before prospective classification, laboratory qualification should determine assay imprecision and measurement uncertainty relative to the spacing between adjacent tier boundaries, including values near the assay quantification limit. The locked assessment rules should specify how to adjudicate values close to a boundary when analytical uncertainty prevents a reliable tier distinction; unsupported precision must not be imposed by rounding.

After the first postnatal week, Domain 1 receives the highest provisional tier among the required age-applicable CYP isoforms. A missing or uninterpretable required isoform prevents final domain classification.

2.5. Domains 2 and 3: Hepatic CRP and 4-HNE Immunohistochemistry

Domains 2 and 3 assess complementary inflammation-associated and oxidative-stress-associated hepatic markers using separate immunohistochemical stains on FFPE liver tissue.

Domain 2 measures hepatic CRP immunoreactivity as an indicator of acute-phase-associated tissue expression. This measurement does not directly quantify circulating CRP, interleukin-6, or systemic inflammatory activity.

Domain 3 measures hepatic 4-HNE protein-adduct immunoreactivity as an indicator of lipid-peroxidation-associated protein modification. This measurement does not directly establish mitochondrial energetic impairment or the overall extent of oxidative injury.

For each marker, a qualified liver or pediatric anatomic pathology reader assesses the percentage of evaluable hepatocytes exhibiting qualifying immunoreactivity. The proposed provisional classifications for both immunohistochemical domains, also summarized in Table 2, are Tier 0—no qualifying hepatocyte staining, Tier 1—greater than 0% but less than 10%, Tier 2—at least 10% but less than 50%, and Tier 3—at least 50%.

These percentages are provisional classification boundaries, not validated age-specific physiological reference intervals or injury-severity grades.

The laboratory qualification process must establish staining procedures, appropriate controls, minimum tissue adequacy, assessment regions, qualifying hepatocyte staining criteria, and reader-adjudication procedures. Initial and final assessments should be documented to permit evaluation of inter-reader agreement. Inadequate tissue, failed controls, or unresolved interpretation discrepancies result in NS rather than Tier 0.

The locked assessment guide should additionally prescribe minimum evaluable hepatocyte counts or a justified qualified percentage-estimation approach, the number and distribution of assessment fields, the definition of a qualifying positive hepatocyte, and adjudication of values near the 0%, 10%, and 50% boundaries. Qualification should evaluate agreement for the recorded continuous percentage or estimate as well as the resulting categorical Tier 0–3 assignment. Background or nonspecific signal alone must not be counted as a nonzero biological finding.

2.6. Domain 4: Finalized Routine Forensic Toxicology

Domain 4 addresses documented xenobiotic exposure through contextual interpretation of finalized routine forensic toxicology findings. No additional research toxicology testing is required by the operational framework.

A qualified forensic pathologist or designated reviewer evaluates the completed toxicology report, including the analytical panel, specimen matrices, confirmed findings, detection limits, and relevant clinical and postmortem circumstances [20,21,22].

The proposed classifications are:

• Tier 0: Completed-negative findings within an adequate investigative panel
• Tier 1: Confirmed positive exposure without evidence of impairment
• Tier 2: Potentially impairing or biologically relevant findings
• Tier 3: Toxicologically significant or potentially explanatory findings

The highest eligible finding determines the domain tier. Positive detection alone does not establish functional impairment or causation. Inadequate panel coverage or unresolved findings that could affect classification ordinarily result in NS. As a restricted ceiling-rule exception, a confirmed, context-adjudicated Tier 3 finding may establish D4 = 3 despite another unresolved assessment, provided the outstanding information cannot change the decisive finding’s eligibility or classification. Such exceptions must be explicitly documented and flagged.

Potentially explanatory toxicology findings remain subject to independent medicolegal assessment and do not establish a research-derived cause of death.

Before use, the contextual toxicology assessment guide should define panel adequacy and substance eligibility, provide representative Tier 1–3 interpretation scenarios, and specify how treatment-related findings, uncertain matrices or concentrations, and disputed assessments are adjudicated. Independent review or documented consensus should be used for consequential ambiguity, including any invocation of the restricted Tier 3 ceiling rule. A negative result is limited to the substances and analytical sensitivity of the completed panel and does not establish absence of exposure.

Table 2 summarizes the provisional Tier 0–3 classification criteria and required evidence for all four domains, including the distinction between scorable classifications and missing or not-scorable (NS) evidence.

2.7. Domain Integration and Provisional Classification

Each of the four domains receives a provisional Tier 0–3 classification when the required evidence is sufficient for interpretation. For complete four-domain profiles, the unweighted composite MVI score, ranging from 0 to 12, is calculated as the sum of the four individual domain tier classifications. This composite is a provisional descriptive research index whose physiological significance has not been established. Higher scores reflect greater accumulated numerical tier assignments but cannot presently be interpreted as demonstrating greater physiological vulnerability, metabolic impairment, or risk of sudden infant death. Equal weighting of the four domains is an operational convention rather than an empirically validated representation of their relative biological contributions.

Each domain also contributes to a descriptive four-domain archetype. Tier 0 is encoded as binary 0, while Tiers 1–3 are encoded as binary 1, generating 16 possible four-bit configurations. The four positions follow the fixed order of CYP450 abundance, CRP immunoreactivity, 4-HNE immunoreactivity, and routine toxicology. These configurations identify which domains contain nonzero findings without distinguishing their individual tier magnitudes. They represent descriptive combinations of candidate physiological vulnerability-related findings, not established mechanisms of impairment or death.

A complete composite score and four-bit archetype require scorable classifications in all four domains. Missing or unevaluable evidence is designated not scorable (NS), never Tier 0. First-week developmental Tier 0 assignments for Domain 1 remain explicitly identified to distinguish them from assay-derived classifications. Original laboratory measurements, staining percentages, toxicological findings, complete Tier 0–3 classifications, and supporting assessment data are retained independently of the composite score and binary configuration, preserving the underlying evidence for subsequent quantitative analyses and empirical refinement of classification thresholds.

Future adequately powered investigations will be required to determine whether the composite MVI score or individual four-bit configurations have reproducible biological significance and whether alternative domain weighting, classification thresholds, or analytical approaches are warranted.

The original quantitative and qualitative observations and the complete four-domain tier vector are the principal descriptive research outputs. The unweighted 0–12 sum and the 16 binary patterns are secondary operational summaries. They are not interchangeable with the original data, and equal unit increments across biologically distinct domains should not be assumed to represent equal biological effects. Future analyses should examine alternative thresholds, weightings, and continuous-data approaches without retrospectively altering the prespecified classifications used to assess initial operational feasibility.

2.8. Data Documentation and Quality Assurance

The standardized case record preserves specimen identifiers, collection and preservation conditions, laboratory measurements, age applicability, adult-reference information, quality-control outcomes, histological assessments, toxicology findings, domain classifications, and reasons for missing or unevaluable evidence.

Selected contextual variables include infant age, sex, gestational age, prematurity, illness, medications, resuscitation and agonal factors, specimen conditions, and relevant circumstances documented during the investigation. These variables do not contribute points to the MVI classification.

3. Results

3.1. Defined Outputs of the Four-Domain Operational MVI

The proposed framework produces four principal methodological outputs: standardized domain classifications, an unweighted composite MVI score, descriptive four-domain archetypes, and documented categories of specimen and measurement completeness.

These outputs describe the structure and intended operation of the methodology. They do not represent results from enrolled infants or empirically validated physiological findings.

3.2. Provisional Domain Classifications and Composite Scores

The framework assigns each scorable domain one of four provisional classifications, ranging from Tier 0 to Tier 3, according to the measurement and interpretive rules specified in Methods.

For cases with complete four-domain profiles, the unweighted composite score ranges from 0 to 12. The score summarizes the assigned domain classifications but does not represent a validated scale of metabolic impairment or individual vulnerability.

The individual domain values remain essential for interpretation because different combinations of findings may produce the same composite score.

3.3. Sixteen Descriptive Four-Domain Archetypes

Each completed four-domain profile is also assigned to one of 16 possible descriptive archetypes, representing the different combinations of domains with and without nonzero findings.

For this purpose, a domain assigned Tier 0 is represented by 0, while any domain assigned Tier 1, 2, or 3 is represented by 1.

For example, the configuration 1010 indicates nonzero findings in Domain 1 (CYP450 abundance) and Domain 3 (4-HNE immunoreactivity), with Tier 0 classifications in Domain 2 (CRP immunoreactivity) and Domain 4 (routine toxicology).

Similarly, 1111 indicates nonzero findings in all four domains, whereas 0000 indicates Tier 0 classifications across all four domains.

Table 3 presents the 16 possible configurations and their descriptive interpretations. These archetypes provide a standardized way to identify and compare candidate patterns of physiological vulnerability. They do not establish independent mechanisms, causal convergence, or a diagnosis. Their potential biological significance requires subsequent investigation and validation.

Table 3. Proposed four-domain descriptive archetypes: marker-specific interpretations and limits.

Pattern (D1 D2 D3 D4)	Marker-specific descriptive archetype	Descriptive marker interpretation
0000	No nonzero domain finding	All four domains assigned Tier 0.
0001	Isolated toxicology finding	Eligible routine-toxicology finding without other nonzero domains.
0010	Isolated hepatic 4-HNE staining	Hepatic lipid-peroxidation-associated staining.
0011	Hepatic 4-HNE–toxicology pattern	Co-occurring hepatic 4-HNE staining and eligible toxicology finding.
0100	Isolated hepatic CRP staining	Hepatic acute-phase-associated staining.
0101	Hepatic CRP–toxicology pattern	Co-occurring hepatic CRP staining and eligible toxicology finding.
0110	Hepatic inflammatory–oxidative pattern	Concurrent hepatic acute-phase-associated and lipid-peroxidation-associated staining.
0111	Hepatic inflammatory–oxidative pattern with toxicology	Concurrent hepatic CRP and 4-HNE staining with toxicology context.
1000	Isolated low hepatic CYP abundance	At least one age-applicable CYP isoform below its provisional abundance minimum.
1001	Low CYP abundance–toxicology pattern	Low hepatic CYP abundance with an eligible toxicology finding.
1010	Low CYP abundance–oxidative pattern	Low hepatic CYP abundance with 4-HNE staining.
1011	CYP abundance–oxidative–toxicology pattern	Low CYP abundance and hepatic 4-HNE staining with toxicology context.
1100	Low CYP abundance–inflammatory pattern	Low hepatic CYP abundance with CRP staining.
1101	CYP abundance–inflammatory–toxicology pattern	Low CYP abundance and hepatic CRP staining with toxicology context.
1110	Three-domain hepatic co-occurrence	Low CYP abundance with hepatic CRP and 4-HNE staining.
1111	Four-domain co-occurrence	Low CYP abundance, hepatic CRP and 4-HNE staining, and eligible toxicology finding.

Note: D1 = hepatic CYP450 protein abundance; D2 = hepatic CRP immunohistochemistry; D3 = hepatic 4-HNE immunohistochemistry; D4 = routine forensic toxicology. Binary 0 represents Tier 0; binary 1 represents Tiers 1–3. Archetypes describe co-occurring domain findings and do not establish physiological impairment, mechanistic convergence, or causation. First-week age-assigned D1 Tier 0 is distinguished from assay-derived Tier 0. Incomplete profiles containing a not-scorable (NS) domain do not receive a complete archetype.

Table 3. Proposed four-domain descriptive archetypes: marker-specific interpretations and limits.

Pattern (D1 D2 D3 D4)	Marker-specific descriptive archetype	Descriptive marker interpretation
0000	No nonzero domain finding	All four domains assigned Tier 0.
0001	Isolated toxicology finding	Eligible routine-toxicology finding without other nonzero domains.
0010	Isolated hepatic 4-HNE staining	Hepatic lipid-peroxidation-associated staining.
0011	Hepatic 4-HNE–toxicology pattern	Co-occurring hepatic 4-HNE staining and eligible toxicology finding.
0100	Isolated hepatic CRP staining	Hepatic acute-phase-associated staining.
0101	Hepatic CRP–toxicology pattern	Co-occurring hepatic CRP staining and eligible toxicology finding.
0110	Hepatic inflammatory–oxidative pattern	Concurrent hepatic acute-phase-associated and lipid-peroxidation-associated staining.
0111	Hepatic inflammatory–oxidative pattern with toxicology	Concurrent hepatic CRP and 4-HNE staining with toxicology context.
1000	Isolated low hepatic CYP abundance	At least one age-applicable CYP isoform below its provisional abundance minimum.
1001	Low CYP abundance–toxicology pattern	Low hepatic CYP abundance with an eligible toxicology finding.
1010	Low CYP abundance–oxidative pattern	Low hepatic CYP abundance with 4-HNE staining.
1011	CYP abundance–oxidative–toxicology pattern	Low CYP abundance and hepatic 4-HNE staining with toxicology context.
1100	Low CYP abundance–inflammatory pattern	Low hepatic CYP abundance with CRP staining.
1101	CYP abundance–inflammatory–toxicology pattern	Low CYP abundance and hepatic CRP staining with toxicology context.
1110	Three-domain hepatic co-occurrence	Low CYP abundance with hepatic CRP and 4-HNE staining.
1111	Four-domain co-occurrence	Low CYP abundance, hepatic CRP and 4-HNE staining, and eligible toxicology finding.

Note: D1 = hepatic CYP450 protein abundance; D2 = hepatic CRP immunohistochemistry; D3 = hepatic 4-HNE immunohistochemistry; D4 = routine forensic toxicology. Binary 0 represents Tier 0; binary 1 represents Tiers 1–3. Archetypes describe co-occurring domain findings and do not establish physiological impairment, mechanistic convergence, or causation. First-week age-assigned D1 Tier 0 is distinguished from assay-derived Tier 0. Incomplete profiles containing a not-scorable (NS) domain do not receive a complete archetype.

3.4. Specimen Evaluability and Standardized Reporting Outputs

The framework distinguishes successful specimen acquisition from laboratory acceptance, analytical evaluability, and final interpretive scorability. This permits incomplete or unsuccessful assessments to be documented without treating missing evidence as a normal finding.

Standardized reporting preserves individual domain measurements, provisional classifications, composite scores, archetype assignments, specimen-quality information, and reasons for incomplete profiles.

Together, these methodological outputs provide a defined structure for prospective feasibility investigations and subsequent studies examining the reproducibility and possible biological significance of candidate vulnerability patterns.

4. Discussion

The four-domain operational MVI establishes a methodological foundation for investigating candidate physiological vulnerabilities that are not systematically assessed during routine medicolegal autopsies. Its scientific development requires a sequential research pathway, beginning with operational feasibility, progressing through measurement calibration and validation, and ultimately evaluating whether reproducible biological patterns demonstrate meaningful concordance with contextual variables. Each stage addresses a distinct question and must be interpreted within its evidentiary limitations.

Operational feasibility and measurement reproducibility. The initial research priority is to determine whether qualified liver specimens can be obtained consistently during routine infant autopsies without materially interfering with medicolegal procedures. Feasibility investigations must establish specimen-acquisition yield, preservation and transport performance, laboratory acceptance, analytical reproducibility, histological interpretive agreement, complete-profile yield, operational burden, and incremental costs. Successful implementation would demonstrate that the proposed measurements can be collected and classified reproducibly, but would not establish that the provisional classifications identify physiological impairment or vulnerability.

Measurement calibration and validation. Following demonstration of operational feasibility, larger investigations should characterize continuous age-related distributions of hepatic CYP3A4, CYP2C19, and CYP2D6 protein abundance using appropriately selected reference or comparison specimens. Developmental maturation, prematurity, interindividual variability, and postmortem preservation conditions must be considered when establishing and independently evaluating age-applicable reference limits and provisional Tier 0–3 boundaries. CYP450 protein abundance was selected as the principal postmortem measurement because catalytic activity may decline rapidly and substantially after death, whereas measurable enzyme protein can remain relatively stable for longer periods under appropriate preservation conditions [14,15]. Future studies should empirically calibrate age-specific abundance thresholds and evaluate assay reproducibility, isoform-specific postmortem protein stability, and classification consistency across developmental ages and specimen conditions. Although protein abundance may provide an indirect correlate of metabolic capacity, it does not directly establish an individual’s antemortem catalytic activity or degree of physiological impairment.

Parallel investigations should qualify hepatic CRP and 4-HNE immunohistochemical measurements, establish reproducible staining and interpretation criteria, evaluate inter-reader agreement, and determine whether proposed percentage-based classifications have defensible biological significance. Appropriate comparison specimens and independently evaluated laboratory procedures will be essential. Calibration of numerical thresholds alone does not establish that a marker represents pathological impairment, and analytical validation must be distinguished from validation of biological or diagnostic utility.

Biological concordance and future hypothesis generation. The scientific value of the four-domain framework may extend beyond individual measurements to their potential biological interrelationships. Developmentally limited CYP450-mediated metabolic capacity (Domain 1) may be further constrained by inflammation-associated processes reflected indirectly by hepatic CRP immunoreactivity (Domain 2), since inflammatory signaling can alter CYP expression and catalytic function [8,9,11]. CYP-mediated reactions and other cellular processes may contribute to reactive oxygen species generation and lipid peroxidation, reflected indirectly by hepatic 4-HNE protein-adduct immunoreactivity (Domain 3), while oxidative conditions may themselves influence CYP function [10,12]. Documented xenobiotic exposures (Domain 4) may impose additional substrate-specific metabolic demands, inhibit CYP activity, or contribute to reactive metabolite formation, potentially exceeding available metabolic capacity [10,12]. These potentially interacting and sometimes bidirectional processes provide a rationale for investigating whether recurring multidomain patterns exhibit biological concordance. However, the proposed measurements do not independently establish impaired catalytic function, metabolic saturation, the direction of biological interactions, or causation.

Once measurement characteristics and classification performance are sufficiently established, subsequent controlled investigations could evaluate whether recurring four-domain patterns demonstrate biological concordance with developmental, clinical, environmental, toxicologic, and other documented contextual variables. Such investigations should account for potential confounding by age, prematurity, illness, terminal events, specimen conditions, and other relevant factors. Comparisons between deaths remaining unexplained and those assigned established causes may help identify recurring biological patterns associated with unexplained deaths. However, infants who die from established causes may also exhibit abnormalities in one or more of the four domains and therefore should not automatically be considered representative of normal infant physiology.

Importantly, biological concordance requires more than temporal proximity between an exposure or contextual event and an infant’s death. A documented exposure occurring shortly before death does not establish that the exposure contributed to a physiological vulnerability. Stronger mechanistic hypotheses would require reproducible associations supported by biologically relevant measurements, appropriate exposure characterization, plausible physiological relationships, and independent replication. Even such concordance would not, by itself, establish individual causation.

Appendix A provides a structured means of preserving contextual information potentially relevant to these future investigations. Not all fields are necessary for the initial feasibility study; additional variables may be selected prospectively for subsequent analyses. Contextual findings remain separate from the numerical MVI scoring system.

The proposed workflow is therefore an initial methodological step rather than a validated assessment of physiological vulnerability. Its ultimate scientific value will depend on whether empirically calibrated, reproducible, and independently validated measurements can identify recurring biological patterns that may help characterize previously unrecognized physiological vulnerabilities in infant deaths that would otherwise remain unexplained following conventional medicolegal investigation.

5. Conclusions

The four-domain operational Metabolic Vulnerability Index (MVI) translates the broader five-domain conceptual framework into a streamlined, specimen-adaptive methodology for standardized postmortem research in initially unexplained sudden infant deaths. By prioritizing accessible hepatic specimens and existing forensic toxicology findings, the workflow is designed to be minimally disruptive to routine medicolegal investigations while enabling reproducible measurement of complementary physiological markers. If operational feasibility and measurement reproducibility are demonstrated, subsequent adequately powered studies will be necessary to calibrate age-specific CYP450 abundance thresholds empirically, validate provisional immunohistochemical scoring conventions and classification boundaries, and investigate whether recurring multidomain patterns demonstrate biological concordance with relevant contextual variables beyond temporal proximity alone. Such investigations may identify previously unrecognized physiological vulnerabilities and provide additional scientific context for infant deaths that otherwise remain unexplained following conventional medicolegal investigation. The framework remains exploratory and does not establish physiological impairment, diagnostic validity, or individual causation of death.

2.9. Prospective Evaluation of Feasibility, Reproducibility, and Costs

Future feasibility investigations should prospectively evaluate specimen acquisition, laboratory acceptance, analytical performance, interpretive reproducibility, complete-profile yield, personnel burden, and incremental costs.

Outcomes should distinguish eligible and authorized cases, attempted collections, laboratory-accepted specimens, analytically evaluable measurements, scorable domains, and completed four-domain profiles. Post-enrollment specimen or laboratory failures should remain visible in the applicable denominators rather than being excluded from analysis.

Operational assessments should document additional personnel time, collection and preservation requirements, specimen shipment, storage, laboratory processing, repeat testing, and any effects on autopsy completion or body release. Incremental costs should include specimen supplies, packaging, shipping, CYP450 quantification, CRP and 4-HNE immunohistochemistry, specialist interpretation, quality control, and unsuccessful or repeated procedures.

Measurement reproducibility should be evaluated through laboratory quality-control performance, technical replicate agreement, and immunohistochemical reader agreement. The frequency and reasons for NS classifications should be reported separately.

A future study must establish its own enrollment targets, acceptance criteria, operational benchmarks, and progression rules before implementation. The present methodology does not prescribe a universal completion threshold or infer biological validity from successful specimen acquisition and laboratory measurement.