Guidelines

in pathologic and forensic-medical diagnostics of sudden infant death syndrome (SIDS) and sudden intrauterine unexplained death syndrome (SIUDS)


SAMPLING METHODS

The procedure for the accurate anatomo-clinical study of SIDS and SIUDS cases includes the examination of the clinical history data, of the scene of death in case of SIDS, as well as the anatomo-pathological investigations.
Thus, the guidelines include:
a) dispatch of the clinical history, together with the clinical and pathological information form opportunely filled;
b) dispatch of the organs and tissues, according to the anatomo-pathological protocol described below.
The anatomo-pathological protocol includes in particular the in-depth study of the autonomic nervous system. The morphological study is completed by investigations of molecular genetics.
Furthermore, it is important to dispatch a lock of hair and organ samples for the investigation of the etiopathogenetic role of environmental risk factors (cigarette smoke, drugs, alcohol, air pollution, pesticides).

Send the samples for the histopathologic, genetic and toxicological examinations to:
Centro di Ricerca "Lino Rossi"
Università degli Studi di Milano
Via della Commenda 19 – 20122 Milano, Italy
Tel. 02-+39-02-50320821; Fax: 02-+39-02-50320823


PROTOCOL FOR EXAMINATION OF THE CENTRAL AUTONOMIC NERVOUS SYSTEM

The anatomopathologic study of the central autonomic nervous system includes an in-depth study of the brainstem, cerebellum, and spinal cord, where the main structures participating in control of the vital functions are located (cardiorespiratory, arousal, upper digestive tract, etc.).

1- Molecular Genetics
Before fixing, take one fresh sample of cerebral cortex (0.5-1 cm3 thick).
Place the sample in a sterile test-tube according to one of the following procedures:

Use dry ice to send the samples to the laboratory of Genetics of the "Lino Rossi" Research Center.
Note: a further sample of cerebral cortex must be taken for the environmental factor investigations (see Toxicological examination).


2- Neuropathology
Brainstem
Examination of the brainstem includes sampling of three specimens, as shown in Figure 1 (in red).
The first specimen, ponto-mesencephalic, includes the upper third of the pons and the adjacent portion of mesencephalon. The second extends from the upper third of the medulla oblongata to the portion adjacent to the pons. The third specimen takes as reference point the obex and extends 2-3 mm above it and below it.
Figure 2 shows the histological sections obtained from the three fragments, indicating the main nuclei and structures to be examined.

Figure 1 - Brainstem sampling: ventral (left) and dorsal (right) surface

Figure 2 – Diagram of the histological sections obtained with the three sampling procedures
of the brainstem for the atomo-pathologic examination

Spinal cord (cervico-thorax tract)
removal of the first 5 thoracic levels of the spinal cord enables examination of the orthosympathetic intermediolateral nucleus, as shown in Figure 3.

Figure 3 – Section of spinal cord with localization of the intermediolateral nucleus

Cerebellum
In order to examine both the cerebellar cortex and the nuclei (dentate, etc.), a specimen of hemisphere extending all along the major diameter should be sampled, as shown in the Figure 4.

Figure 4 – Schematic section of cerebellum


CARDIAC CONDUCTION SYSTEM

Heart Removal
At autopsy, the heart is removed in the usual way, but taking the utmost care to sever the great vessels very close to the pericardial reflections; particularly, the superior vena cava is better cut a couple of cms above the pericardial sac, to secure examination of a possibly "high" vein. It is important to save the pacemaker by avoiding the customary cut at the right heart margin driving the incision laterally into (or towards) the superior vena cava, and dividing the intercaval bridge lengthwise. Indeed, with this technique the sino-atrial node may be diagonally slashed, together with the Crista Terminalis. The aorta should be always opened by cutting the mitral aortic valve.

Figure 5 – Cardiac conduction system sampling

Removal of conduction tissue blocks
The conduction system, although fairly constant in layout and structure, is subject to noteworthy individual variations; hence, only histological examination (with spatial reconstruction) by serial sections in each case is expected to provide the necessary data on both topography and pathology of the specialized atrio-ventricular (AV) pathways.

Two blocks of heart tissue are to be removed for paraffin embedding, as follows:

  • Block 1 - Contains the sinu-atrial (SA) node, its atrial approaches and the Crista Terminalis, and the SA node gangliar plexus. The main visual reference for the removal is centered upon the Sulcus-Crista Terminalis. Two longitudinal cuts are driven, parallel to the sulcus-crista line, through the atrial wall, with a medial prolongation on the right side to encompass the anterior aspect of the inlet of the superior vena cava; on the left side, the cava-cava bridge has to be sectioned very medially,  prolonging the cut on the superior vena cava wall. Of the two transverse cuts, the superior one is oriented to remove as much as possible of the cava funnel (in between the longitudinal sections). The inferior cut removes, more or less distally (according to the atrial volume), the fan of  pectinate muscles that radiate from the Crista Terminalis.
  • Block 2 - Contains the AV system with its atrial approaches: the pinpoints of the excision are, on the right side, the outlet of the coronary sinus and the pars membranacea septi. Holding the already opened heart so as to expose the interventricular septum against a fairly intense light source, one can clearly spot the transparent area of the pars membranacea and pin it between thumb and index; thereupon, one proceeds to excise the interventricular septum together with the central fibrous body, the lowermost part of the atrial septum and the adjacent segments of the AV fibrous annuli. The cuts will be driven as follows:
    1. an inferior, longitudinal incision through the posterior part of the septum, across the AV annulus fibrous and up to the superior margin of the coronary sinus ostium;
    2. an anterior longitudinal incision parallel to the former, through the superior part of the septum, extending to the aortic valvular ring;
    3. two cuts perpendicular to a. and b. to remove the tissue block, with its upper (atrial-aortic) margin about 1.5 cm above the AV ring and its lower (ventricular apex) margin encompassing the base of the medial tricuspid papillary muscle and (possibly) the moderator band.

After  removal, block 2 can be trimmed to regularize its shape by removing the valvular leaflets with the chordae, and the useless tracts of aortic wall and pulmonary conus muscle, well above the pars membranacea.
Both blocks 1 and 2 are routinely fixed in 10% buffered formalin that, besides simplicity, has the advantage of allowing for further silver impregnations of the nervous system, whenever needed. On paraffin embedding, it is advisable to place the medial margin of block 1 and the posterior-inferior of block 2 at the bottom. The resulting section plane of block 1 is perpendicular to the atrial endocardial surface and parallel to the major axis of the Crista Terminalis. The block 2 section is also perpendicular to both endocardial surfaces, along the major septal axis. Besides the generic differences from these oversimplified models, individual variations must be expected, due to the remarkable variability of the conduction system and to the slight discrepancies in the excision of the heart fragments, from case to case.


TOXICOLOGICAL EXAMINATION

To search for xenobiotics (nicotine components, drugs, alcohol, air pollution, pesticides) in hair and nervous tissue samples.

How to take hair
Collect a lock of hair of the victim cutting with scissors near the scalp, and tying as shown in Figure 5. Conserve in glass test-tubes, well sealed, at room temperature.

Figure 5 – How to take hair

How to take nervous tissue samples
Take, before fixing, a fresh sample of cerebral cortex (size approx. 1 cm3).
This sample should be placed in test-tubes without adding any fixative and kept at -20 °C.

DOWNLOADABLE FORMS

Perinatal death form
SIDS form
Post-mortem examination form