Tissue Selection




Tissue Selection





Chapter 13 Selection of Brain Areas for Microscopic Examination


Mirna Lechpammer and Rebecca Folkerth



Introduction

Tissue specimens collected during the autopsy are largely used for diagnostic purposes; however, portions of obtained samples are also frequently preserved for research/teaching with adequate consent. Therefore the selection of a sampling protocol largely depends on the autopsy objectives. Regardless of the primary objective, the examiner should always keep in mind that inadequate tissue sampling during the autopsy may have medical and/or legal consequences, especially when a detailed report is required. Neuropathological examination is no exemption to this rule, being usually guided by the specific diagnostic question, although brain autopsy may be performed for brain banking and future research purposes [1].


Here we summarize the recommended tissue sampling procedure for microscopic brain examination and tissue banking purposes, while technical considerations in perinatal forensic, metabolic, and genetic autopsy are addressed in the following chapters.



Brain Tissue Sampling Procedure

In advance of the brain cutting, a neuropathologist should communicate with the multidisciplinary team consisting of neonatologist/pediatrician, neuro-radiologist, and autopsy/pediatric pathologist to obtain and understand the following information about each perinatal or pediatric case [1, 2]:




  • Clinical course, including sex and age of the patient; onset of symptoms (in utero / perinatal); history of seizures; developmental delay; gross physical malformations; maternal exposure to environmental toxins, infections and/or use of illicit substances during pregnancy; positive family history for inherited systemic diseases.



  • Neuroradiology reports and images, which may be suggestive of neuronal migration disorders, hydrocephalus, as well as vascular and/or neoplastic lesions, including MRI or sonographic signal alterations in specific anatomical regions.



  • General autopsy findings and cause of death, (e.g., sudden unexpected infant death or death caused by a known condition); state of the other major organ systems including birth deformities and anomalies; and presence of inflammatory, infectious or neoplastic diseases.



I. Recommended Steps for Handling of the Whole Brain

Pathology laboratories may receive brains either unfixed or fixed in 4% paraformaldehyde (PFA) or 10% neutral buffered formalin. Handling of the unfixed brain is preferred, as it allows expert sampling for diagnostic and research purposes, where the following approaches may be considered:




  • Recommended approach would be to make first a sagittal cut through one hemisphere in the midline of the brainstem (while brain is positioned dorsally on the cutting surface), followed by the cut through the corpus callosum. In this approach, one hemisphere can be snap-frozen, while the other can be fixed. The shortcoming of this approach is that some abnormalities can be asymmetric, as well as that focal lesions in the frozen hemisphere will be missed during the neuropathological examination of the other, fixed hemisphere [2].



  • An useful pitfall to prevent the possibility of overlooking major pathology in the unfixed hemisphere is to perform a rapid sampling of the representative fragments of the frontal, temporal and occipital cortex, hippocampus, basal ganglia, thalamus, brainstem, and cerebellum, and fixing those tissues in formalin before freezing of the whole hemisphere. Fixed samples from the frozen hemisphere then undergo the neuropathological examination along with the formalin-fixed contra-lateral hemisphere [2].



II. Gross Examination of the Brain and Spinal Cord

Recommended steps for the gross examination are the same for fixed and unfixed brains and spinal cord [3, 4]:




  • Examine the dura and leptomeninges (document location and size of any hemorrhages or presence of the cerebral venous sinus thrombosis).



  • Document the brain size and shape; describe gyral pattern or malformations.



  • Examine the circle of Willis, optic chiasm, and other cranial nerves.



  • Describe gross surface lesions, including palpable areas of brain tissue softening or firmness; document herniations (i.e., uncal and tonsillar), as well as the presence of any surgical prostheses (i.e., shunts and reservoirs).



  • After coronal cutting of the brain and spinal cord, describe any visible abnormalities focusing on the cortical ribbon, white matter, hippocampus, amygdala, basal ganglia, thalamus, cerebellum, brainstem (including cerebellar peduncles and pontine basis), and spinal cord. Document abnormalities in any region.



III. Microscopic Examination of the Brain

The diagnostic level of microscopic examination in general varies according to the number of examined blocks, thus ranging from full diagnostic to an indication of a pathological disorder that warrants further metabolic and/or genetic analyses [4, 5]. To yield high-quality diagnostics, at least 20 blocks should be sampled, preferentially with additions of some blocks from the contralateral hemisphere, as specified before [2]. If not already included in these blocks, additional sampling should be done from all regions with detected gross alterations.


Sudden unexpected infant deaths (SUID) include sudden infant death syndrome (SIDS), accidental suffocation in a sleeping environment, and other deaths from unknown causes. In the United States, approximately 3,500 infants die suddenly and unexpectedly each year, requiring a thorough pathological investigation to establish the cause of death [6]. Given the severity of the problem that SUID and SIDS pose for the health system and the neuropathology practice in particular, here we present key brain sections for the SUID diagnostic protocol (Figure 13.1) and summarize recommended selection of brain areas for microscopic examination, including differentiation of anatomical regions that should be examined for diagnostic and/or screening purposes (Table 13.1) [7].





Figure 13.1 Key brain sections per the condensed SUID protocol.


(From the archives of Rebecca Folkerth.)



Table 13.1 Selection of brain areas for microscopic examination of SUID and sudden unexpected death in childhood


























































Section Brain region in the paraffin block
1 Posterior cingulate gyrus, including corpus callosum * #
2 Right insula and superior temporal gyrus
3 Left insula and superior temporal gyrus
4 Amygdala *
5 Right hippocampus at level of lateral geniculate nucleus * #
6 Left hippocampus at level of lateral geniculate nucleus *
7 Basal ganglia (caudate, internal capsule, globus pallidus, putamen) * #
8 Hypothalamus (mamillary bodies)
9 Thalamus, posterior, including internal capsule * #
10 Calcarine cortex
11 Parietal cortex and white matter at atrium of lateral ventricle * #
12 Midbrain * #
13 Pons * #
14 Medulla (rostral and caudal) and spinal cord * #
15 Cerebellar hemisphere including dentate nucleus and cerebellar vermis *
16 Other (if clinically indicated):


  1. a) Eyes and/or optic nerves *



  2. b) Cervical nerve roots, bone, soft tissue *



  3. c) Dura, other microscopic lesions *



Legend: (*) Anatomical regions that should be examined for diagnostic purposes; and (#) for screening purposes, in an appropriate clinical setting [7].


(Modified with permission from [7])

















1 – Medulla; half of pons; spinal cord (cervical, thoracic, lumbosacral).
2 – Left hippocampus; half of midbrain.
3 – Right hippocampus; amygdala.
4 – Parietal cortex and periventricular WM.
5 – Basal ganglia; thalamus.
6 – Cerebellum (vermis, dentate, WM, cortex); anterior cingulate gyrus, corpus callous, and periventricular WM.



References

1.Kovacs GG, Budka H. Current concepts of neuropathological diagnostics in practice: neurodegenerative diseases. Clin Neuropathol 2010;29(5):271–88.

2.Stefanitis H, Budka H, Kovacs GG. Asymmetry of neurodegenerative disease-related pathologies: a cautionary note. Acta Neuropathol 2012;123(3):449–52.

3.Samarasekera N, Al-Shahi Salman R, Huitinga I, Klioueva N, McLean CA, Kretzschmar H, et al. Brain banking for neurological disorders. Lancet Neurol 2013;12(11):1096–105.

4.Vonsattel JP, Del Amaya MP, Keller CE. Twenty-first-century brain banking. Processing brains for research: the Colombia University methods. Acta Neuropathol 2008;115(5):509–32.

5.Ernst LM, Sondheimer N, Deardorff MA, Bennett MJ, Pawel BR. The value of the metabolic autopsy in the pediatric hospital setting. J Pediatr 2006;148(6):779–83.

6.Centers for Disease Control and Prevention. Sudden unexpected infant death and sudden infant death syndrome. Available at www.cdc.gov/sids/about/index.htm (page last reviewed: Dec. 31, 2018).

7.Folkerth RD, Nunez J, Georgievskaya Z, McGuone D. Neuropathologic examination in sudden unexpected deaths in infancy and childhood: Recommendations for highest diagnostic yield and cost-effectiveness in forensic settings. Acad Forensic Pathol 2017;7(2):182–99.



Chapter 14 Technical Considerations in Perinatal CNS Tissue Processing



Mirna Lechpammer



Introduction

Traditionally, autopsy comprises of gross and microscopic examination, which routinely involves tissue fixation, processing, and paraffin embedding. Perinatal autopsies present with a variety of challenges, not the least of which involves the removal and examination of the immature brain. Even if great care is taken during brain removal, one may be left with an amorphous, semifluid mass of softened tissue by the time the brain is ready for fixation and further processing [1]. Therefore, in Appendix 1, we summarize the preparation of recommended fixative, manual, and automated procedures for the processing of perinatal brain tissue, which in our experience help to preserve CNS morphology and allow further downstream tissue studies.


The fresh brain should be immediately immersed in the 4% paraformaldehyde (PFA) or if not available 10% neutral buffered formalin should be used. The fetal and infant brain may be suspended in a surgical bouffant cap, unlike older pediatric and adult brains which are typically suspended by the basilar artery. The recommended time of fixation is two weeks.


Nonetheless, for evaluation of functional abnormalities, metabolic and genetic autopsies should be considered. Such procedure requires that harvesting of tissues and running of diagnostic tests happens as soon as possible after death to avoid autolysis. Figure 14.1 shows the algorithm of proposed steps in decision-making for metabolic autopsy, which has a great value if there is a diagnostic suspicion for inborn errors of metabolism (IEM) [2, 3].





Figure 14.1 Recommended pre-mortem and post-mortem studies.


(Adapted from [2].)


Metabolic Autopsy

Post-mortem samples of tissues and body fluids are recommended to be collected and processed/preserved as follows (Table 14.1) [4]:




  • Tissue samples – Ideally should be collected 2–4 hours post mortem. Open biopsies are the first choice; however if not manageable, two to three needle biopsies should be taken, and samples wrapped in aluminum foil and snap-frozen in liquid nitrogen (N2) and stored at –70°C or in liquid N2. Organs to be considered for tissue sample collection include brain, spinal cord, skeletal muscle, cardiac muscle, liver, and kidneys.



  • Blood – Spot of a few drops of the whole blood on the paper card should be collected. Blood sample can be obtained by cardiac puncture and when more blood is available, 10 mL of heparinized blood should be taken, separated and preserved: plasma at –20°C, and cells at +4°C. For DNA analysis, 5 mL of whole blood immediately frozen at –20°C is recommended; while for the assessment of intermediary metabolites, 5 mL of blood in a fluoride oxalate tube needs to be obtained.



  • Urine – At least 100 μL should be collected by catheterization or suprapubic puncture and transferred into a container. Alternatively, the bladder may be washed with a small quantity of sterile saline. Urine contaminated with blood needs to be centrifuged, and the sample should be frozen and stored at –20°C.



  • Vitreous fluid – Needle aspiration should be performed with immediate transfer into a fluoride vial and stored at –20°C.



  • Bile – Should be collected as spot on the paper card or collected into a plain sterile tube and stored at –20°C.



  • Skin – Two punch biopsies should be obtained at different sites and placed in sterile vials with cell culture medium. Another option is the collection of two 3×3 mm full-thickness biopsies. Skin biopsy should be performed at the beginning of the autopsy by collecting small samples, to avoid contamination. Samples should be either transferred to the laboratory for the analysis immediately after the biopsy or stored overnight at +4°C.




Table 14.1 Tissue sampling for metabolic autopsies























































Tissue Substrate Technique
CNS and other tissue* snap-frozen in liquid nitrogen and stored at -70°C Tricarboxylic acid cycle and electron chain complexes • Spectrophotometry
Tissue* in glutaraldehyde • Electron microscopy
Blood Amino acids • Ion exchange chromatography
Acylcarnitines • Tandem mass spectrometry
Chromosomes
DNA extraction
Urine Organic acids • Gas chromatography mass spectrometry
Amino acids • Ion exchange chromatography
Acylcarnitines • Tandem mass spectrometry
Vitreous fluid Organic acids • Gas chromatography mass spectrometry
Bile Acylcarnitines • Tandem mass spectrometry
Skin Skin fibroblasts • Fibroblast culture




* Tissue = brain, skeletal muscle, cardiac muscle, liver, and kidneys.


(Adapted from [2])


Genetic Autopsy

In the case of stillborn infants, the possibility of the existence of a metabolic disorder should be explored. However, for a genetic disorder to be ruled out, a routine chromosome evaluation with microarray analysis should be performed on samples of umbilical cord tissue or placenta. Genetic autopsy is comprised of several steps and starts with the consultation with a geneticist and obtaining of detailed family history, clinical photographs, and complete skeletal survey. Perimortem laboratory investigation of genetic metabolic disorders is summarized in Table 14.2 [5]. At the present time, a variety of molecular tests can be run on formalin-fixed paraffin-embedded (FFPE) tissue, collected during a longer postmortem period. Nevertheless, attempts should be made to collect, preserve, and transport samples as soon as possible.


Aug 7, 2021 | Posted by in PEDIATRICS | Comments Off on Tissue Selection

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