Approach to nerve biopsy

1. NERVE BIOPSY
Dr. Laishram Mrinalini Devi
3rd year resident
Dept of Pathology, NMC

2. OVERVIEW
• INDICATIONS
• SITES
• SURGICAL PROCEDURES
• NEUROPATHOLOGICAL WORK UP
• SKIN BIOPSY
• PITFALLS
• WHAT TO LOOK FOR IN A NERVE BIOPSY

3. INDICATIONS
1. To gain information about therapeutic options –
inflammatory neuropathy is considered
2. As part of the therapeutic decision if
inflammation/ interstitial pathology
 vasculitis, granulomatous inflammation,
amyloidosis or typical CIDP – suspected
3. To detect pathological immunoglobulin deposits

4. INDICATIONS. . .
4. Differential diagnosis of hereditary neuropathies
with atypical presentation
5. Combined etiologies
Microangiopathic/diabetic and inflammatory
Hereditary and inflammatory

5. PREWORK UP
• Complete clinical
• Electrophysiological
• Laboratory work up
• It should be performed by medical
professionals
• Histological processing
Benefits > discomfort and side effects

6. SITES
• Specimen should be obtained from the
affected nerve
• Most neuropathies show distal accentuation
• Sural nerve – most frequently chosen for
biopsy
• Superficial peroneal nerve
• Superficial radial nerve

7. SITES. . .
• Obturator nerve biopsy –differentiate motor
neuropathies and lower motor neuron
diseases
• Larger, mixed sensory and motor nerves
guided by MRI and/or sonography – focal
lesions

8. SURGICAL PROCEDURES
• Nerve segment- excised inflicting minimal
mechanical injury
• Squeezing / stretching
• Excessive removal of fat or connective tissue
• Nerve fibres – sensitive to mechanical injury

9. “Toothpaste”
artifact

10. “Pseudo-
tomaculous”
fibre

11. SURGICAL PROCEDURES
• Proximal nerve cut- performed first
• Do not place proximal stump immediately
under the skin or fix it to skin by a suture
Neuroma
Fixation of the proximal stump in the
gastrocnemic muscle - suggested

12. SURGICAL PROCEDURES
• Biopsy of the complete cross section –
recommended
• Recommended length – 5 cm

13. NEUROPATHOLOGICAL WORK UP

14. Frozen without fixation
in isopentane cooled by
liquid nitrogen
Prox 1.5-2cm
fixed in buffered 10% formalin
Distal 1.5-2cm
3.9 %
glutaraldehyde

15. SURGICAL PROCEDURES
• Frozen sections can be cut and stained
immediately for rapid initial screening
• IHC
• To perform IHC
• Good source for RNA and protein studies
• Frozen material – store at -80 degree celsius

16. CUT UP AND SECTIONING
• Formalin-fixed nerve segment is dissected in
2-4 pieces
• Arranged transversely and longitudinally in a
paraffin block
• 3-4 micrometre thickness cut sections
• Serial sectioning of 3-4 levels or alternatively
30 consecutive sections- recommended if
inflammatory neuropathy- suspected

17. TINCTORIAL STAINS
• H&E
• Congo red
• Pearl’s
• Thioflavin S or T
• Gomori trichrome
• Ladewig
• Luxol fast blue
• IHC stains for myelin and axon proteins
• Semithin section resin histology>>

18. IMMUNOHISTOCHEMISTRY
• LCA, CD3, CD8
• CD4, CD20, SMA – vascular alterations
• PGP9.5 – axons
• Transthyretin , EMA – cells with perineural
differentiation
• S100- Schwann cells
• Myelin basic protein – myelin sheaths
• Specific types of endoneurial lymphocyte
infiltration – confirmed by IHC examination of
frozen sections

19. SEMITHIN RESIN CROSS AND
LONGITUDINAL SECTIONS
• Provide comprehensive and detailed picture
• Higher resolution, morphological accuracy of
relevant structures than paraffin sections
• Toluidine blue and methylene blue-azure II
Better contrast, detection of metachromatic
material
• Longitudinal – nodes of Ranvier and adjacent
internodes

20. TEASED FIBRE PREPARATION

21. TEASED FIBRE PREPARATION
• Extent and progression of fibre degeneration
• Regeneration, axonal atrophy, axonal
swellings, de- and remyelination and tomacula

22. TRANSMISSION ELECTRON
MICROSCOPY
• TEM of ultrathin sections contrast-enhanced
with uranyl acetate & lead citrate
• Changes of unmyelinated fibers - denervated
Remak bundles, collagen pockets (non-
myelinating Schwann cells ensheathing
bundles of collagen fibers )
• abnormal processes of non-myelinating
Schwann cells, as found in CMT4C

25. TRANSMISSION ELECTRON MICROSCOPY
• Uncompacted / decompacted
• Focally folded myelin
• Patholological inclusions
(adrenomyeloneuropathy)

26. MORPHOMETRY
• Determine extent of nerve fibre loss
• Axonal vs myelin sheath degeneration
• Not use in routine diagnosis

27. NEUROLOGICAL SKIN BIOPSY
• Used to examine various nerve fiber of
epidermis and the dermis- small,
unmyelinated epidermal nerve fibers
• 3 – 4 mm punch biopsies
• Standard location- 10 cm proximal to lateral
malleolus and at the proximal thigh
• Fixed in Zamboni solution / buffered
paraformaldehyde
• most frequent indication -suspected small
fiber neuropathy.

28. NEUROLOGICAL SKIN BIOPSY
• to examine :
epidermal nerve fiber density and morphology
density of the subepidermal plexus
sweat gland innervation
40 – 50 µm cryostat sections are stained
immunohistochemically using a PGP9.5 antibody

29. NEUROLOGICAL SKIN BIOPSY
• Gives valuable information - whether the
neuropathy is length-dependent or not
• IHC for inflammatory cells -tool to detect
vasculitis
• Less invasive than sural nerve biopsy and can
be repeated

30. PITFALLS
1. Blood vessel – mistaken for sural nerve
2. Biopsied nerve segment is mechanically
damaged due to inadequate handling of the
biopsy
3. Handling artefacts: shrinkage due to
hyperosmolar fixative or freezing the nerve
prior to or after fixation

31. PITFALLS
• If entire nerve biopsy is immediately placed
in formalin, fixation - suboptimal for semithin
sections and electron microscopy
• a mixture of formalin and glutaraldehyde
severe artefacts
Myelin splitting

32. PITFALLS
• glutaraldehydefixed tissue is less suitable for
most immunohistochemical stains
Antigen masking

33. Features To Be Described Routinely In
A Nerve Biopsy Report
Status of the epineurium including blood vessels
 Alterations of the perineurium (thickening, fibrosis,
calcification)
Endoneurial edema
Density of large and small myelinated nerve fibers
Extent of axonal degeneration and atrophy
 Frequency of bands of Büngner and macrophages containing
myelin debris
 Number of macrophage clusters (CD68 staining)
Regeneration clusters
Demyelinated/remyelinated fibers
Onion bulb formations
Inflammatory infiltrates
Presence/absence of amyloid

34. WHAT TO LOOK FOR
IN NERVE BIOPSY?

35. VASCULAR CHANGES
• Microangiopathy
• Atherosclerosis of small epineurial arteries
• Media calcification
• Granular osmiophilic deposits of cerebral
autosomal dominant angiopathy with
subcortical infarcts and leukoencephalopathy
(CADASIL) can be detected in sural nerve
biopsies by EM

36. DIABETIC NEUROPATHY
• CLUSTERS OF
REGENERATING NERVE
FIBRES
• MARKED THICKENING OF
ENDONEURIAL VESSEL
WALL

37. INFLAMMATORY ALTERATIONS
Guillain-Barré syndrome (GBS)
• Multifocal and randomly distributed
juxtanodal areas of demyelination
• focally accentuated lymphocytic infiltration of
the endoneurium
• endoneurial edema
• Sural nerve biopsy is rarely performed

38. GBS

39. INFLAMMATORY ALTERATIONS
• Chronic neuritis
• chronic inflammatory demyelinating
neuropathy (CIDP)
• chronic inflammatory axonal neuropathy
(CIAP)
endoneurial edema, endoneurial macrophage
clusters, ↑numbers of CD8-immunoreactive
cytotoxic T lymphocytes

40. CIDP
ENDONEURAL CLUSTERS OF
CD8 IMMUNOREACTIVE T
CELLS

41. CIDP
ENDONEURIAL CLUSTERS OF
CD68 IMMUNOREACTIVE
MACROPHAGES

42. SARCOIDOSIS
• Non caseating granulomatous lesions - in
epineurium
• Chronic necrotizing vasculitis
• Inflammatory infiltration -mainly associated
with axonal neuropathy
• Small fiber neuropathy - complication

43. INFECTIOUS DISORDERS
Borreliosis
• lymphocytic infiltration of epineurial blood
vessel walls
• perineurial thickening and fibrosis
• axonal neuropathy
• characteristic pattern of perineurial TNF-α,
C5b-9, and ICAM-1 expression -in sural nerve

44. • HIV infection - non-inflammatory, mostly sensory
neuropathy
• GBS , chronic neuritis
• Leprosy
• Histological diagnosis -skin biopsy
• nerve biopsy
• Lepromatous leprosy -chronic inflammatory infiltrates with
masses of acid-fast bacilli in histiocytes- which can occur in
any compartment of the nerve
• bacilli rarely detected in the granulomatous lesions of
tuberculoid leprosy
• Painful chronic inflammatory neuropathy with acid-fast
bacilli

45. AMYLOIDOSIS
• Both primary (AL) amyloidosis and familial
ATTR amyloidosis affect peripheral nerve
• Congo red stain - screening method
• Thioflavin S
• toluidineblue stained semithin sections
• focally distributed
serial sections of multiple blocks should be
searched for deposits
amyloid neuropathy

46. AMYLOIDOSIS

47. AMYLOIDOSIS
• IHC
• transthyretin, amyloid A component,
immunoglobulin and light chain antibodies
• Luminescent-conjugated Polymer
Spectroscopy

48. TOXIC NEUROPATHIES
• Alcoholic neuropathy -axonal loss -small nerve
fibers
• Axonal neuropathy- large fibers
• Chronic alcoholic neuropathy -clusters of
regenerating nerve fibers
• Drugs – taxol, vincristine, chloroquine

49. NEUROPATHIES ASSOCIATED WITH
NEOPLASIAS
• Paraneoplastic neuropathy - small-cell lung
cancer
• rapid nerve fiber breakdown with numerous
myelin ovoids
• Bands of Büngner and endoneurial
macrophages

50. NEUROPATHIES ASSOCIATED WITH NEOPLASIAS

51. NEUROPATHIES ASSOCIATED WITH NEOPLASIAS
• Direct infiltration of peripheral nerves by
carcinomas is a common feature of advanced
tumor progression
• diffuse infiltration of peripheral nerves -
malignant lymphomas

52. HEREDITARY NEUROPATHIES
• Molecular genetic testing
• Nerve biopsy findings can help to narrow
down the potential disease gene
• Mutations ˃ 50 genes - hereditary sensory and
motor and hereditary sensory and autonomic
neuropathy (HSMN and HSAN, respectively)
• Demyelinating hereditary neuropathies - focal
myelin thickenings -tomacula

53. TOMACULA

54. HEREDITARY NEUROPATHIES
Onion bulb formations
• surplus Schwann cell processes
• Schwann cell basal laminae
chronic hereditary demyelinating neuropathy:
CMT1A and B , CIDP

55. HEREDITARY NEUROPATHIES

56. HEREDITARY NEUROPATHIES
• NEFL mutations -prominent swellings of axons
• similar to the large focal axonal distensions
found in giant axon neuropathy

58. COMPRESSION INJURY
• Chronic nerve compression - fibrosis of epi-,
peri- and endoneurium
• endoneurial edema
• de- and remyelination
• axonal loss
• Endoneurial deposits of mucoid substance

59. COMPRESSION INJURY
• Renaut bodies
• sparse, concentrically arranged, elongated
fibroblast-like cells surrounded by ample
mucoid extracellular matrix that contains
precursors of elastic fibers
• found in a subperineurial
• at sites of chronic nerve compression, i.e., in
median nerve.
• D.D -organized nerve infarcts.

60. RENAUT BODIES

61. REFERENCES
• ANDERSON
• Clinical Neuropathology, Vol. 31 – No. 1/2012
(7-23)
• Bruno C, Bertini E, Federico A, Tonoli E, Lispi
ML, Cassandrini D, Pedemonte M, Santorelli
FM, Filocamo M, Dotti MT, Schenone A,
Malandrini A, Minetti C.