Nerve biopsy

Approach to nerve biopsy

1. NERVE BIOPSY Dr. Laishram Mrinalini Devi 3 rd 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 To gain information about therapeutic options – inflammatory neuropathy is considered 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. F rozen 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

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

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

25. NEUROLOGICAL SKIN BIOPSY Used to examine various nerve fiber of epidermis and the dermis - small, unmyelinated epidermal nerve fibers 3 – 4 mm punch biopsies S tandard 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.

26. 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

27. 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

28. PITFALLS Blood vessel – mistaken for sural nerve Biopsied nerve segment is mechanically damaged due to inadequate handling of the biopsy H andling artefacts: shrinkage due to hyperosmolar fixative or freezing the nerve prior to or after fixation

29. 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

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

31. 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

32. WHAT TO LOOK FOR IN NERVE BIOPSY?

33. VASCULAR CHANGES Microangiopathy Atherosclerosis of small epineurial arteries M edia calcification G ranular osmiophilic deposits of cerebral autosomal dominant angiopathy with subcortical infarcts and leukoencephalopathy (CADASIL) can be detected in sural nerve biopsies by EM

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

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

36. GBS

37. 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

38. CIDP ENDONEURAL CLUSTERS OF CD8 IMMUNOREACTIVE T CELLS

39. CIDP ENDONEURIAL CLUSTERS OF CD68 IMMUNOREACTIVE MACROPHAGES

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

41. 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

42. 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

43. 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

44. AMYLOIDOSIS

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

46. TOXIC NEUROPATHIES Alcoholic neuropathy -axonal loss -small nerve fibers A xonal neuropathy- large fibers C hronic alcoholic neuropathy - clusters of regenerating nerve fibers Drugs – taxol , vincristine, chloroquine

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

48. NEUROPATHIES ASSOCIATED WITH NEOPLASIAS

49. 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

50. 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

51. TOMACULA

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

53. HEREDITARY NEUROPATHIES

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

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

56. 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.

57. RENAUT BODIES

58. 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.