i

A CLINICAL STUDY ON THE UTILITY OF NERVE

BIOPSY IN PERIPHERAL NEUROPATHY

Thesis submitted for the partial fulfilment for the requirement of

the degree of DM Neurology

DR. JITESH GOEL

DM NEUROLOGY RESIDENT

2014–2016

DEPARTMENT OF NEUROLOGY

SREE CHITRA TIRUNAL INSTITUTE FOR MEDICAL

SCIENCES AND TECHNOLOGY, TRIVANDRUM, KERALA 695011

ii

DECLARATION

I, Dr Jitesh hereby declare that the thesis “A CLINICAL STUDY ON THE UTILITY OF

NERVE BIOPSY IN PERIPHERAL NEUROPATHY” was undertaken by me under the

guidance and supervision of Dr MD Nair, Senior Professor and Head of Department,

Department of Neurology at the Sree Chitra Tirunal Institute for Medical Sciences and

Technology, Thiruvananthapuram.

Dr.Jitesh Goel

Thiruvananthapuram Senior Resident

Date: Dept. of Neurology

SCTIMST

Thiruvananthapuram

iii

CERTIFICATE

This is to certify that the thesis titled “A CLINICAL STUDY ON THE UTILITY OF NERVE

BIOPSY IN PERIPHERAL NEUROPATHY”, is the bonafide work of Dr Jitesh Goel, Senior

Resident, DM Neurology and has been done under my direct guidance and supervision

at the Sree Chitra Tirunal Institute for Medical Sciences and Technology,

Thiruvananthapuram. He has shown keen interest in the research project and actively

participated in all its phases.

Thiruvananthapuram Dr MD Nair (Guide)

Date: Senior Professor and Head of Department

Department of Neurology,

SCTIMST. Thiruvananthapuram

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CONTENTS

Sl. No. Title Page No.

1 Introduction 1

2 Review of Literature 3

3 Aim of The Study 32

4 Materials And Methods 32

5 Results 34

6 Discussion 62

7 Conclusion 72

8 References 75

9 Annexures 84

IEC Approval

Proforma

1

INTRODUCTION

Peripheral neuropathy is among the common disorders in patients attending

neuromuscular clinic. Systematic approach comprising a comprehensive clinical

history, thorough neurological and systemic examination, nerve conduction studies,

EMG and relevant biochemical tests should be undertaken in all cases.

Nerve biopsy is indicated with a strong suspicion of disorders, like amyloidosis,

vasculitis, leprosy, and tumor infiltration. Nerve biopsy is useful for atypical

presentations of CIDP, and is helpful in exclusion of other etiologies. The yield of nerve

biopsy result is dependent on number of factors, including appropriate selection of

patients for biopsy, expertise of the laboratory, and techniques used in the analysis.

A prospective study by Gabriel etal has shown altered management in nearly 60 % of

cases after nerve biopsy, and nerve biopsy was more diagnostic in severe

demyelinating, distal asymmetric, and multifocal type of neuropathy 1, similar results

have been shown in another retrospective study 2. The yield of nerve biopsy in

vasculitic neuropathy is around 20 %, as observed in some other studies 3,4.

Combined nerve and muscle biopsy has shown improved yield in vasculitic neuropathy,

due to the more frequent involvement of the peroneal nerve and the involvement of

muscular arteries in vasculitis neuropathy. Higher yield in vasculitis neuropathy has

2

been shown to be useful by performing a biopsy of the superficial peroneal nerve

combined with a peroneus brevis muscle biopsy, confirmed in a multicenter

prospective study5. Nerve biopsy is more contributive in the diagnosis in multifocal

neuropathy than in the other patterns of neuropathy.

3

REVIEW OF LITERATURE

In a community-based study conducted in Bangalore, the age adjusted prevalence rate

of neuropathy in population was found to be 0.067 % 6, and in another study

conducted in Parsi community in Bombay, prevalence rate was found to 2.3 % 7.

Prevalence of peripheral neuropathy in Community based surveys in Italy have been

reported between 1-3% 8. In the Italian field screen study for distal symmetrical

polyneuropathy, out of 4191 subjects aged more than 55 years, 888 patients had

peripheral neuropathy and of these nearly 47.3 % of patients had diabetes mellitus.

Detailed analysis revealed a prevalence of distal symmetric polyneuropathy to be 3.3

– 3.6 per 100 population. Most common symptoms reported by these patients were

distal paraesthesias and muscle cramps. Most common signs reported were impaired

reflexes and impaired sensations9.

The approach to patients with suspected polyneuropathy starts with a comprehensive

clinical history and identification of risk factors. Detailed neurologic examination and

electro-diagnostic studies are used to identify the distribution of the neuropathy and

to characterize the process as sensory (large or small fiber types, or mixed), motor,

sensorimotor, or autonomic, and as axonopathic, demyelinating, or combined. The

decision for nerve biopsy is taken on the basis of these results. Nerve biopsy remains

a useful diagnostic tool in cases of multifocal, asymmetrical, painful or autonomic

neuropathies where vasculitis, sarcoidosis and amyloidosis are diagnostic possibilities.

4

Besides, nerve biopsy is also useful to detect or confirm histologically other causes

of peripheral neuropathy such as infections (e.g. leprosy, cytomegalovirus infection),

tumors like lymphoma or neurinoma, and granulomatous diseases.

In a prospective study by C M Gabriel etal, diagnostic utility of sural nerve biopsy in 50

consecutive patients with peripheral neuropathy was studied. Nerve biopsy was

useful in nearly 60 % cases, especially in cases with demyelinating neuropathy and

multiple mononeuropathy, besides helpful in confirmation of clinical diagnosis in

nearly 70 % cases. Nerve biopsy revealed an otherwise unsuspected diagnosis in 14%

of the patients and in 16% the biopsy findings were non - contributory. This study also

showed that the yield of nerve biopsy done after site selection as guided by clinical

and electrodiagnostic findings are higher 1. In another prospective study of 38 patients

who underwent nerve biopsy, nerve biopsy proved to be useful in defining the etiology

in 14 patients (37%). The diagnostic yield of nerve biopsy was highest in acute/ sub

acute symmetric and sub acute asymmetric neuropathies, followed by chronic

symmetric and chronic asymmetric neuropathies. The biopsy was diagnostic in 6

patients (16%), in cases where histopathological features were suggestive of

vasculitis, and was supportive of diagnosis in 8 patients (21%) 10.

In the current scenario, due to availability of newer genetic, and pathological

diagnostic modalities, and recognition of newer diagnostic entities, the percentage of

cases of neuropathy of undetermined etiology has considerably decreased. However,

5

inspite of rapid advances, etiology of neuropathy remains elusive in approximately

20% cases, especially axonal neuropathies.

Indications for nerve biopsy

1. Vasculitic neuropathy: Nerve biopsy is indicated in vasculitic neuropathy, to

establish definitive diagnosis before starting treatment. It has been seen that

in vasculitis, nerves are more commonly involved than other readily biopsied

structures like skin and muscle, and hence a search for vasculitis will have

higher yield with a nerve biopsy. Peripheral neuropathy is reported in nearly

52-60 % cases with vasculitis 11.

2. Diabetic neuropathy, especially in cases where superimposed CIDP or vasculitis

is suspected.

3. Toxic neuropathies (amiodarone)

4. Infections (HIV, Leprosy)

5. CIDP, and paraproteinemic neuropathies (deposits of IgM in the nerve usually

precede IgM gammopathy in serum)

6. Amyloidosis.

7. Hereditary neuropathies with negative appropriate genetic tests. Nerve biopsy

can also be helpful by identification of characteristic features, thereafter

planning appropriate genetic tests, for example MPZ gene mutations with

failure of myelin compaction, MTMR2 gene mutations with numerous myelin

6

outfoldings, or MFN2 mutations with abnormalities of intraaxonal

mitochondria.

8. Diagnostic etiology of neuropathy is not established even after a detailed

investigation. In a study of 365 nerve biopsies studied in patients with

undetermined etiology, Shin J. Oh etal found clinically relevant information or

helpful information in nearly 45 % cases. In the same study, specific diagnosis

was reached in 24 % of cases. A diagnosis of vasculitis was established in 12 %

cases, making it the most common diagnosis among those with specific

diagnosis 12.

Selection of nerve for biopsy

Selection of nerve for biopsy including sural nerve, superficial peroneal nerve,

superficial branch of radial nerve, dorsal cutaneous branch of ulnar nerve is done

depending on the clinical scenario. Biopsy should preferably be obtained from a nerve

indicating clinical and electrophysiological abnormalities. The nerve to be biopsied

may also be indicated by imaging techniques (MRI and ultrasonography)

demonstrating affected nerve segments. Sural nerve is usually preferred for biopsy,

due to long length of the nerve, pure sensory distribution, and protection behind the

lateral malleoli and easily testable electro physiologically. Because of the above

mentioned reasons, the yield of sural nerve biopsy is more with only mild sensory loss

as a sequalae and free from compression artefacts 12. Superficial peroneal nerve

7

biopsy combined with peroneus brevis muscle biopsy is preferred in cases of

vasculitic neuropathy. Studies have demonstrated that such combined muscle and

nerve biopsy has a moderately increased yield in demonstrating vasculitis in

comparison to nerve biopsy alone 13, 14, however, study by Bennette et al showed no

significant increase in yield with combined biopsy compared to nerve biopsy alone 15.

Other sensory nerves such as the superficial femoral, superficial radial and the

antebrachial cutaneous nerves may also be biopsied. The nerve specimen should be

processed in a specialized laboratory that analyzes at least paraffin and embedded

(plastic) semi-thin sections. Ultrastructural studies are sometimes required, and

teased fiber preparation is helpful, especially in assessing demyelination and

remyelination.

Procedure of nerve biopsy

The patient is positioned, nerve if palpable marked, and site is cleaned and prepared.

The skin is infiltrated with Lidocaine and incision made. The nerve is identified by

glistening appearance and differentiated from veins by branching pattern. The nerve is

cut taking care not to produce crush artifacts. Skin is closed with vicryl after securing

hemostasis. A bandage is applied to prevent oozing or edema formation and dressing

is done anti-septic ointments.

8

Parameters affecting the diagnostic yield of nerve biopsy

Various parameters affect the yield of nerve biopsy. In a retrospective

Clinicopathological study done by Deprez et al, in 355 patients to evaluate the clinical

and neuropathological parameters affecting the yield of nerve biopsy, it was seen that

contributive biopsies formed nearly 35.5% of total cases16. Clinical parameters

affecting the yield of nerve biopsy were:

(a) Pre-biopsy diagnosis: Greater yield was associated with clinically suspected

vasculitis, inflammatory demyelinating neuropathy and hereditary

sensorimotor neuropathies.

(b) Distribution of symptoms: Contributive findings were more often reported

with multifocal or asymmetrical presentations.

(c) Interval between disease onset and biopsy: Contributive findings were more

often reported with onset-to-biopsy interval of less than 6 months.

(d) Neuropathological techniques used: Serial sections on frozen, paraffin-

embedded and resin-embedded material improved sensitivity for interstitial

pathology; Combined muscle biopsy increased sensitivity in the detection of

vasculitis; and teasing of nerve fibers added critical information to the

classical techniques in 4% cases.

9

Inflammatory Neuropathies

Clinical, electro diagnostic, and cerebrospinal fluid findings are usually diagnostic in

most cases of acute and chronic inflammatory demyelinating polyneuropathies (CIDP).

The role for nerve biopsies is specially important in patients with neuropathy

detected to have only subtle evidence of a demyelinating component on

electrophysiologic studies but clinical features are strongly suggestive of CIDP. The

diagnostic criteria as proposed by American Academy of Neurology (AAN) research

criteria

(Ad Hoc Subcommittee of the American Academy of Neurology AIDS Task Force, 1991)

are tabulated in Table -1 17 . Subsequently, EFNS/PNS consensus guidelines (Joint Task

Force of the EFNS and the PNS, 2010) 18 were designed which provide more even

specificity and sensitivity to the diagnostic criteria (Table – 2).

According to American Academy of Neurology (AAN) criteria, biopsy evidence of

demyelination in sural nerve is mandatory for diagnosis of CIDP, which requires

teased fiber preparation and electron microscopy. While subperineurial edema,

inflammatory cell infiltration, onion bulb formation, and variation in fascicular

involvement are considered supportive of the diagnosis. Variability in the pathological

findings is determined by duration of disease, response to treatment, and the nerve

chosen for biopsy. Various autopsy studies have demonstrated that inflammation and

demyelination more often involves the spinal radicals in a patchy multifocal manner

and it may be completely lacking in the segment of distal (e.g., sural) nerve sampled.

10

The frequency of detection of various specific pathological features appear to be

highly variable in different studies [Table - 3]

In a study done in NIMHANS, in forty-six patients with idiopathic CIDP satisfying AAN

clinical and electrophysiological criteria for CIDP, 32 patients had a progressive clinical

course and 14 with relapsing-remitting course. The frequency of four supportive

pathological alterations - demyelination, inflammation, onion bulb formation, and

axonal changes in sural nerve biopsies were seen in nearly 100% of cases.

Electrophysiological abnormalities were detected in 90.8%, suggesting that supportive

histologic AAN criteria are helpful in diagnosis of CIDP. Besides, endoneurial

inflammation was frequent in the relapsing-remitting form and epineurial

inflammation and axonal changes in those with progressive course. Greater disability

at presentation, poor response to immunomodulation, and lower CSF protein levels

was seen in those with axonal pathology 19.

Onion bulbs, a characteristic feature in CIDP have been reported in 15%-40% of cases.

Dyck and Engelstad 20 reported a mixed pattern of distribution, with well-developed

large onion bulbs intermixed with smaller evolving forms as a characteristic finding in

CIDP.

In a study done by Molenaar et al to investigate the additional diagnostic value of sural

nerve biopsy in 64 CIDP patients, multivariate logistic regression analysis was used

to study the additional diagnostic value of sural nerve biopsy along with six clinical

11

features (remitting course, symmetric sensorimotor neuropathy in arms and legs,

areflexia, raised CSF protein concentration, nerve conduction studies consistent with

demyelination, and absence of co morbidity or relevant laboratory abnormalities). This

study showed that CSF protein concentration >1 g/l and NCS studies suggestive of

demyelination were strong predictors of CIDP. In this study, an independent predictive

value of sural nerve biopsy could not be confirmed 21. Due to the presence of

significant overlap in histopathological findings between CIDP and chronic idiopathic

axonal neuropathies (CIAP), sural nerve biopsy found found to have limited

diagnostic value in CIDP.

More specific alterations in nerve biopsies to differentiate CIDP from other

inflammatory

neuropathies, particularly vasculitis have been described; which include signs of T

cell activation, detection of matrix metalloproteinases 2 and 9, chemokine receptors

and interferon-γ- inducible protein (IP-10), and up regulation of Th1 cytokine IL-17 and

IFN-γ 22. Study using macrophage differentiation antigens and ‘macrophage clustering’

(defined as presence of three or more macrophages around a blood vessel) around

endoneurial vessels have been used to differentiate between inflammatory and

hereditary neuropathies 23.

12

Table 3: Comparison of pathologic findings in CIDP in published literature

Author, No. of cases

Inflammation Demyelination Onion bulbs

Axonal changes

Mixed Normal

Dyck et al 20

(n=26)

54% NA 15%

More common than demyelination

NA

NA

Prineas and MacLeod

24

(n=23)

No significant inflammation

26% 40% NA NA 24%

Barohn et al

25

(n=60) (56 biopsied)

10% 48% NA 21% 12% 17.9%

Krendel et al

26

(n=14)

29%

50%

36%

NA

NA

NA

Bouchard et al 27 (n=100)

18% 68% NA 5% 20% NA

Haq et al 28

(n=24)

36% 50% (teased fiber) 79% (EM), 93% (semithin)

58% 71% NA Nil

Rizzuto et al

29

(n=105)

25% 100% 48% NA NA NA

Vital et al 30

(n=42)

100% 40.4% NA 9.5% 76.1% NA

Vallat et al 31

(n=44)

75% 100% Variable Variable NA NA

Kulkarni et al

19

(n=46)

58.7% 82.8% 28.3% 8.7% 23.9% Nil

NA – Information not available.

13

Vallat et al. retrospectively studied 44 consecutive patients diagnosed clinically as

CIDP. Sensory findings predominated in all the cases. Eight cases did not have a

clear-cut electrophysiologic diagnosis of CIDP, but they satisfied the pathologic

features suggestive of CIDP. Five of these eight patients responded to

immunosuppressive therapy. Thus, nerve biopsy provided unequivocal evidence of

CIDP with no diagnostic EPS findings 31. However, study by Bosboom et al. reported

limited diagnostic utility of sural nerve biopsy in CIDP. In this study, sural nerve biopsy

specimens were taken from 21 consecutive patients who met established criteria for

diagnosis of CIDP, as well as sural nerves from 13 patients with idiopathic axonal

polyneuropathies and six autopsy nerves were taken as controls. There was no

difference in demyelinating features between patients with CIDP and axonal

neuropathies, besides evidence of axonal degeneration was found in both groups 32.

Role of nerve biopsy in diabetic patients and CIDP : There is also an important role

for nerve biopsy in diabetic patients with demyelinating polyneuropathy. The

importance is underlined by possible benefit of immunomodulatory therapy if there

are histopathological features similar to CIDP. Haq et al.28 retrospectively reviewed 10

patients with CIDP (nine of whom had diabetic polyneuropathy) and at least one of the

proposed electrodiagnostic criteria for demyelination, and 21 diabetic patients with

axonal polyneuropathy who underwent sural nerve biopsy. The diabetic patients with

demyelinating polyneuropathy had similar clinical, electrophysiologic, and histologic

14

features as the patients diagnosed with CIDP alone. The majority of patients in both

groups exhibited subperineurial and endoneurial edema, and only 2% to 20% had

inflammatory cells. Onion bulbs were seen in 88% of patients with diabetes and

demyelinating polyneuropathy; thinly myelinated and “naked” axons were frequently

present, but myelin stripping was very uncommon. Six patients with diabetes and

demyelinating polyneuropathy were treated with immunomodulatory therapy and

showed favorable response.

Use of inflammatory markers in nerve biopsy specimens to differentiate CIDP in

diabetes patients from typical diabetic peripheral neuropathy: Certain inflammatory

markers are useful to differentiate the two conditions. One such marker described is

MMP-9, Jann et al. 33 found increased immunoreactivity for MMP-9 in endoneurial

vessels and in epineurial T cells in diabetic CIDP nerves diabetic peripheral neuropathy

nerves. Patients with MMP-9 reactive nerve responded better to intravenous

immunoglobulin.

Peripheral neuropathy has been described with hepatitis C infection, with or without

cryoglobulinemia. Nemni et al. 34 provided an excellent report of 51 patients with

hepatitis C infection. Forty of 51 had cryoglobulinemia. Significant polyneuropathy was

more prevalent in the cryoglobulinemia patients; however, mononeuropathy or

multiple mononeuropathy were more prevalent in the cryoglobulin-negative patients.

Cranial neuropathies occurred in five of 11 (46%) patients with neuropathy who were

cryoglobulinemia-negative and in three of 40 (7.5%) patients who had

15

cryoglobulinemia. Nerve biopsy evidence of vasculitis was present in one third of

patients with cryoglobulinemia and in two out of three cryoglobulinemia-negative

patients. Differential fascicular axon loss suggestive of ischemia was present in 30% to

40% of all biopsies. Axonopathic changes were seen in majority, and combination of

demyelination and axon loss was present in 28% of the specimens from

cryoglobulinemia patients. Peripheral neuropathy is rare in sarcoidosis. Said et al. 35

studied 11 patients undergoing nerve biopsy that revealed epineurial granulomas

and perineuritis. The neuropathies varied from focal to multifocal and included a

patient with multifocal neuropathy with conduction blocks and one with a Guillain-

Barre-like presentation, and chronic, symmetrical, sensory, and sensorimotor

polyneuropathies, facial neuropathies. Multinucleated giant cells were found in eight

of 11 specimens, and vasculitis was present in seven. Muscle biopsy specimens from

10 patients showed inflammatory infiltrates and granulomas in nine patients and

necrotizing vasculitis in two. This study showed that necrotizing vasculitis with

ischemia may be a major mechanism for nerve injury in sarcoid neuropathies 35.

Pathological criteria for demyelination proposed by Ad hoc committee of AAN AIDS

task force (table – 1):

A. Mandatory : Nerve biopsy specimen showing unequivocal evidence of

demyelination and remyelination : Demyelination by either electron

microscopy(> 5 fibres), or teased fibre study (> 12 % of teased fibres), minimum

of 4 internodes each, demonstrating demyelination and remyelination.

16

B. Supportive criteria :

a. Subperineurial or endoneurial edema.

b. Mononuclear cell infiltration.

c. Onion-bulb formation.

d. Prominent variation in degree of demyelination between the fascicles.

C. Exclusion: vasculitis, neurofilamentous swollen exons, amyloid deposits,

intracytoplasmic inclusions in Schwann cells or macrophages indicating

adrenoleucodystrophy, Metachromatic leucodystrophy, globoid cell

leucodystrophy or other evidence of specific pathologic process.

European Federation of Neurological Societies (EFNS)/Peripheral Nerve Society (PNS)

Guideline Electrodiagnostic Criteria [Joint Task Force of the EFNS and PNS, 2010]

table - 2.

I. Definite: at least one of the following :

A. At least 50% prolongation of motor distal latency above the upper limit of normal

values in two nerves, or

B. At least 30% reduction of motor conduction velocity below the lower limit of

normal values in two nerves, or

C. At least 20% prolongation of F-wave latency above the upper limit of normal values

in two nerves (>50% if amplitude of distal negative peak compound muscle action

potential [CMAP] <80% of lower limit of normal values), or

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D. Absence of F-waves in two nerves if these nerves have amplitudes of distal negative

peak CMAPs at least 20% of lower limit of normal values + at least one other

demyelinating parameter in at least one other nerve, or

E. Partial motor conduction block: at least 50% amplitude reduction of the proximal

negative peak CMAP relative to distal, if distal negative peak CMAP at least 20% of

lower limit of normal values, in two nerves, or in one nerve + at least one other

demyelinating parameter in at least one other nerve, or

F. Abnormal temporal dispersion (>30% duration increase between the proximal and

distal negative peak CMAP) in at least two nerves, or

G. Distal CMAP duration (interval between onset of the first negative peak and return

to baseline of the last negative peak) of at least 9 ms in at least one nerve + at least

one other demyelinating parameter in at least one other nerve II.

Probable : At least 30% amplitude reduction of the proximal negative peak CMAP

relative to distal, excluding the posterior tibial nerve, if distal negative peak CMAP at

least 20% of lower limit of normal values, in two nerves, or in one nerve + at least one

other demyelinating parameter in at least one other nerve III.

Possible : As in ‘I’ but in only one nerve.

Vasculitic neuropathy

Systemic vasculitis involving small to medium-sized arteries commonly affects

epineurial vessels in vasa nervorum and thereby produce peripheral neuropathies.

18

Peripheral neuropathy can either be the sole manifestation of vasculitis, or it may

represent one of the manifestations of systemic necrotizing vasculitis 36.

Classification system for classifying vasculitides associated with peripheral neuropathy

was proposed by Dyck et al 36, which recognized two broad categories based on

etiopathogenesis.

(a) Vasculitis resulting from direct infection: Microorganisms related vasculitis

related to direct cytopathic effect of peripheral neurons are HSV and CMV.

Viruses associated with persistent replication such as HBV, HCV, HIV and HTLV-

1 have strongest association with vasculitic neuropathy 37.

(b) Vasculitis resulting from immunological mechanism

a. Systemic necrotizing vasculitis

i. Classic PAN: Peripheral neuropathy occurs in 50-75 % of patients

37. Vasculitic neuropathy mostly presents as mononeuritis

multiplex, or rarely as distal polyneuropathy affecting most

frequently the lower extremities. Cranial nerve involvement

occurs in nearly 2 % of patients.

ii. Microscopic polyangitis: Peripheral neuropathy has been

described in 20-60 % of patients 37. Mononeuritis multiplex is

the most common neurological manifestation, caused by

inflammation of epineurial arterioles, leading to ischemia of

both sensory and motor nerves.

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iii. Churg-Strauss syndrome: Peripheral neuropathy is the second

most common manifestation after pulmonary involvement,

occurring in nearly 70 % of patients 38. The most common

pattern is mononeuritis multiplex which tends to evolve into

polyneuropathy, and symmetrical polyneuropathy is seen in

advanced disease. Necrotizing vasculitis of epineurial arterioles,

eosinophilic infiltration and granuloma formation are the

characteristic features of vasculitic neuropathy associated with

Churg-Strauss syndrome.

iv. Wegener’s granulomatosis: Peripheral neuropathy occurs in 15-

44 % of patients 39. The most common presentation is

Mononeuritis multiplex, the onset of symptoms is usually

sudden with involvement of one or more peripheral nerves,

followed by widespread sensori-motor polyneuropathy. Multiple

cranial neuropathies most commonly involving optic, abducent

and facial nerves occurs in about 10 % of patients 40.

v. Connective tissue disorders

1. Rheumatoid arthritis: Approximately 20 % patients have

vasculitis, and vasculitic neuropathy occurs in nearly 10-

25 % of cases with Rheumatoid arthritis 41. Most patients

have slowly progressive distal symmetrical sensory or

20

sensori-motor polyneuropathy and approximately half

are predominantly sensory neuropathies 42.

2. SLE: Approximately 5-10 % patients develop vasculitic

neuropathy, and most common pattern is multifocal

neuropathy at onset which evolves into symmetrical

polyneuropathy 41.

3. Sjogrens syndrome: Prevalence of peripheral neuropathy

is around 25 % 43. Distal symmetrical sensory

predominant pattern is seen in nearly 75 % of

neuropathies. Sensory neuronopathy caused by dorsal

root ganglionitis is a distinctive feature accounting for

15-20 % of neuropathies.

4. Scleroderma : Peripheral neuropathy is seen in 20-25 %

patients with scleroderma. Motor predominant

neuropathy is common and trigeminal sensory

neuropathy is described in 3 % of scleroderma patients

44. Vasculitic neuropathy is seen in 0.5 % of scleroderma

patients and 1 % of patients with CREST syndrome.

5. Behcet disease: Peripheral neuropathy is seen in 0.5 % of

patients 45.

21

b. Hypersensitivity vasculitis: Essential mixed cryoglobulinemia, serum

sickness with radiculoneuropathy as manifestation of Hypersensitivity

vasculitis has been described 46.

c. Giant cell arteritis: Casseli et al (1988) have described peripheral

neuropathy in nearly 15 % of patients, and there is 15-20 % risk of

permanent vision loss from anterior ischemic optic neuropathy 47.

d. Non Systemic Vasculitic neuropathy (NSVN) : The term NSVN was first

described by Dyck et al 1987, who described 20 such patients. NSVN

refers to localized form of vasculitis, mediated by immune response

against tissue specific antigens (peripheral nervous system), with

different pathogenesis and prognosis compared to systemic

vasculitides. NSVN has overlapping features with systemic vasculitides

including presence of constitutional symptoms, elevated ESR, anemia,

leucocytosis, thrombocytosis, presence of auto antibodies in 20-40 % of

patients, detection of vasculitic changes in skeletal muscles, and better

therapeutic response with combination therapy36.

22

Diagnostic criteria for NSVN:

Inclusion criteria:

1. Clinical evidence of neuropathy by history and examination

2. Electro diagnostic findings consistent with neuropathy

3. Nerve or nerve/muscle biopsy diagnostic of necrotizing

vasculitis.

Exclusion criteria:

1. Clinical, laboratory, radiological or pathological evidence of organ

involvement outside the peripheral nervous system (except muscle)

2. Identified etiological agent(drug exposure, infections especially HIV,

HBV, HCV, CMV or HZV).

3. Underlying systemic conditions predisposing to vasculitis

(connective tissue disease, malignancy, diabetes mellitus, mixed

cryoglobulinemia).

23

Clinical profile of Primary vasculitides associated with peripheral neuropathy 48(Schaublin et al 2005)

Peripheral Nerve

Upper Respiratory

Lower Respiratory

Renal GIT Arthralgias Cardiac Skin CNS C-ANCA P-ANCA

Microscopic polyangiitis

60 – 70 % -- 20-80 % 70-90% 30% 50% 10-20% 50-70% 10-20% 10-50% 50-80%

PAN 50-70% -- -- -- 20-60% 50-80% 5-30% 25-50% 5-20% -- --

Churg-strauss syndrome

60-80% 50-60% 40-70% 10-40% 30-60% 50% 10-40% 50-60% 5-30% 3-35% 2-50%

Wegeners Granulomatosis

40-50% 95% 70-85% 70-80% <5% 60-70% 10-25% 40-50% 5-10% 75-90% 5-20%

Profile of Secondary vasculitides associated with peripheral neuropathy48

Neuropathy Clinical pattern Associated features

Laboratory findings Electrophysiology Nerve pathology

Rheumatoid arthritis Uncommon Sensory/sensorimotor Joint-100%, skin 70%

RF 90%, ESR(elevated) 85%

Distal sensori-motor axonal, mononeuritis multiplex

Necrotising arteritis; IgM deposits

SLE Rare Distal sensorimotor; mononeuritis multiplex , cranial

Skin 90%, joint 85 %, renal 50 %

ANA 90-100% DSDNA 60%

Distal sensori-motor axonal Asymmetric

Necrotising arteritis; Complement deposits

Sjogrens syndrome Common Distal sensory cranial; ganglionopathy

Sicca 50-90%, skin 75%

ANA 90%,RA 60%,SSA/SSB 50%

Axonopathy; ganglionopathy

Perivascular Infiltrates, axonal loss

Systemic Sclerosis

Rare Distal symmetrical; cranial; mononeuritis multiplex

Fibrotic skin, Raynauds phenomenon, calcinosis

ANA 100% Anti-centromere antibody 70%

Distal motor predominant neuropathy

Endoneurial vasculopathy; Multifocal axonal loss

Sarcoidosis Uncommon Distal symmetrical; cranial; mononeuritis multiplex; radiculopathy

Pulmonary 90%, skin 25%

ACE 70%,ESR(elevated) 50%

Distal sensori-motor axonal Asymmetric

Noncaseating granuloma

Cryoglobulinemia Common 20-80%

Asymmetrical sensori-motor; mononeuritis multiplex

Purpura 90% Skin ulcers

Cryoglob 100% HCV 50-70%

Asymmetric sensori-motor; symmetric

Necritizing arteritis, immune complex deposits

24

Pathological diagnosis:

Definite diagnosis of vasculitic neuropathy is dependent on histopathology. In

vasculitic neuropathy, the yield of peripheral nerve biopsy is much more than skin

or muscle biopsy 41. The yield of peripheral nerve biopsy is higher than muscle

biopsy in cases of NSVN, whereas both muscle and nerve biopsies have almost

similar yield in systemic vasculitis 20. In a study by Vrancken et al 49, the additional

yield of combined nerve and muscle biopsy compared to nerve biopsy alone is

about 5% in patients with clinically suspected vasculitis, and around 15% in patients

with vasculitic neuropathy.

The comparison of diagnostic sensitivities in vasculitic neuropathy are highlighted

in table –

Comparison of diagnostic sensitivities in vasculitic neuropathy (Collins et al 2005)

Biopsy site Reference Vasculitis sensitivity

Sural nerve Collins et al(2003) NSVN 14/30(47%)

Hattori et al(2002) Microscopic polyangitis

20/26(80%)

Clausen et al (2000)

Systemic & NSVN 27/45(60%)

Dyck et al (1987) Systemic & NSVN 31/65 (48%)

Hattori et al(2002 Churg Strauss Syndrome

16/30 (53%)

Combined total Systemic & NSVN 109/196 (56%)

Peroneus brevis & superficial peroneal nerve

Collins et al (2000) Systemic 15/25 (60%)

Collins et al (2003) NSVN 11/19 (58%)

Chia et al ( 1996) Systemic & NSVN 23/33 (70%)

Combined total Systemic & NSVN 49/77 (64%)

25

Histopathological classification of vasculitic neuropathy

Definite vasculitis 50:

Active lesion: Inflammatory infiltrate within the vessel wall and one or more signs

of vascular destruction such as fibrinoid necrosis, vascular/perivascular

hemorrhage, or endothelial cell disruption.

Chronic lesion with signs of repair or healing: Nerve biopsy showing mononuclear

inflammatory cells in the vessel wall and one or more of the following – intimal

hyperplasia, fibrosis of media, adventitial/periadventitial fibrosis; chronic

thrombosis with recanalisation.

No evidence of another primary disease process that may mimic vasculitis

pathologically such as lymphoma, lymphomatoid granulomatosis or amyloidosis.

Probable / suspicious vasculitis 50:

Predominant axonal changes and perivascular inflammation accompanied by signs

of active or chronic vascular damage or perivascular inflammation plus one or more

of the following: Asymmetrical/multifocal nerve fibre loss, hemosiderin deposits

(Perls stain for iron); Vascular deposition of complement; Ig M or fibrinogen by

direct IF; active axonal degeneration, myofibre necrosis, regeneration, or infarcts in

concomitant peroneus brevis muscle biopsy.

Possible vasculitis 50:

Presence of predominant axonal changes and inflammation in vessel wall without

other signs of definite vasculitic neuropathy, in absence of above criteria for

definite and probable vasculitis OR

26

One or more signs of active/chronic vascular damage or pathological criteria of

definite vasculitic neuropathy without vessel wall or perivascular inflammation.

Diagnostic criteria for clinically probable vasculitic neuropathy in patients lacking

biopsy proven necrotizing vasculitis 51

1. Clinical presentation typical for a vasculitic neuropathy: asymmetrical or

multifocal painful sensorimotor neuropathy; Acute/sub acute relapsing,

progressive or relapsing progressive course.

2. Laboratory evidence of a systemic inflammatory state, for example elevated

ESR.

3. Electro diagnostic evidence of an active asymmetrical axonal sensori-motor

neuropathy.

4. Clinical response to immunosuppressive therapy.

5. Suggestive neuromuscular pathology: vascular thickening narrowing or

obliteration of vascular lumen , thrombosis, periadventitial capillary

proliferation, hemosiderin deposits, asymmetrical nerve fibre loss or

wallerian like degeneration.

6. Clinicopathological evidence of a systemic/ secondary etiology : concurrent

condition known or suspected to predispose to vasculitis ( connective tissue

disease, infections, certain drugs, cryoglobulinemia, malignancy/

paraproteinemias); simultaneous multi-organ non peripheral nerve

involvement; Biopsy proven vasculitis in other tissues.

27

At least 3 of first 5 criteria required for NSVN, criterion 6 is mandatory for systemic

vasculitis.

Neuropathies Associated with Infections

1. HIV infection: Polyneuropathies in HIV infection include sensori - motor

neuropathies affecting large and small myelinated and unmyelinated fibers.

Mechanism of HIV-mediated neuropathy includes inflammatory,

demyelinating, mononeuritis-multiplex, cytomegalovirus-induced

neuropathy, as well as mixed neuropathy as part of the diffuse

inflammatory lymphocytosis syndrome. Macrophage activation in the

presence of pro-inflammatory cytokines mediates axonal injury. Patients

with toxic neuropathies due to antiretroviral therapies have similar

pathologic findings, and mitochondrial disruptions are described. Diffuse

infiltrative lymphocytosis syndrome is painful, usually symmetrical, and

acute or sub acute. There is axon loss and marked CD8 lymphocytic

angiocentric infiltration in nerve biopsy specimens in the epineurium and

endoneurium, along with a massive HIV proviral load. Mononeuritis

multiplex due to vasculitis has also been reported. Other infections in HIV

like CMV, HCV, and Syphilis are also responsible for neuropathy 52.

2. West Nile virus: Usually presents with a new paralytic illness affecting

anterior horn cells and motor roots. Histopathological studies have shown

presence of perivascular chronic inflammation in the spinal cord, along with

28

loss of anterior horn cells, and neuronophagia. There is presence of

inflammation around lumbar spinal cord nerve roots 53.

3. Leprosy :

Paraproteinaemic Neuropathies

Anti–myelin-associated glycoprotein (anti-MAG) IgM monoclonal gammopathies:

Gammopathies are characterized by myelin abnormalities including the presence of

tomacula, increased myelin loops, redundant myelin infolding or outfolding,

enlargement of the adaxonal space, and tomacula in fibers containing paranodal or

internodal demyelination. Eurelings etal. have described the presence of T-cell

inflammation in monoclonal gammopathy, and thus usefulness of nerve biopsy

in identification of these patients with superimposed vasculitis who will respond

favourably to immunotherapy 54.

Amyloidosis

Peripheral neuropathy is the cardinal clinical feature of familial amyloid

polyneuropathies, and occurs in 15% to 35% of patients with acquired amyloidosis.

Peripheral nerve amyloidosis is typically characterized by accumulations of amyloid

in the endoneurium, epineurium, perineurium, and in blood vessel walls. There is

preferential loss of small myelinated and unmyelinated fibers, with characteristic

clinical features of painful neuropathy with autonomic symptoms 55. In a

retrospective study done by Andrews etal to compare the utility of sub-

cutaneous fat aspiration and nerve biopsy in diagnosing amyloidosis in patients

29

with isolated sensorimotor peripheral neuropathy, only 6% of patients had a

positive aspirate for amyloid, and most of these patients had a monoclonal protein

or other clinical findings associated with amyloidosis. Thus concluding that the

yield of subcutaneous fat aspirate in patients with isolated peripheral neuropathy is

low and this biopsy should be reserved for patients with systemic amyloidosis 56.

Hereditary Neuropathies

Nerve biopsies are not indicated if hereditary neuropathy is clinically suspected.

However, nerve pathology is helpful in sporadic cases and it provides relevant

information regarding the genetic study to be performed in selected cases.

Longitudinal study by Gabreëls-Festen 57 in 25 patients with Dejerine-Sottas

syndrome, patients with PMP22 mis-sense mutations had thin myelin sheaths,

suggestive of hypomyelination and patients with PMP22 duplications had increased

myelin thickness, more age-related pathology, with early active demyelination,

followed by onion bulb formation and stabilization, and then late axon loss.

Inflammation plays an important role in neuropathic attacks in hereditary

neuropathies, including hereditary brachial plexus neuropathy. Klein et al. 58

examined upper extremity nerve biopsy specimens from four patients during

exacerbations of hereditary brachial plexus neuropathy. These patients had

evidence of active axonal degeneration and prominent perivascular inflammation

with disruption of vessel walls; however teased fibers showed no evidence of

reduplicated myelin (tomacula). It was concluded that inflammation, probably

30

immune mediated, was the cause of these attacks, and use of immune modulation

was indicated.

Giant axonal neuropathy is a rare autosomal recessive disorder, affecting both the

peripheral and central nervous system. Nerve biopsy shows evidence of axonal

swellings with tightly packed filaments, and uniform onion bulbs. Kuhlenbäumer et

al. 59 investigated two generations of a family with five members affected by giant

axonal neuropathy. The authors identified two novel mutations in the gigaxonin

gene.

Toxic Neuropathies

Several toxic agents and drugs have characteristic histo-pathological changes.

Amiodarone is one such drug that has characteristic histopathology. Pulipaka et al.

60 reported three patients with amiodarone-induced mixed polyneuropathies and

vacuolar myopathy. Pathological changes are characterized by presence of

lysosomal inclusions in the endoneurium, especially in the cytoplasm of Schwann

cells and in endothelial cells and fibroblasts, and presence of osmiophilic inclusions,

best seen on semi-thin plastic sections and on ultrastructural studies. Chloroquine

is also described to be associated with osmiophilic inclusions. Most other toxins,

such as thalidomide and alcohol, cause nonspecific, length-dependent

sensorimotor axonal degeneration. Small axons are primarily affected in alcoholic

neuropathy, which is not associated with thiamine deficiency 61.

31

Acquired Metabolic Neuropathy

Diabetes mellitus remains the most common cause of neuropathy with

multifactorial pathogenesis. Nerve biopsy is usually indicated in diabetic patients

with distal polyneuropathy with consideration of vasculitis and CIDP. In patients

with diabetic radiculoplexus neuropathy, biopsies often reveal perivascular

inflammation or vasculitis and features suggestive of nerve ischemia. The

administration of immunomodulatory therapy in this group of patients is useful.

Peripheral neuropathy, likely due to thiamine deficiency, occurs after gastrectomy

or gastrostomy. Evaluation of 12 patients with post-gastrectomy sensorimotor

polyneuropathy, and 7 of whom had features suggestive of autonomic dysfunction;

there was electrophysiological evidence of an axonal neuropathy with mild

demyelination. Sural nerve biopsy specimens revealed loss of large and small

myelinated and unmyelinated fibers, active axonal degeneration, and rare

segmental demyelination and remyelination, with subperineurial “edema.” These

patients had evidence of thiamine deficiency and they improved with thiamine

administration62.

32

AIMS AND OBJECTIVES

1. To study the utility of nerve biopsy in providing diagnostic, therapeutic or

prognostic information that aid in clinical management of patients with

peripheral neuropathy.

2. To study the clinical and demographic profile of patients undergoing nerve

biopsy.

INCLUSION CRITERIA

Patients with established diagnosis of peripheral neuropathy who

underwent nerve biopsy.

EXCLUSION CRITERIA

Nerve biopsy samples in which the diagnostic labeling was not possible.

METHODS

All patients admitted in Neurology ward – Sree Chitra Tirunal Institute of Medical

Sciences and Technology, Trivandrum between 2010 and 2015, with established

diagnosis of peripheral neuropathy who underwent nerve biopsy were included in

the study.

Retrospective analysis of patients who have undergone nerve biopsy during period

2010 – 2015, was undertaken. A detailed clinical, socio-demographical data for all

patients, essential investigations (routine hematology, blood glucose and other

biochemical parameters, serological tests for HIV and paraproteinemia, and

vasculitis workup), including CSF Study and electrophysiological study was

33

collected. Detailed histopathological studies was done on all the specimens, and

special stains were done in selected cases, if indicated. An attempt was made to

correlate histopathological features of biopsy with electrophysiological study.

Indications for nerve biopsy

Based on clinical evaluation, indications for biopsy were classified into:

1. Suspected vasculitis, CIDP, amyloidosis, HMSN, or other inherited metabolic

diseases;

2. Multiple potential causes of neuropathy, biopsies used to narrow

differential diagnosis;

3. Absence of presumptive etiology (no working diagnosis, NWD);

STATISTICAL ANALYSIS : The demographic details and outcomes of the study

population was entered in Microsoft Excel sheet, and descriptive analysis was done

using SPSS 17.0. Data was presented as numbers and percentages.

34

RESULTS

In this study, nerve biopsies of 84 patients done between the period 2010 -2015

were studied.

Demographic details:

The subjects whose nerve biopsy results were included in the study included 53

males and 31 females (Figure – 1). Mean age of onset of symptoms was 58.6 +/-30.2

years (range 2 - 75 years) and the mean age at the time of nerve biopsy was 59.8 +

/-35.2 years. Mean duration of symptoms was 12.6 months (range 5 days – 240

months).

The age distribution of patients is shown in Figure – 2. Majority of patients (19) were

in between the age group 60-70 years, in addition 11 patients were between age

group 5-60 years and 10 each in between the age group 10-20 years and 30-40

years.

35

Figure – 1

Figure – 2

Males : 53(63.8%)

Females : 31 (36.2%)

Gender distribution

0

2

4

6

8

10

12

14

16

18

20

< 10 y 10-20 y 20-30 y 30-40 y 40-50 y 50-60 y 60-70 y > 70 y

Age distribution

36

Clinical profile of patients:

The course of symptom progression was acute in 7 (7.6 %), sub acute in 11(12.7 %),

and chronic in 66 (79.7 %) patients. The onset involving lower limbs in 64, upper

limbs in 11, and simultaneous upper and lower limbs in 9. The presenting symptoms

are summarized in Table – 1. Majority of patients presented with positive sensory

symptoms in form of paraesthesias

(n – 34, 43 %). Negative sensory symptoms were present in 18 patients (22.8 %).

Motor complaints in lower limbs and upper limbs as presenting symptoms were

seen in 26(23.4%) and 10(12.5 %) patients respectively (Table – 1).

The clinical features of these patients are summarized in Table 2.

Motor system abnormalities included weakness in 67 (84.8 %) and muscle wasting in

15(19 %) patients. Cranial neuropathy was seen in 5 patients, in the form of facial

nerve weakness in 5 patients, trigeminal nerve involvement in 4 patients, bulbar

palsy in 2 and external ophthalmoplegia in 2 patients. In addition, 2 patients had

evidence of SNHL. Features of autonomic dysfunction in form of orthostatic

hypotension, sweating abnormalities and gastrointestinal abnormalities were noted

in 11 patients. Asymmetric onset of symptoms was seen in 19 patients and

asymmetric clinical profile was seen was noted in 58 patients. Thickened nerves

were noted in 8 patients. Most common sites of nerve thickening was ulnar nerve,

in addition, dorsal cutaneous branch of ulnar nerve, superficial branch of radial

nerve and superficial peroneal nerve were thickened in 2 patients each.

37

Presenting symptoms(Table-1)

1. Positive sensory symptoms in lower limbs 26 (32.9 %)

2. Negative sensory symptoms in lower limbs 14 (17.7 %)

3. Motor weakness in lower limbs – proximally 19 (14.5 %)

Motor weakness in lower limbs – distally 7 (8.9 %)

4. Motor weakness in upper limbs – proximally 2 (2.5 %)

Motor weakness in upper limbs – distally 8 (10 %)

5. Positive sensory symptoms in upper limbs 8 (10.1 %)

6. Negative sensory symptoms in upper limbs 3 (2.6 %)

7. Cranial neuropathy 1 (1.3 %)

Clinical features (Table-2)

1. Motor weakness in lower limbs Proximal : 21 (30 %)

Distal : 48 (64.8 %)

Proximal and distal : 67 (84.8 %)

2. Motor weakness in upper limbs Proximal : 12 ( 14.5 %)

Distal : 58 ( 68.6 %)

Proximal and distal : 65 (82.3 %)

3. Negative sensory symptoms 58 (73.4 %)

4. Positive sensory symptoms 41 (51.9 %)

5. Skin and hair changes 50 (63.3 %)

7. Wasting Upper limbs :11 (13.9 %)

Lower limbs :4 (5.1 %)

Both upper and lower limbs : 13 (16.5 %)

8. Cranial neuropathy 10 ( 12.7 %)

38

Examination (Table – 3)

1. Motor weakness in lower limbs Proximal: 18 (23.4 %)

Distal: 54 (71.3 %)

Proximal and distal: 61 (77.2 %)

2. Motor weakness in upper limbs Proximal: 14 (17.2 %)

Distal: 56 (68.6 %)

Proximal and distal: 60 (75.9 %)

3. Loss of pinprick sensations 48 (60.8 %)

4. Loss of temperature sensations 40 (45.6 %)

5. Loss of touch sensation 32 (40.4 %)

6. Loss of JPS, vibration sensations 36 (45.6 %)

7. Orthostatic hypotension 13 (16.5 %)

8. Peripheral nerve thickening 8 (10.2 %)

9. Deformities Kyphoscoliosis: 2 (2.6 %)

Pes cavus : 11 (13.9 %)

Hammer toes : 1(1.3 %)

Clawing : 8 (10.1 %)

10. Positive Rhombergs sign 33 (41.8 %)

11. Deep tendon jerks 38 (48.1 %)

12. Wasting Lower limbs distally : 8.9 %

Upper limbs distally : 12.7 %

Both upper and lower limbs : 17.7 %

13. mRS at presentation 0 : 1 (1.3 %)

1 : 1 (1.3 %)

2 : 10 (12.7 %)

3 : 26 (32.9 %)

4 : 37(46.8 %)

5 : 3(3.8 %)

Examination finding are summarized in Table-3. Most common findings were motor

weakness in lower limbs (77.2 % patients), followed by motor weakness in upper

limbs (75.9% cases). Evidence of peripheral nerve thickening was present in 8 cases

(10.2 %), in 3 cases of CIDP, and 5 cases with Hansens disease. Most common nerves

thickened were bilateral ulnar nerves (5 cases), 3 cases showing involvement of

peroneal nerves, and one case each with involvement of great auricular nerve and

superficial radial nerve. The clinical features of patients with a diagnosis of CIDP and

Vasculitis are summarized in Table – 4 and 5 respectively.

39

Clinical features of patients with diagnosis of CIDP on nerve biopsy(Table – 4)

Feature Observation ( n- 23)

Duration of symptoms(months) 9.25 +/-13.6

Nerve biopsy delay duration(days) 36 +/-12

Initial / onset neuropathic symptom Sensory Sensori-motor Motor

19 (82.6 %) 2 (8.5 %) 3 (13 %)

Onset of neuropathic symptoms Upper limbs Lower limbs

2 (8.8 %) 21 (91.2 %)

Clinical pattern of neuropathy Symmetric sensori-motor neuropathy Asymmetric sensori-motor neuropathy Symmteric sensory neuropathy

11 ( 30.4 %) 25 (69.6 %) 1(4 %)

Clinical features Positive sensory symptoms Negative sensory symptoms Weakness in BLL Weakness in BUL, BLL Areflexia Loss of pain, temperature sensations Loss of JPS, Vibration sensations History of falls Rhombergs sign Cranial nerve involvement

20 (87 %) 18 (78.3 %) 23 (100 %) 21 (91.3 %) 18(78.3 %) 12 (52.2 %) 20 (87 %) 12(52.2 %) 18 (78.3 %) 1 (4.3 %)

Clinical features of patients with diagnosis of vasculitic neuropathy on nerve

biopsy(Table – 5)

Feature Observation (n – 18)

Presence of systemic symptoms 8 (44.4 %)

Duration of systemic symptoms (months) 16.87 +-13.66

Duration of neuropathic symptoms(months) 9.25 +-13.6

Initial neuropathic symptom Positive sensory symptoms in LL Positive sensory symptoms in UL Motor weakness in UL Motor weakness in LL

7 (38.9 %) 3 (16.73 %) 1 (5.6 %) 7 (38.9 %)

Onset of neuropathic symptoms Upper limbs Lower limbs

5 ( 13.6 %) 31 ( 86.4 %)

Clinical pattern of neuropathy Mononeuritis multiplex Diffuse polyneuropathy

11(61.1 %) 7 (38.9 %)

Sensori-motor and sensory neuropathy(clinical) Sensori-motor Pure sensory

28 ( 77.8 %) 8 ( 22.2 %)

Cranial nerve involvement 4 (22.3 %)

40

Co-morbidities(Table – 6)

S. No Co-morbidities N ( percentage)

1. Diabetes mellitus 21 ( 25.5 %)

2. Systemic Hypertension 8 (7.5 %)

3. Lymphoma 1(1.3% )

4. Malignancy 1 (1.3%)

5. Connective Tissue disorder 1 (1.3%)

6. Hypothyroidism 4 (5.1 %)

7. Tuberculosis 3 (3.9 %)

8. Coronary Artery disease 5 (6.5 %)

9. No co-morbidities 43 ( 54.4 %)

Common co-existing systemic illnesses are summarized in Table – 6. The most

common co - existing illnesses were Diabetes mellitus and Systemic hypertension in

21(25.5 %) and 8(7.5 %) patients respectively. History of malignancy was obtained in

2 patients. These included Non-Hodgkin’s lymphoma and carcinoma tonsil

(operated-hemimandibulectomy done 2 years back) – one case each detected 3

years and 2 years prior to the onset of neuropathic symptoms and hence considered

unrelated to neuropathy. Other co-existing medical illnesses included Tuberculosis

in 3 patients (3.9 %), Hypothyroidism in 4 patients (5.1 %). Past history of Hansen’s

disease was present in 2 patients, who had presented with neuropathic symptoms.

History of Undifferentiated polyarthritis was present in 4 patients, and Rheumatoid

arthritis was present in 2 patients.

EPS

Most common electrophysiological pattern was axonopathic neuropathy(n-62, 73.8

%), followed by mixed demyelinating and axonopathic neuropathy(n-16, 19 %) and

pure demyelinating neuropathy(n-6, 7.1 %).

41

Laboratory investigations:

The results are summarized in table 8. CSF study was done in 55 patients, and 42

cases had evidence of elevated CSF protein, with mean value of 91.4 mg/dl (range:

24 - 492 mg/dl). CSF albumin-cytological dissociation was present in 19 cases, out of

total of 24 cases (79.2 %) diagnosed as CIDP.

Positive ANA profile was present in 5 patients, in 3 cases positivity was seen for Anti-

Ro antibodies, and one case each showing positivity for Anti-Smith, and Anti-

PM/SCL. Serum protein electrophoresis was performed in 42 patients and none

revealed an M spike. Non-specific abnormalities in form of polyclonal gamma-

globulinemia and hypo-albuminemia were noted in 7 and 8 patients respectively.

None of patients showed the presence of BJP in urine.

Summary of investigations (Table 7)

ESR (a) 10 – 20 mm fall Ist hr (b) 20 – 30 mm fall Ist hr (c) > 30 mm fall Ist hr

27 (34.2 %) 13 (16.5 %) 37 (46.8 %)

CSF Albumino-cytological dissociation( CIDP) 19/24

CSF protein (N-55) Mean : 91.40 mg/dl (range : 24-492 mg/dl)

Positive ANA Profile (N-6) Anti-Ro – 3

Anti-Smith, Jo-1 – 1

Anti-PM/Scl – 1

HBAIC > 6.5 % 10

42

HISTOPATHOLOGY STUDIES

Most common biopsied nerve was the sural nerve in 75 patients. 3 patients

underwent biopsy of dorsal cutaneous ulnar nerve (sural nerve study normal

electrophysiological), and one patient biopsy of radial cutaneous nerve. Sites of

biopsies are summarized in Table – 8.

Table - 8

Biopsy – site N

Sural nerve 75

Dorsal cutaneous ulnar nerve 3

Radial cutaneous nerve 1

Combined nerve and muscle biopsy 10

Skin biopsy 12

Lip biopsy 6

CIDP

The diagnosis of CIDP was established in 24 cases on the basis of available clinical,

electrophysiological results (as per EFNS 2010 guidelines). Evidence of CSF albumin-

cytological dissociation was present in 19 cases (79.2 %). Sural nerve biopsy findings

were consistent with diagnosis of CIDP in 16 cases, 3 cases showed features

suggestive of vasculitis. 2 cases showed non-specific chronic axonopathic changes,

with no evidence of inflammatory cell infiltration or vasculitis, and 2 cases showed

evidence of diabetic neuropathy with no inflammatory cell infiltration. In addition, 3

cases which had shown evidence of acquired demyelination in form of multiple

conduction block and temporal dispersion, and CSF study showing albumino-

cytological dissociation, there was no evidence of inflammation on sural nerve

biopsy and there was uniform distribution of onion bulbs ( suggestive of CMT). The

43

findings are summaried in Table – 9. Myelin alteration was demonstrated in all

patients, with most common histopathological finding being non-uniform loss of

myelinated fibres in 78.3 % cases and evidence of demyelination/remyelination,

followed by sub-endoneurial edema, in 71.3 % cases, endoneurial inflammation in

43.5 % cases and non-uniform distribution of onion bulbs in 28 % cases. Features

suggestive of diabetic microangiopathy, co-existent with inflammation were seen in

6 cases. Epineurial inflammation and axonopathic changes co-related with

progressive and severe course of CIDP, seen in 4 cases.

Figure 1 shows HPE features seen in a case of CIDP, characterized by nonuniform

loss of myelinated fibers and several fibers show thinned out myelin rings,

perivascular inflammation and endoneurial edema within the fascicle, chronic de-

and re-myelination leads to concentric ‘onion bulb’ Schwann cell hyperplasia.

Figure 4 showing nerve biopsy changes in a case of Diabetic neuropathy, showing

thickened perineurial sheath, and endoneurial hyaline arteriosclerosis, a feature of

diabetic microangiopathy. 4E shows a case Diabetic neuropathy with CIDP,

showing endoneurial lymphocytic inflammation, and nerve fibre loss, involving

small more than large myelinated fibers.

9 cases showed evidence of symmetric predominantly demyelination sensori-motor

neuropathy, the mean CSF protein in these cases was 93.9 mg/dl(range 28 – 273

mg/dl) compared to 11 cases showing demyelination and axonopathic changes

(mean CSF protein 80.8 mg/dl, range 24 – 158 mg/dl). 3 cases showing evidence of

acquired demyelination in form of multiple conduction blocks and temporal

44

dispersion, and CSF study showing albumin-cytological dissociation, there was no

evidence of inflammation on sural nerve biopsy and there was uniform distribution

of onion bulbs ( suggestive of CMT).

HPE findings in CIDP (Table – 9)

Nerve biopsy findings Observation (n-24)

Sub-endoneurial edema 17 (71.3%

Myelinated fibre loss ( non-uniform) 18 (78.3 %)

Acute axonal breakdown 30.4 %

De / remyelination Uniform: 10 (43.5 %)

Non-uniform: 12 (52.2 %)

Onion bulb formation 10 (43.5 %)

Bands of Bungner 4 (18.5 %)

Fibrosis Epineurial Endoneurial Perineurial

6 (26.1 %) 10 (43.5 %) 5 (21.4 %)

Epineurial inflammation Endoneurial inflammation Perineurial inflammation

4 (16.7 %) 10 (43.5 %) 6 (26.1 %)

Diabetic micro-angiopathy 6

Endoneurial vascular thickening 39.1 %

Vasculitic neuropathy

Histopathological evidence of vasculitic neuropathy was present in 26 nerve

biopsies. This included 16 patients with definite vasculitis, and 10 patients with

probable vasculitis (as per Collins criteria). The findings are summarized in Table –

10.

Definite vasculitis was demonstrated in 10 patients, with evidence of fibrinoid

necrosis in medial coat seen in 10(55.6 %) cases, best demonstrable in Massons

trichrome stains. There was predominant involvement of nutrient vessels in

45

epineurium, showing segmental infiltration of vascular walls with irregular dilatation

and narrowing, peri-vascular cuffing by lymphocytes, macrophages and plasma cells

in surrounding epineurial small arterioles and venules. 10 cases with probable

vasculitis showed presence of chronic vascular changes and ischemia with medial

hypertrophy and fibrosis narrowing the lumen. 6 cases had mild endoneurial or

epineurial inflammation, along with vascular changes and sectoral loss. Within the

endoneurium, axonal changes were prominent with acute axonal degeneration

being seen in 13(58.8 %) cases, reflecting acute changes, while axonal regeneration

accompanying chronicity was evident in 10(43.5 %) cases. Myelinated fibre

depletion was seen in 12(66.7 %) cases, non-uniform involvement with fascicle to

fascicle variation was common reflecting ischemia, with some cases demonstrating

sectoral loss in central/perifascicular distribution. Presence of sectoral loss of

myelinated fibres and hemosiderin deposits reflected ongoing vascular injury.

The pre-biopsy diagnosis in these cases was mononeuritis multiplex (n–13), CIDP (n-

3), and neuropathy with undetermined etiology (n-10).

Figure 1 shows nerve biopsy in a case of vasculitic neuropathy, showing inflamed

nutrient blood vessel (large arteriole) in the epineurium exhibiting marked vascular

wall thickening, striking luminal narrowing, and dense perivascular and transmural

inflammation (inflammation extending through the vessel wall). Acute axonal

breakdown (Wallerian degeneration) is evident as clear spaces caused by acute

axonal swelling and granular breakdown of the axoplasm, and the fibre loss is

sectorial and non uniform.

46

Muscle biopsy showed evidence of vasculitis in 3 cases, involving most frequently

small vessels in perimysium or epimysium. Lip biopsy revealed evidence of

sialadenitis in 2 cases, with a clinical diagnosis of Sjogrens syndrome.

HPE results in vasculitis (Table – 10)

Nerve biopsy findings Observation (n-23)

Sub-endoneurial edema 2 (2.7%)

Myelinated fibre loss ( non-uniform) 12 (66.7 %)

Acute axonal breakdown 58.8 %

De / remyelination Uniform : 1 (5.6 %)

Non-uniform : 16 (88.9 %)

Fibrosis Epineurial Endoneurial Perineurial

4 (22.2 %) 6 (33.3 %) 4 (22.2 %)

Epineurial inflammation Endoneurial inflammation Perineurial inflammation

16 (88.8 %) 13 (72.3 %) 15 (83.4 %)

Fibrinoid necrosis of epineurial vessels 10 (55.6 %)

Epineurial vascular changes Vascular thickening Neovascularisation

6 (33.3 %) 2(11.8 %)

Endoneurial vascular thickening Neovascularisation

5.9 % 41.2 %

Hansen’s disease

Nerve biopsy was helpful in 6 cases, to confirm the diagnosis of Hansens disease.

Out of these 6 cases, 4 cases were clinically diagnosed as Hansens disease –

presentation with mononeuritis multiplex, and evidence of hypopigmented skin

patches. One case was a known case of Hansens disease, who had completed course

of MDT nearly one year back, who now presented with ulnar mononeuropathy.

47

Nerve biopsy was helpful to confirm diagnosis of chronic leprous neuritis, in a case

presenting with proximal weakness and areflexia, with evidence of hyper pigmented

skin patches and NCS showing demyelinating neuropathy.

Figure 2 shows features of nerve biopsy in Hansen’s disease, showing expanded

nerve fascicles infiltrated by dense inflammation, forming prominent perineurial

cuffs. 2B shows Hansen’s Neuritis, borderline tuberculoid showing the presence of

a large epithelioid granuloma rimmed by lymphocytes, within a nerve fascicle. 2C

shows case of Hansen’s Neuritis with variable fascicular involvement. 2D shows a

case of borderline lepromatous neuropathy, Fite Faraco stain showing numerous

acid fast Lepra bacilli. 2E is a case of Chronic Hansens disease showing extensive

nerve fibre loss in the myelin stain.

CMT

Three patients who were clinically suspected to have CMT – 1, on the basis of

deformities (champagne-leg deformity, pes cavus, hammer-toes) and there was

evidence of uniform slowing, uniform demyelination had evidence of CMT – 1 on

sural nerve biopsy (uniformly distributed onion bulbs, uniform demyelination).

Three patients, who showed step-wise deterioration with a long history of sensor-

motor complaints, and there was evidence of acquired demyelination (multiple

conduction blocks), inflammatory neuropathy (CSF showing albumin-cytological

dissociation), showed biopsy features suggestive of HMSN-1(lack of inflammation,

and uniformly distributed onion bulbs). CMT – 2 was confirmed in 4 cases

48

presenting clinically as hereditary neuropathy, showing axonopathic changes in

NCS.

Figure 2(F-H) shows histopathological features in a case of HMSN 1, showing

expanded fascicle filled with uniformly distributed concentric Schwann cell hyper

plastic units (‘onion bulbs’), Masson’s trichrome stain for collagen highlighting the

layered pattern of the ‘onion bulbs’ due to alternating layers of collagen and

Schwann cell membrane and uniform reduction in myelinated fibers. Figure 2(I-J)

showing histopathological features in a case of HMSN 2, showing atrophic nerve

fascicle with no ‘onion bulbs’, uniform fiber loss with a striking involvement of large

diameter fibers, and presence of axonal sprouting indicating few regenerating

clusters (three or more myelinated fibers close together).

Figure 4 showing 3 cases : First cases (4G – H), a clinically compatible case of CIDP

showing features of hypertrophic demyelinating neuropathy mimicking a

hereditary etiology, with relatively uniformly distributed ‘onion bulb’, Schwann cell

units, absence of inflammation and mildly thickened epineurial vessels with no

inflammation or vasculitis. Second case (4 I-J). showing a clinically suspected case

of CIDP with histology mimicking HMSN (axonal). Third case (4 K-L), showing a

nerve fascicle with no onion bulbs or inflammation, and a moderate degree of

relatively uniform fiber loss, large fiber loss is somewhat more prominent than

small.

49

Undiagnosed/Idiopathic neuropathies :

Thirty-one patients, with peripheral neuropathy in whom the etiological diagnosis

could not be ascertained, based on the available clinical and laboratory

investigations, underwent nerve biopsy. In these patients, vasculitis was diagnosed

in 7, demyelination in 3 and diabetic neuropathy in 3 cases, and two cases with

Hansens disease. Hence, the nerve biopsy was helpful to yield information which

enabled initiation of definite treatment in 15 out of 31 patients (48.4 %).

Figure 3(C-F) shows nerve biopsy in Metachromatic Leucodystrophy, displaying

several macrophages with red granular material, demyelination evident on myelin

stain, and metachromatic storage material (sulfatide) showing golden brown color

on staining with cresyl violet (metachromasia). Figure 3(G-K) showing nerve biopsy

in a case of Giant axonal neuropathy (GAN) which displays several enlarged,

markedly distended axons, and thinned out myelin sheaths surround the swollen

axons (secondary demyelination).

50

Co-relation of pre- and post-biopsy diagnosis(Table-11)

Clinically suspected diagnosis

N Biopsy diagnosis N

CIDP 24 CIDP 15

Vasculitis 2

Diabetic polyneuropathy

2

CMT 3

Chronic neuropathy 1

Hansens disease 1

Mononeuritis multiplex

23 Definite vasculitis 9

Probable vasculitis 12

Hansens disease 2

Hansens disease 5 Chronic leprous neuritis

1

BT 2

BL 2

Metachromatic leucodystrophy

1 MLD 1

Undetermined etiology

31 Vasculitis 7

Diabetic neuropathy 3

Hansens disease 2

Chronic axonopathy 15

Demyelination 3

Giant axonal neuropathy

1

CLINICO-PATHOLOGICAL CORELATION

The best possible pre-biopsy diagnosis was arrived at based on the available clinical,

laboratory and electrophysiological data. Clinico-pathological co-relation and

treatment initiated subsequently is summarized in Table – 11. Mononeuritis

multiplex was the most common indication for biopsy in 30 patients. Nerve biopsy

was helpful to confirm the clinical diagnosis of vasculitis in 16 cases, Hansen’s

disease in 2 patients, and establish histopathological diagnosis of vasculitis in 7

patients with undifferentiated neuropathy. These included 3 patients, positive for

51

Anti-Ro, Anti-Jo and Anti-SSA antibodies. 24 patients with clinical diagnosis of CIDP,

nerve biopsy was helpful to confirm the diagnosis in 16 cases, 2 cases had features

suggestive of vasculitis, and 2 cases showed histopathological features of diabetic

neuropathy. Nerve biopsy was thus helpful to delineate the treatment plan in these

groups of patients. 3 cases presenting with long standing neuropathy and

deformities, NCS showing multiple conduction blocks and presence of CSF albumino-

cytological dissociation, Nerve biopsy showed no evidence of inflammation and

uniformly distributed onion bulbs. These patients showed improvement following

immunomodulatory treatment with IVMP, oral steroids.

Utility of nerve biopsy:

In peripheral neuropathy with undetermined etiology, nerve biopsy revealed

diagnostic features in 15 patients, and proved essential for management. This

included cases with vasculitis (n-7), Hansen’s disease (n-2), diabetic neuropathy (n-

3), demyelinating neuropathy with no inflammation(n-3). Nerve biopsy was helpful,

to confirm the clinical / pre-biopsy diagnosis, in 44 cases, these cases included CIDP

(n-16), and mononeuritis multiplex (n-16), Hansens disease (n-5), CMT (n-5),

Metachromatic leucodystrophy and Giant axonal neuropathy one case each. In 3

cases with a pre-biopsy diagnosis of CIDP, 2 turned out to be Diabetic neuropathy,

and one was a non-specific chronic axonopathy. In 16 patients, nerve biopsy was

suggestive of chronic axonopathy, with undetermined etiology. Thus, nerve biopsy

provided some form of information that was helpful in initiating therapy or guided

focused investigation in 63 out of 84 patients(79.7 %).

52

Treatment details following biopsy (Table – 12)

Clinically suspected diagnosis

Biopsy diagnosis Treatment

CIDP(N-24) CIDP(N-16) PLEX (6)

IVIG(5)

IVMP(10)

Oral steroids(6)

Azathiopurine(6)

Cyclophosphamide(1)

MMF(1)

Vasculitis(N-2) IVMP, Oral steroids

Diabetic polyneuropathy(N-2)

Supportive treatment

CMT(N-3) IVMP, oral steroids

Chronic neuropathy(N-1) --

Hansens disease(N-1) MDT

Mononeuritis multiplex (N-23)

Definite vasculitis(N-9) IVMP(16)

Oral steroids(10)

Probable vasculitis(N-11) IVMP(16)

Oral steroids(10

Hansens disease(N-2) MDT

Hansens disease(N-5) Chronic leprous neuritis(N-1)

MDT

BT(N-2) MDT

BL(N-2) MDT

Metachromatic leucodystrophy(N-1)

MLD(N-1) --

Undetermined etiology (N-31)

Vasculitis(N-7) Oral steroids

Diabetic neuropathy (N-3) --

Hansens disease(N-2) MDT

Chronic axonopathy(N-15) --

Demyelination(N-3) --

Giant axonal neuropathy(N-1)

--

Outcomes on follow-up of patients (following initiation of definite treatment)

Response to treatment was decided on the basis of data available in MRD. The

follow-up was available in 62 patients. Among 16 patients diagnosed with CIDP, 4

patients have a gradually progressive course with frequent relapses, on plasma

exchanges. These patients are on regular follow-up, while being on immune-

53

modulation (steroids and azathiopurine). Biopsy was helpful to delineate the

diagnosis of vasculitis in 7 cases with undetermined etiology, thereby initiation of

definite treatment in these cases. However, in 19 cases no definite diagnosis could

be established after biopsy, 12 of these cases are on regular follow-up (10 have

remained status quo, while 2 have shown improvement gradual improvement).

54

FIGURE 1

55

FIGURE 2

56

FIGURE 3

57

FIGURE 4

58

FIGURE 1:

A- E: Chronic inflammatory demyelinating polyneuropathy (CIDP). A: An expanded

nerve fascicle is surrounded by thickened perineurium. B: Perivascular

inflammation and endoneurial edema within the fascicle. C: The myelin special

stain reveals a nonuniform loss of myelinated fibers and several fibers show

thinned out myelin rings indicating demyelination. D-E: Chronic de- and re-

myelination leads to concentric ‘onion bulb’ Schwann cell hyperplasia which in this

figure is prominent throughout the fascicle mimicking a hereditary etiology,

however the myelin stain reveals the strikingly nonuniform affection of the fascicle

indicating the acquired nature of the underlying pathology (E).

F-M: Vasculitic neuropathy. F-G: The inflamed nutrient blood vessel (large arteriole)

in the epineurium exhibits marked vascular wall thickening, striking luminal

narrowing, and dense perivascular and transmural inflammation (inflammation

extending through the vessel wall) (G). H: Vasculitis involving the smaller calibre

vessels. The adjacent nutrient vessel is not involved. I-J: Acute axonal breakdown

(Wallerian degeneration) is evident as clear spaces caused by acute axonal swelling

and granular breakdown of the axoplasm. This is seen in the longitudinal section (J)

as myelin ovoids formed by secondary collapse and breakdown of the myelin

consequent to acute axonal loss of integrity. K: Chronic vasculitis with past micro

bleeds may lead to hemosiderin pigment formation (K) whose detection is made

easier by the Perl’s Prussian blue stain (inset). L: Shows a previously thrombosed

59

vessel with reparative recanalisation of the lumen. M: The fibre loss is sectorial and

non uniform (M).

FIGURE 2:

A-E: Hansen’s Neuritis. A: Expanded nerve fascicles are infiltrated by dense

inflammation, forming prominent perineurial cuffs. B: Hansen’s Neuritis,

borderline tuberculoid. A large epithelioid granuloma rimmed by lymphocytes, is

seen within a nerve fascicle. C: Hansen’s Neuritis. Variable fascicular involvement is

evident. D: Fite Faraco stain shows numerous acid fast Lepra bacilli in a case of

borderline lepromatous neuropathy. E: Extensive nerve fibre loss in Hansen’s

neuritis in the myelin stain.

F-H: Hereditary demyelinating neuropathy - HMSN 1. F: The fascicle is expanded

and filled with uniformly distributed concentric schwann cell hyperplastic units

(‘onion bulbs’).

G: Masson’s trichrome stain for collagen highlights the layered pattern of the

‘onion bulbs’ due to alternating layers of collagen and Schwann cell membrane. H:

Uniform reduction in myelinated fibers- a feature characteristic of hereditary

neuropathy.

I-J: Hereditary axonal neuropathy - HMSN 2. I: The nerve fascicle is mildly atrophic

with no ‘onion bulbs’. J: Uniform fiber loss with a striking involvement of large

diameter fibers. Few regenerating clusters (three or more myelinated fibers close

60

together) are present indicating axonal sprouting as a reparative reaction of

chronic axonopathy.

(Stains- A,B,C,F,I : H&E. G: Masson’s trichrome. D: Fite Faraco for lepra bacilli. E,H:

Kpal for myelin.

Original magnification: D: x400; F,G, E,H,J: x200; B,C,I: x100; A x40).

FIGURE 3:

A-B: Hereditary neuropathy with liability to pressure palsies (HNPP). A: A fiber with

very thick myelin sheath stands out amidst fibers with myelin sheaths of normal

thickness. B: The myelin stain highlights two such hypermyelinated fibers

(‘tomaculi’).

C-F: Metachromatic Leukodystrophy. C-D: Longitudinal sections display several

macrophages with red granular material. E: The myelin stain reveals thin myelin

sheaths indicating demyelination. F: Metachromatic storage material (sulfatide)

shows golden brown colour on staining with cresyl violet (metachromasia).

G-K: Giant axonal neuropathy (GAN) G-I: Transverse sections display several

enlarged, markedly distended axons. J: Longitudinal section of the segmentally

bloated axon.

K: Thinned out myelin sheaths surround the swollen axons (secondary

demyelination).

61

(Stains- C,D,G,H,I,J: H&E. A: Masson’s trichrome. E&K: Kpal for myelin. F: Cresyl

violet.

Original magnification: A,K,F: x400; B,C,D,E,J,K: x200; H x100; G x40).

FIGURE 4:

A-F: Diabetic neuropathy. A: Low power view of a nerve fascicle bordered by

thickened perineurial sheath. B-C: Endoneurial hyaline arteriosclerosis, a feature of

diabetic microangiopathy. D: Chronic axonopathy. The endoneurium contains

numerous ‘Bands of Bungner’ - empty Schwann cell units. E: Diabetic neuropathy

with CIDP. Endoneurial lymphocytic inflammation. F: Diabetic neuropathy. Fiber

loss, involving small more than large myelinated fibers.

G-J: A clinically compatible case of CIDP showing features of hypertrophic

demyelinating neuropathy mimicking a hereditary etiology. G: Moderately

expanded nerve fascicles. H: Relatively uniformly distributed ‘onion bulb’, Schwann

cell units. Note the absence of inflammation. I: Thinly myelinated fibers. J: Mildly

thickened epineurial vessels with no inflammation or vasculitis.

K-L: A clinically suspected case of CIDP with histology mimicking HMSN (axonal)

K: Nerve fascicle with no onion bulbs or inflammation. L: A moderate degree of

relatively uniform fiber loss, large fiber loss is somewhat more prominent than

small.

(Stains- A,B,D,E,G-K: H&E. C: Masson’s trichrome. F&L: Kpal for myelin.

Original magnification: I: x400; B,C,D,E,H, K: x200; A, F, L: x100; G x40).

62

DISCUSSION

The diagnostic work-up in patients with peripheral neuropathy is tailored to each

individual based on the presenting history and examination, topographic pattern

and evolution of clinical signs. Even after detailed evaluation, in 20 % of axonal

neuropathies, the etiology remains elusive. 63 In this study, we studied the clinical-

pathological data of 84 subjects with established diagnosis of peripheral

neuropathy who underwent nerve biopsy.

Clinical features

A male pre-ponderance of patients was noted in our study, with Male: Female ratio

being 53 : 31. Study by Chia etal, also reports majority of subjects being males 64.

Majority of cases (88 %) presented with a chronic progressive clinical course,

similar to study by Chia et al, who also reported a chronic course in 68 % of the

patients at the time of biopsy 64. Majority of patients (19) were in between the age

group 60-70 years, in addition 11 patients were between age group 50-60 years

and 10 each in between the age group 10-20 years and 30-40 years. In nearly 54

cases (77 %), the symptom onset was in lower limbs (77 %, n – 54), with sensory

disturbances in form of paraesthesias in 32.9 %, and sensory loss in 17.7 % of

patients. Proprioceptive loss was documented in 36 (45.6 %) patients, impaired

pain and temperature in 38 (47.5 %) patients. Pan-sensory loss involving all

modalities was present in 57.6 % patients. Motor system involvement in form of

wasting was noticed in 21.6 % of patients and weakness was noticed in 77.4 %

63

patients. Study by Chia et al, reported motor deficits in 71 %, paraesthesias of

varying severity in 76 %, and sensory loss in 83 % of patients, and most patients

had distal predominant type of neuropathy, documented in nearly 40.2 %64.

The most common pattern was symmetrical sensori-motor axonopathic

neuropathy (29.1 %), followed by asymmetric sensori-motor axonopathic

neuropathy(20.3 %) and mononeuropathy multiplex (17.7 %). Sensori-motor

demyelinating neuropathy with superimposed axonopathic changes were present

in 27.8 % cases. Six patients had evidence of conduction blocks and temporal

dispersion. Chia et al reported distal and symmetrical pattern in 56 % of patients,

distal asymmetrical in 15 % of the patients, and focal/multifocal in 29 % of the

patients 64.

Co-morbidities

The co-morbidities of these patients were noted to assess the role of these factors

in the causation or contribution in each case. Diabetes mellitus was present in 25.5

% of total number of cases in this study. Chia et al reported 6 patients with

diabetes mellitus out of 100 patients who underwent nerve biopsy for disabling

neuropathy 64. Other co-morbidies included Systemic hypertension (7.5 %),

Coronary artery disease (6.5 %), and hypothyroidism (5.1 %). A history of

malignancy was present in 2 cases, in whom a paraneoplastic etiology was

suspected, and 4 cases had history suggestive of undifferentiated polyarthritis in

past.

64

Vasculitic neuropathy

Nerve biopsy was indicated in 23 cases presenting with clinical profile suggestive of

mononeuritis multiplex. Among these patients clinically suspected to have

vasculitis (n- 23), nerve biopsy confirmed the presence of vasculitis in 20 patients

including 9 patients with definite vasculitis and 11 patients with probable vasculitis.

Thus 86.9 % of patients in whom vasculitis was clinically suspected had biopsy

confirmation. Hansen’s disease was diagnosed in one patient from the group with

clinically suspected vasculitis.

Overall, 30 patients had a biopsy diagnosis of vasculitis, including 10 patients with

definite vasculitis and 20 patients with probable vascultis. This is lower when

compared to the study by Chia et al where 35 patients had evidence of vasculitis in

nerve biopsy 64. In a retrospective review of 355 patients, Deprez etal found that

vasculitis was suspected in 10 % of patients, and nerve biopsy provided helpful and

essential information in 50-60 % of patients, this study showed that findings

specific or highly suggestive of necrotizing vasculitis were found in 18 patients with

a mean age of 58 years16. Prayson reported that a significant proportion of

vasculitic neuropathies occurs in the setting of systemic vasculitis, most commonly

polyarteritis nodosa, or may be confined to peripheral nervous system 65. In

patients with systemic vasculitis, a polyneuropathy picture is the most common, in

contrast to isolated peripheral nervous system vasculitis, where a mononeuropathy

multiplex pattern is seen 65. Rheumatoid arthritis is one of the common causes of

systemic vasculitis. The histological findings overlap with those of polyarteritis

65

nodosa, and include necrotizing vasculitis of epineurial vessels42. Peripheral

neuropathy complicates SLE in nearly 5 – 22 % cases, and Sjogrens syndrome in 5 –

40 % of cases. Neuropathy in SLE is usually a distal symmetrical polyneuropathy.

Sjogrens syndrome commonly manifests as sensory ganglionitis, autonomic

neuropathy, or polyneuropathy 43. Peroneal nerve is the most frequently affected

peripheral nerve in vasculitic neuropathy. Sural nerve is invariably affected in

length dependant neuropathies like diabetic or alcoholic neuropathy; it is not the

most common site of affection by vasculitic neuropathy 66. In fact, sural nerve

yields unequivocal evidence of vasculitis in only 20 % of cases compared to

peroneal nerve biopsy that has a sensitivity of 60 %32, 49. Sural nerve biopsy was

done in 24 cases. In 4 patients with predominant upper limb involvement, biopsy of

dorsal cutaneous branch of ulnar nerve was done in 3 patients, and superficial

branch of radial nerve biopsy in one patient. In ten patients, a combined nerve –

muscle biopsy was done, out of a pre-biopsy diagnosis of vasculitis was offered in

23 patients. Combined nerve-muscle biopsy was additionally useful to diagnose

vasculitis in 3 cases. Previous studies have shown that combined superficial

peroneal nerve and peroneus tertius muscle biopsy increases the diagnostic yield

for vasculitis by 15 – 40 % 16, 32, 49. Patients with isolated vasculitis of the peripheral

nervous system have a better prognosis and response to immunomodulatory

therapy compared to those with systemic vasculitis. Some patients with vasculitis

restricted to peripheral nervous system may eventually develop evidence of

systemic disease 65. Of the 30 patients, in the present cohort who was proven to

have vasculitis by biopsy, 4 had a systemic vasculitic disorder characterized by

66

positive auto antibodies. In the remaining 26 patients, non-systemic vasculitis or

isolated vasculitis of peripheral nervous system was considered. The nerve biopsy

has an unchallenged role in the diagnosis of these patients for whom a diagnosis of

vasculitis in the nerve cannot be obtained by any other means. This large

proportion of patients (32 %), who are potentially treatable with immune-

modulators, justifies the role of nerve biopsy in the management.

Hansen’s disease was seen in 6 patients in our study. This is higher than that

reported in other studies of neuropathy. This is probably a reflection of the endemic

nature of the disease. Out of these 6 cases, 4 cases were clinically diagnosed as

Hansen’s disease – presentation with mononeuritis multiplex, and evidence of

hypopigmented skin patches. Nerve biopsy was helpful to confirm leprous neuritis in

2 cases, with neuropathy and undetermined etiology.

Diabetic neuropathy

Two cases had superimposed changes suggestive of diabetic neuropathy and CIDP,

characterized by endoneurial lymphocytic inflammation, and fiber loss, involving

small more and large myelinated fibers. Three cases showed features of diabetic

neuropathy, with thickened perineurial sheath, and endoneurial hyaline

arteriosclerosis, a feature of diabetic microangiopathy. Diabetes mellitus was

present in 21(25.5 %) cases in our study. In the largest clinical studies of

neuropathy in the elderly, diabetes was found to be the commonest cause of

67

neuropathy accounting for 41 % of all cases. This declined with age, commensurate

with an increased proportion of idiopathic neuropathies 67.

CIDP

The diagnosis of CIDP was established in 24 cases on the basis of clinical,

electrophysiological results (as per EFNS 2010 guidelines). Sural nerve biopsy

findings were consistent with diagnosis of CIDP in 16 cases. The most common

histopathological findings were non-uniform loss of myelinated fibres in 78.3 %

cases and evidence of demyelination/remyelination, followed by sub-endoneurial

edema, in 71.3 % cases, endoneurial inflammation in 43.5 % cases and non-

uniform distribution of onion bulbs in 28 % cases. Epineurial inflammation and

axonopathic changes co-related with progressive and severe course of CIDP, seen

in 4 cases. In a study done by Vallat et al, out of 44 suspected cases of CIDP, nerve

biopsy examination was helpful for diagnosis of CIDP in 8 patients (demyelination,

loss of myelinated fibers, cellular infiltrates, and onion bulb proliferation). who

These eight patients had normal results on nerve conduction studies, and based on

histopathology results they were started on immunomodulation, and showed a

significant positive response. 35 In a study done by Kulkarni et al , in forty-six

patients with idiopathic CIDP at NIMHANS, (32 with progressive course and 14 with

relapsing-remitting course) satisfying AAN clinical and electrophysiological criteria

for CIDP, frequency of four supportive pathological alterations - demyelination,

inflammation, onion bulb formation, and axonal changes in sural nerve biopsies

were seen in nearly 100% of cases. Electrophysiological abnormalities were

68

detected in 90.8%, suggesting that supportive histologic AAN criteria are helpful in

diagnosis of CIDP 37.

Endoneurial inflammation was frequent in the relapsing-remitting form and

epineurial inflammation and axonal changes in those with progressive course.

Greater disability at presentation, poor response to immunomodulation, and lower

CSF protein levels was seen in those with axonal pathology. In addition, 3 cases

which had shown evidence of acquired demyelination in form of multiple

conduction block and temporal dispersion, and CSF study showing albumin-

cytological dissociation, there was no evidence of inflammation on sural nerve

biopsy and there was uniform distribution of onion bulbs ( suggestive of CMT).

Patients with no pre-biopsy diagnosis

31 patients had no pre-biopsy diagnosis. Nerve biopsy revealed vasculitis in 7

patients, with 1 case showing definite vasculitis, and 6 cases showing features of

probable vasculitis. A diagnosis of Hansen’s disease was made in 2 cases and

features suggestive of diabetic neuropathy were present in 3 cases. One case

showed features suggestive of Giant axonal neuropathy on nerve biopsy. In 15

cases, chronic axonopathy was found without further clues from the nerve biopsy.

Thus nerve biopsy proved to be essential in 3 cases, and helpful in 12 cases out of

31 cases with un-determined etiology. In 15 cases (52 %), the biopsy was either

essential (n-3) or helpful (n-12). Hence, nerve biopsy findings were useful in this

group with no definite pre-biopsy diagnosis despite investigations and had been

69

useful for guiding treatment and prognosis in 15 out of 31 cases. This would be

helpful in resource limited settings, wherein financial and human resources are

limiting factors.

Nerve biopsy: utility and side effects

The indication for performing nerve biopsy should be balanced against the

expected diagnostic yields and post-surgical complications after biopsy. The

diagnostic yield of nerve biopsy ranges from 24 – 94 % 1, 12, 16, 68. Nerve biopsy is

associated with residual complications and morbidity in 33 % of patients 1.

20 patients who underwent nerve biopsy were diagnostic in our study including 9

patients with definite vasculitis, 5 patients with Hansens disease and 6 patients

with features of inflammatory demyelinating neuropathy. These biopsies confirmed

the diagnosis in each case. Another 40 patients with nerve biopsies, biopsy was

helpful, since the findings helped to confirm, or exclude the clinical diagnosis.

These included 18 cases with probable and possible vasculitis, 13 cases with

inflammatory demyelinating neuropathy, 5 cases with Hansens disease, 2 cases

with diabetic neuropathy and chronic axonopathy(with past history of nutritional

neuropathy). Nerve biopsy was not helpful in 16 patients.

The current study shows that nerve biopsy proved to be essential and helpful in 20

patients and 40 patients respectively. Several studies have shown varying results

of yield of nerve biopsy in peripheral neuropathies. Study on 44 cases of peripheral

neuropathy undergoing nerve biopsy, Gabriel et al concluded that nerve biopsy

70

contributed in the therapeutic management in nearly 60 % of patients and

confirmed the suspected diagnosis in 70 %. The biopsy altered the diagnosis in 14

%, and was non-contributory in 16 % 1. Another study by Neundorfer et al showed

that 27 % of nerve biopsies were essential, by establishing definite diagnosis, and

37 % of biopsies were helpful, by providing valuable diagnostic information

confirming the clinical diagnosis68. Similarly, study by Oh et al reported that 24 %

of biopsies established specific diagnosis and provided helpful or diagnostic

information in 45 % 12. Chia et al showed a much higher yield of nerve biopsy, being

diagnostic in 94 % of patients, by making use of combination of histological and

clinical data64 .

Several clinical and histo-pathological factors affect the diagnostic yield of nerve

biopsy16. The pre-biopsy diagnosis of vasculitis, inflammatory demyelinating

disease, HMSN, distribution of symptoms in a multifocal and asymmetrical fashion

and symptom onset to biopsy interval of less than 6 months are associated with a

better yield. The higher yield in multifocal and asymmetric neuropathies is related

to a high proportion of vasculitis in these patients 16. Combined muscle nerve

biopsy increased the diagnostic yield for vasculitis, and serial sections on frozen

paraffin embedded and resin embedded material improves the sensitivity for

diagnosis of interstitial pathology.

In our study, 14 patients had axonal neuropathy of unknown etiology. Previous

studies have shown that despite extensive evaluation, the etiology of neuropathy

remains undiagnosed in 10 to 20 % of patients. These patients most commonly

71

have axonal polyneuropathy, this entity is called Chronic Idiopathic Axonal

Polyneuropathy (CIAP)69. This disorder presents with an insidious onset, slowly

progressive distal symmetrical sensory or sensori-motor symptoms in the 6th

decade, and is not associated with severe disability even after 5-10 years into the

disease process69.

72

SUMMARY AND CONCLUSION

This retrospective study analyzed 84 patients with established diagnosis of

peripheral neuropathy who had undergone nerve biopsy at SCTIMST during the

period 2010-2015, to determine the clinical, electrophysiological and pathological

profile of peripheral neuropathy and further examine the utility of nerve biopsy in

providing diagnostic, therapeutic or prognostic information in the clinical

management of patients with peripheral neuropathy.

Majority were males (M: F= 53:31), with a mean age at nerve biopsy of 59.8

years. The mean duration of symptoms(prior to nerve biopsy) was 12.6

months.

Majority of patients (n-19) were in the age group 60-70 years, 11 patients

were in the age group 50-60 years and 10 each in the age group 10-20 years

and 30-40 years, respectively.

The majority of patients had chronic progressive clinical course, seen in 79.7

% patients. Motor involvement (weakness in 84.8 % and wasting in 19 %)

was more common than sensory symptoms in the form of paraesthesias (43

%), impaired sensations (22.8 %).

Most common co-morbidities seen in this group of patients were Diabetes

mellitus(25.5 %), and Hypertension(7.5 %) cases.

Most common clinical and electrophysiological pattern of neuropathy

observed was Symmetric sensori-motor axonopathic neuropathy (29.1 %),

73

sensori-motor predominantly demyelinating neuropathy (27.8 %), followed

by mononeuritis multiplex (17.7 %) cases.

53 (63 %) patients had a pre-biopsy etiological diagnosis on the basis of

available data. The pre-biopsy diagnosis was CIDP (n-21), vasculitis(n-20),

Hansens disease(n-5), Metachromatic leucodystrophy(n-1). In the rest (n-

31), no definite etiological cause for neuropathy could be established

before biopsy.

Most common histopathological features seen in patients with CIDP were

non-uniform loss of myelinated fibres (78.3 %), followed by sub-endoneurial

edema (71.3 %), endoneurial inflammation (43.5 %) and non-uniform

distribution of onion bulbs (28 %) cases. Epineurial inflammation and

axonopathic changes co-related with progressive and severe course of CIDP,

seen in 4 cases.

Most common histopathological features in patients with definite vasculitis

were fibrinoid necrosis in medial coat (55.6 %), peri-vascular cuffing by

lymphocytes, macrophages and plasma cells in surrounding epineurial small

arterioles and venules (51.6 %), medial hypertrophy and fibrosis narrowing

the lumen(30.6 %) and evidence of acute axonal degeneration (28.8 %)

cases.

Among 24 cases with pre-biopsy diagnosis of CIDP, nerve biopsy

histopathology confirmed the clinical diagnosis in 15 cases,

histopathological diagnosis in the remaining 9 cases was: vasculitis (n-2),

diabetic polyneuropathy(n-2), CMT(n-3), Hansens disease(n-1) and chronic

74

neuropathy(n-1). Histopathological diagnosis in 23 cases with prebiopsy

diagnosis of mononeuritis multiplex was definite vasculitis(n-9), probable

vasculitis(n-12), and Hansens disease(n-2). Nerve biopsy confirmed the

clinical diagnosis of Hansens disease(n-5), and Metachromatic

leucodystrophy(n-1).

In 31 cases(37 %) without definite diagnosis, despite extensive work-up,

nerve biopsy proved to be essential in 3 cases (definite vasculitis-1,

Hansen’s disease-2). Besides, nerve biopsy was helpful in 12 cases (probable

vasculitis-6, diabetic neuropathy-3, Giant axonal neuropathy-1,

demyelinating neuropathy without inflammation-2). Thus, nerve biopsy

proved to be essential and helpful in 16 out of 31 cases with no pre-biopsy

diagnosis (51.6 %).

Our study suggests that nerve biopsy becomes useful in potentially

treatable peripheral neuropathies, particularly in patients with

Mononeuritis multiplex, or in patients with undetermined etiology. Also it

clarified the etiology in patients presenting with demyelinating CIDP.

75

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study of the usefulness of sural nerve biopsy. J Neurol Neurosurg Psychiatry

69: 442-446.

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84

PROFORMA

1.Identification information

1.1 Serial number ---------------------------

1.2 Unique identification number ---------------------------

2. Demographic data

2.1 Age --------------------------- years

2.2 Sex --------------------------- 1. Male 2. Female

2.3 Occupation ------------------------

2.4 Year of registration ------------------

3. History(1 = Yes, 0 = No)

3.1 Age of onset -----------------------

3.2 Duration of illness ------------------------

3.3 Developmental delay -------------------------

3.3.1 If yes, specify------------------------

3.4 Poor athletic performance -------------------------

3.5 Distal lower limb weakness ------------ Duration ----------------

3.6 Proximal lower limb weakness --------- Duration ----------------

3.7 Distal upper limb weakness ------------ Duration ----------------

3.8 Proximal lower limb weakness --------- Duration ----------------

3.9 Gait difficulty --------- Duration ------------------

85

3.10 Falls--------------- Duration --------------------

3.11 Neck weakness ---------------

3.12 Trunk weakness ---------------

3.13 Respiratory difficulty --------------

3.14 Cranial nerve involvement -----------

3.14.1 I yes, specify -------------------

3.15 Sensory symptoms --------------- Duration ----------------

3.15.1 If yes, specify ---------------

3.16 Pain ----------------

3.17 Cramps-----------------

3.18 Deformities----------------

3.19 Functional status -----------------

(0 = normal, 1 = mild difficulty, independent, 2= moderate difficulty, independent,

3 = partly dependent, 4 = bed/ wheel chair bound)

4.Other history ( 1= Yes, 0 = No)

4.1 Other illnesses ---------------------------------

4.1.1 If yes, details ---------------------------------

4.2 Family history ---------------------------------

4.2.1 If yes, details ---------------------------------

4.3 Consanguinity ----------------------------------

86

4.9.1 If yes, details -----------------------------------

5.Physical signs (1 = Yes, 0 = No)

5.1 Systemic signs ---------------- Specify ---------------

5.2 Cranial neuropathy --------------- Specify -----------------

5.3 Deformities ---------------

5.3.1 Pes cavus ----------------

5.3.2 Hammer toes ------------------

5.3.3 Equinovarus ------------------

5.3.4 Claw hands ------------------

5.3.5 Kyphoscoliosis ------------------

5.3.6 Others ------------------

5.4 Contractures ------------------ Specify --------------

5.5 Wasting ------------------ Specify --------------

5.6 Power of lower limbs (Right/ Left)

5.6.1 Toe flexors ------------------

5.6.2 Toe extensors ------------------

5.6.3 Ankle dorsiflexion ------------------

5.6.4 Ankle plantar flexion ------------------

5.6.5 Knee flexion ------------------

5.6.6 Knee extension ------------------

87

5.6.7 Hip abduction ------------------

5.6.8 Hip adduction ------------------

5.6.9 Hip flexion ------------------

5.6.10 Hip extension ------------------

5.7 Power of upper limbs (Right/ Left)

5.7.1 Intrinsic muscles ------------------

5.7.2 Hand grip ------------------

5.7.3 Wrist flexion ------------------

5.7.4Wrist extension ------------------

5.7.5 Elbow flexion ------------------

5.7.6 Elbow extension ------------------

5.7.7 Shoulder abduction ------------------

5.7.8 Shoulder adduction ------------------

5.7.9 Shoulder flexion ------------------

5.7.10 Shoulder extension ------------------

5.8 Axial muscles

5.8.1 Neck flexion ------------------

5.8.2 Neck extension ------------------

5.8.3 Trunk ------------------

5.9 Reflexes

88

5.9.1 Biceps jerk ------------------

5.9.2 Supinator jerk ------------------

5.9.3 Triceps jerk ------------------

5.9.4 Knee jerk ------------------

5.9.5 Ankle jerk ------------------

5.9.6 Abdominal reflex ------------------

5.9.7 Plantar response ------------------

5.10 Sensory involvement ------------------

5.10.1 Pain impairment ------------------

5.10.2 Touch impairment ------------------

5.10.3 Vibration impairment ------------------

5.10.4 Joint position sense impairment ------------------

5.10.5 Pattern ------------------

(1= stocking-glove, 2=stocking, 3= glove, 4=asymmetric)

5.11 Gait (describe) ------------------

5.12 Tremors of upper limbs ------------------

5.13 Other signs (specify) ------------------

6. Electrophysiology and nerve biopsy

6.1 Conduction velocity (right/ left)

6.1.1 Median ------------------

6.1.2 Ulnar ------------------

89

6.1.3 Peroneal ------------------

6.1.4 Tibial ------------------

6.2 CMAP amplitude

6.2.1 Median ------------------

6.2.2 Ulnar ------------------

6.2.3 Peroneal ------------------

6.2.4 Tibial ------------------

6.3 Sensory peak latency

6.3.1 Median ------------------

6.3.2 Ulnar ------------------

6.3.3 Sural ------------------

6.4 Sensory amplitudes

6.4.1 Median ------------------

6.4.2 Ulnar ------------------

6.4.3 Sural ------------------

6.5 EMG ------------------

(0=normal, 1= denervation, 2= reinnervation only, 3= not done)

6.6 Final impression ------------------

(1 = Axonal, 2 = demyelinating, 3= intermediate, 4 = indeterminate)

6.7 Final clinical diagnosis --------------------------------

90

7. Laboratory parameters

7.1 Hemoglobin

7.2 ESR

7.3 Total leucocyte count, Differential leucocyte count

7.4 CSF Study

7.5 ANA Profile

8. Nerve biopsy histopathology

8.1 Endothelial edema

8.2 Endothelial fibrosis

8.3 Sub-epithelial edema

8.4 Sub-epithelial fibrosis

8.5 Transmural necrosis

9. Post-biopsy diagnosis

10. Treatment initiated

11. Follow up

11.1 Date of last follow up ------------------

11.2 Total follow up duration ------------------

11.3 Status at last follow up ---

4%SIMILARITY INDEX

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2

3

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5

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Jitesh Goel_Thesis.docxORIGINALITY REPORT

PRIMARY SOURCES

www.bioline.org.brInternet

Anish, L., M. Nagappa, A. Mahadevan, and A. B. Taly."Neuropathy in elderly: lessons learnt from nervebiopsy", Age and Ageing, 2014.Crossref

Said, Gérard. "Indications and Usefulness of NerveBiopsy", Archives of Neurology, 2002.Crossref

G. said. "Contribution of never biopsy f indings tothe diagnosis of disabling neuropathy in theelderly", Brain, 1996Crossref

ael.physic.ut.eeInternet

Prayson, R.A.. "Clinicopathologic study of 43patients with sural nerve vasculitis", HumanPathology, 200305Crossref

A. Gordon Smith. "A Rational Diagnostic Approachto Peripheral Neuropathy", Journal of ClinicalNeuromuscular Disease, 06/2003Crossref

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