A Clinical Study on the Utility of Nerve Biopsy in Peripheral Neuropathy

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

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

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

iii

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

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

1 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.

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.

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,

4 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

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

6 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.

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.

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.

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.

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

10 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.

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

Author, No. Inflammation Demyelination Onion Axonal Mixed Normal of cases bulbs changes Dyck et al 20 54% NA 15% More NA NA (n=26) common than demyelination

Prineas and No significant 26% 40% NA NA 24% MacLeod 24 inflammation (n=23)

Barohn 10% 48% NA 21% 12% 17.9% et al 25 (n=60) (56 biopsied)

Krendel 29% 50% 36% NA NA NA et al 26 (n=14)

Bouchard 18% 68% NA 5% 20% NA et al 27 (n=100) Haq et al 28 36% 50% (teased 58% 71% NA Nil (n=24) fiber) 79% (EM), 93% (semithin) Rizzuto et 25% 100% 48% NA NA NA al 29 (n=105)

Vital et al 100% 40.4% NA 9.5% 76.1% NA 30 (n=42) Vallat et al 75% 100% Variable Variable NA NA 31 (n=44) Kulkarni et 58.7% 82.8% 28.3% 8.7% 23.9% Nil al 19 (n=46) NA – Information not available.

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

13 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

14 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.

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

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.

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.

18 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

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.

20 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.

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).

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

Peripheral Upper Lower Renal GIT Arthralgias Cardiac Skin CNS C-ANCA P-ANCA Nerve Respiratory Respiratory

Microscopi 60 – 70 % -- 20-80 % 70-90% 30% 50% 10-20% 50-70% 10-20% 10-50% 50-80% c polyangiitis PAN 50-70% ------20-60% 50-80% 5-30% 25-50% 5-20% -- -- Churg- 60-80% 50-60% 40-70% 10-40% 30-60% 50% 10-40% 50-60% 5-30% 3-35% 2-50% strauss syndrome Wegeners 40-50% 95% 70-85% 70-80% <5% 60-70% 10-25% 40-50% 5-10% 75-90% 5-20% Granuloma tosis Profile of Secondary vasculitides associated with peripheral neuropathy48

Neuropathy Clinical pattern Associated Laboratory findings Electrophysiology Nerve pathology features Rheumatoid arthritis Uncommon Sensory/sensorimotor Joint-100%, skin RF 90%, ESR(elevated) Distal sensori-motor Necrotising arteritis; IgM 70% 85% axonal, deposits mononeuritis multiplex SLE Rare Distal sensorimotor; Skin 90%, joint 85 ANA 90-100% Distal sensori-motor Necrotising arteritis; mononeuritis %, renal 50 % DSDNA 60% axonal Complement deposits multiplex , cranial Asymmetric Sjogrens syndrome Common Distal sensory cranial; Sicca 50-90%, skin ANA 90%,RA Axonopathy; Perivascular ganglionopathy 75% 60%,SSA/SSB 50% ganglionopathy Infiltrates, axonal loss Systemic Rare Distal symmetrical; Fibrotic skin, ANA 100% Distal motor Endoneurial vasculopathy; Sclerosis cranial; mononeuritis Raynauds Anti-centromere predominant Multifocal axonal loss multiplex phenomenon, antibody 70% neuropathy calcinosis Sarcoidosis Uncommon Distal symmetrical; Pulmonary 90%, ACE 70%,ESR(elevated) Distal sensori-motor Noncaseating granuloma cranial; mononeuritis skin 25% 50% axonal multiplex; Asymmetric radiculopathy Cryoglobulinemia Common Asymmetrical sensori- Purpura 90% Cryoglob 100% Asymmetric sensori- Necritizing arteritis, immune 20-80% motor; Skin ulcers HCV 50-70% motor; complex deposits mononeuritis symmetric multiplex

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 20/26(80%) polyangitis Clausen et al Systemic & NSVN 27/45(60%) (2000) Dyck et al (1987) Systemic & NSVN 31/65 (48%) Hattori et al(2002 Churg Strauss 16/30 (53%) Syndrome Combined total Systemic & NSVN 109/196 (56%) Peroneus brevis & Collins et al (2000) Systemic 15/25 (60%) superficial Collins et al (2003) NSVN 11/19 (58%) peroneal nerve Chia et al ( 1996) Systemic & NSVN 23/33 (70%) Combined total Systemic & NSVN 49/77 (64%)

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

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.

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

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 subcutaneous fat aspiration and nerve biopsy in diagnosing amyloidosis in patients

28 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

29 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.

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.

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

32 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.

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.

Gender distribution

Males : 53(63.8%)

Females : 31 (36.2%)

Figure – 1

Age distribution 20 18 16 14 12 10 8 6 4 2 0 < 10 y 10-20 y 20-30 y 30-40 y 40-50 y 50-60 y 60-70 y > 70 y

Figure – 2

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.

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 %)

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.

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 19 (82.6 %) Sensori-motor 2 (8.5 %) Motor 3 (13 %) Onset of neuropathic symptoms Upper limbs 2 (8.8 %) Lower limbs 21 (91.2 %) Clinical pattern of neuropathy Symmetric sensori-motor neuropathy 11 ( 30.4 %) Asymmetric sensori-motor neuropathy 25 (69.6 %) Symmteric sensory neuropathy 1(4 %) Clinical features Positive sensory symptoms 20 (87 %) Negative sensory symptoms 18 (78.3 %) Weakness in BLL 23 (100 %) Weakness in BUL, BLL 21 (91.3 %) Areflexia 18(78.3 %) Loss of pain, temperature sensations 12 (52.2 %) Loss of JPS, Vibration sensations 20 (87 %) History of falls 12(52.2 %) Rhombergs sign 18 (78.3 %) Cranial nerve involvement 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 7 (38.9 %) Positive sensory symptoms in UL 3 (16.73 %) Motor weakness in UL 1 (5.6 %) Motor weakness in LL 7 (38.9 %) Onset of neuropathic symptoms Upper limbs 5 ( 13.6 %) Lower limbs 31 ( 86.4 %) Clinical pattern of neuropathy Mononeuritis multiplex 11(61.1 %) Diffuse polyneuropathy 7 (38.9 %) Sensori-motor and sensory neuropathy(clinical) Sensori-motor 28 ( 77.8 %) Pure sensory 8 ( 22.2 %) Cranial nerve involvement 4 (22.3 %)

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 %).

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 27 (34.2 %) (b) 20 – 30 mm fall Ist hr 13 (16.5 %) (c) > 30 mm fall Ist hr 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

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

42 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

43 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 6 (26.1 %) Endoneurial 10 (43.5 %) Perineurial 5 (21.4 %) Epineurial inflammation 4 (16.7 %) Endoneurial inflammation 10 (43.5 %) Perineurial inflammation 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

44 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.

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 4 (22.2 %) Endoneurial 6 (33.3 %) Perineurial 4 (22.2 %) Epineurial inflammation 16 (88.8 %) Endoneurial inflammation 13 (72.3 %) Perineurial inflammation 15 (83.4 %) Fibrinoid necrosis of epineurial vessels 10 (55.6 %) Epineurial vascular changes Vascular thickening 6 (33.3 %) Neovascularisation 2(11.8 %)

Endoneurial vascular thickening 5.9 % Neovascularisation 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.

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

47 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.

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).

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

Clinically suspected N Biopsy diagnosis N diagnosis CIDP 24 CIDP 15 Vasculitis 2 Diabetic 2 polyneuropathy CMT 3 Chronic neuropathy 1 Hansens disease 1 Mononeuritis 23 Definite vasculitis 9 multiplex Probable vasculitis 12 Hansens disease 2 Hansens disease 5 Chronic leprous 1 neuritis BT 2 BL 2 Metachromatic 1 MLD 1 leucodystrophy Undetermined 31 Vasculitis 7 etiology Diabetic neuropathy 3 Hansens disease 2 Chronic axonopathy 15 Demyelination 3 Giant axonal 1 neuropathy

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

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 %).

Treatment details following biopsy (Table – 12)

Clinically suspected Biopsy diagnosis Treatment diagnosis 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- Supportive treatment 2) CMT(N-3) IVMP, oral steroids Chronic neuropathy(N-1) -- Hansens disease(N-1) MDT Mononeuritis Definite vasculitis(N-9) IVMP(16) multiplex Oral steroids(10) (N-23) Probable vasculitis(N-11) IVMP(16) Oral steroids(10 Hansens disease(N-2) MDT Hansens disease(N-5) Chronic leprous neuritis(N- MDT 1) BT(N-2) MDT BL(N-2) MDT Metachromatic MLD(N-1) -- leucodystrophy(N-1) Undetermined etiology Vasculitis(N-7) Oral steroids (N-31) 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-

52 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).

FIGURE 1

FIGURE 2

FIGURE 3

FIGURE 4

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 remyelination 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

58 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

59 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).

(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).

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 %

62 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.

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

64 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

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

66 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

67 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

68 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

69 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

70 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.

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 %),

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

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.

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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 ------

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 ------

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 ------

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

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 ------

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 ------

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 ---

Jitesh Goel_Thesis.docx

ORIGINALITY REPORT

4% SIMILARITY INDEX

PRIMARY SOURCES

1 www.bioline.org.br Internet 74 words — 1%

2 Anish, L., M. Nagappa, A. Mahadevan, and A. B. Taly. 74 words — % "Neuropathy in elderly: lessons learnt from nerve 1 biopsy", Age and Ageing, 2014. Crossref

3 Said, Gérard. "Indications and Usefulness of Nerve 35 words — % Biopsy", Archives of Neurology, 2002. < 1 Crossref

4 G. said. "Contribution of never biopsy findings to 21 words — % the diagnosis of disabling neuropathy in the < 1 elderly", Brain, 1996 Crossref

5 ael.physic.ut.ee Internet 21 words — < 1%

6 Prayson, R.A.. "Clinicopathologic study of 43 18 words — % patients with sural nerve vasculitis", Human < 1 Pathology, 200305 Crossref

A. Gordon Smith. "A Rational Diagnostic Approach 7 15 words — % to Peripheral Neuropathy", Journal of Clinical < 1 Neuromuscular Disease, 06/2003 Crossref