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peroneus brevis muscle biopsy in the diagnosis of vasculitic neuropathy
Journal of Clinical Neuroscience 19 (2012) 1392–1396
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Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Usefulness of superficial peroneal nerve/peroneus brevis muscle biopsy in the diagnosis of vasculitic neuropathy J.B. Agadi a,⇑, Govindarajan Raghav a, A. Mahadevan b, S.K. Shankar b a b
Department of Neurology, Bowring and Lady Curzon Hospital, Bangalore 560001, India Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India
a r t i c l e
i n f o
Article history: Received 17 March 2011 Accepted 6 November 2011
Keywords: Diagnostic yield Perls’ stain Peroneus brevis biopsy Sensitivity Specificity Superficial peroneal nerve Vasculitic neuropathy
a b s t r a c t Sensitivity, specificity, and diagnostic yield of the superficial peroneal nerve (SPN)/peroneus brevis muscle (PBM) biopsy in 43 patients with clinically suspected vasculitic neuropathy was studied. Biopsies were classified as ‘‘definite’’, ‘‘suspicious’’ or ‘‘possible’’ in accordance with established criteria. Vasculitis was detected in 27 patients (21 with non-systemic vasculitis, and six with systemic vasculitis). In patients with ‘‘definite’’ vasculitis (n = 13), the sensitivity of SPN/PBM biopsy was 76.4% with 100% specificity. By including patients suspicious for vasculitis (n = 10), sensitivity increased to 85.1% but the specificity dropped to 87.5%. The overall diagnostic yield of SPN biopsy in those patients with definite vasculitis was 76.9% (10/13), and 53.8% (7/13) for muscle biopsy. The addition of muscle biopsy increased the diagnostic yield by 23%. Asymmetric nerve fiber loss, Wallerian degeneration and presence of hemosiderin were statistically significant markers of probable vasculitis. Muscle tissue was more likely to show hemosiderin (85.7%) than a nerve biopsy (71%). A combined SPN/PBM biopsy offers excellent diagnostic yield in the diagnosis of vasculitic neuropathy. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Vasculitides are a heterogenous group of disorders with systemic or localized involvement of various organ systems which is dictated by the caliber of vessels involved. Several forms of vasculitis involve the peripheral nervous system, either as an isolated process (non-systemic vasculitic neuropathy) without other organ involvement, or more commonly, as part of a systemic disease. Despite the development of newer, non-invasive serological tests for a wide range of auto-antibodies, diagnosis of vasculitis neuropathy often relies on pathological demonstration of vasculitis in involved peripheral nerves suitable for biopsy.1–3 Histological evaluation of the sural nerve is most often used for diagnosis. Several studies have examined the sensitivity of sural nerve biopsy in the diagnosis of vasculitic neuropathy.4–7 Mandatory pathologic criteria for a ‘‘definitive’’ diagnosis require demonstration of transmural inflammatory infiltrates in vessel walls, and evidence of vascular destruction with fibrinoid necrosis, endothelial disruption or hemorrhage. These criteria are, however, met in only 20% of sural nerve biopsies, leaving as many as 80% undiagnosed.8–10 To enhance the diagnostic sensitivity, several workers have used alternative features that reflect ischemic injury (multifocal nerve fibre loss and secondary vascular changes without
⇑ Corresponding author. Tel.: +91 80 25592813; fax: +91 80 25591325. E-mail address: [email protected] (J.B. Agadi). 0967-5868/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2011.11.034
distinct vessel wall destruction) in the presence of perivascular inflammation to define ‘‘suspicious’’, ‘‘probable’’ or ‘‘healed’’ vasculitis. Applying these criteria, the pooled diagnostic sensitivity of sural nerve biopsies in different series reaches 56%.11 Said and Lacroix6 and Fort et al.12 documented the presence of vasculitis in muscle biopsy in 45% to 50% of patients with vasculitic neuropathy, and advocated a combined nerve and muscle biopsy to enhance the diagnostic yield. Subsequent studies have evaluated the usefulness of combined sural nerve and quadriceps, gastrocnemius/tibialis anterior or soleus muscle biopsies.8,13–16 Only a few studies have assessed the diagnostic yield of superficial peroneal nerve (SPN)/peroneus brevis muscle (PBM) biopsy in diagnosing vasculitic neuropathy.6,11,17–20 These studies, however, do not address the sensitivity and specificity of SPN/PBM biopsies in diagnosis of vasculitis. Logically SPN/PBM biopsy would be ideal, first on account of technical ease of obtaining a combined nerve and muscle biopsy through a single incision, and second, the peroneal nerve is clinically the most commonly involved nerve in the lower limbs in vasculitis.21 At our Institute, which is primarily a referral centre for neurological disorders, this procedure has been practiced consistently for over a decade. As an audit, this study was undertaken to determine the sensitivity, specificity, and the diagnostic yield of a combined SPN/PBM biopsy in patients with clinically suspected vasculitic neuropathy. The study also aimed to assess the significance of supportive pathologic features in patients who were ‘‘suspicious’’ for vasculitis.
J.B. Agadi et al. / Journal of Clinical Neuroscience 19 (2012) 1392–1396
2. Material and methods 2.1. Participants and clinical data acquisition This study was undertaken as an audit and was approved by the Institutional Scientific Ethics Committee. The muscle and nerve biopsies were performed as diagnostic procedures following the informed consent of patients. All patients with clinically suspected vasculitis who underwent combined SPN/PBM biopsies between 1998 and 2008 at the neurological services of Bowring and Lady Curzon Hospital, a primary and secondary referral centre in South India, were reviewed. The patient clinical and demographic details as well as laboratory findings were retrieved from the medical records. All patients were evaluated clinically by the neurologist (J.B.A.). Electrophysiologic, biochemical and serological testing was performed to exclude other causes of peripheral neuropathy.
2.2. Biopsy and histopathology A full thickness biopsy of the SPN (1.5–2 cm in length) and PBM were obtained by a single incision in the lower third of the leg.22 The histopathologic analysis was carried out at the Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore. The nerve biopsy was fixed in 2.5% buffered gluteraldehyde and processed for paraffin embedding. Sections (2 lm thick) were stained with hematoxylin and eosin (H&E), Masson trichrome stain for collagen, Kulchitsky-Palstain for myelin, and Perls’ Prussian blue for extravasated hemosiderin (iron) from damaged and leaky vessels. In selected sections, immunohistochemistry was performed by the indirect immunoperoxidase technique to label macrophages (CD68, monoclonal, 1:50, Biogenex, Fremont, CA, USA) and Leukocyte Common Antigen (CD45, polyclonal, 1:50, Biogenex) to detect inflammatory infiltrates. Tissue from the peroneus brevis was divided into two: one part was flash frozen in isopentane-cooled liquid nitrogen. Cryosections (8 lm thick) were stained with H&E, modified Gomori trichrome, oxidative enzymes (succinate dehydrogenase [SDH], nicotinamide adenine dinucleotide-teterazolium reductase [NADH-TR]) and adenosine triphosphatase (ATPase) (pH 9.5 and pH 4.6) stains. The other portion was fixed in 10% buffered formalin and processed for paraffin embedding. Deparaffinised sections were stained with H&E, Masson trichrome stain for collagen and Perls’ Prussian blue to demonstrate hemosiderin deposits.
2.3. Biopsy criteria The histological diagnosis of the nerve biopsies were classified as ‘‘definite’’, ‘‘suspicious’’ or ‘‘possible’’ vasculitis in accordance with previously established criteria.11,19,23 Vasculitis was considered definite if at least one blood vessel had transmural infiltration by inflammatory infiltrates in addition to signs of vascular injury such as fibrinoid necrosis, fragmentation of internal elastic lamina or hemorrhage into the vessel wall24 or in the presence of microvasculitis when inflammatory infiltration involved small arterioles (<30–40 microns) that lacked internal elastic lamina as well as venules.25 The biopsy was considered suspicious of vasculitis if, in the absence of transmural vessel infiltration, at least one vessel in the biopsied nerve was rimmed by inflammatory cells in the presence of one of the following supportive pathologic features: vascular wall thickening and sclerosis, narrowing or obliteration of the lumen, thrombosis with or without recanalization, epineurial capillary proliferation/neovascularization, periadventitial hemosiderin
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deposits, asymmetric nerve fiber loss or active Wallerian-like degeneration.19 Sections that showed only supportive features in the absence of inflammation were classified as possible vasculitis.19 Both the suspicious and possible histologic categories required the presence of clinical, laboratory and electrophysiological criteria to be considered as clinically probable vasculitic neuropathy.19 Nerve biopsies with none of the above features or isolated perivascular lymphohistiocytic infiltrates of variable degrees were considered negative for vasculitis.19 In the muscle biopsies, vasculitis was considered definite in presence of necrotizing vasculitis and probable when transmural or perivascular inflammation was not accompanied by necrosis but demonstrated supporting evidence of vascular injury.19 2.4. Clinical criteria Patients were classified as having systemic vasculitic neuropathy (SVN) in the presence of positive auto-antibodies or clinical, radiologic or biopsy evidence of other systemic organ involvement. Patients were classified as having non-systemic vasculitic neuropathy (NSVN) in the absence of the above, according to the criteria of Collins et al.11 2.5. Statistical analysis Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software (version 12) (SPSS, Chicago, IL, USA). A bivariate analysis using Fisher’s exact test was performed to determine the odds ratio and statistical significance of the supportive histological features in the suspicious and possible categories of vasculitis biopsies. 3. Results A total of 43 patients were analyzed in the study: 27 revealed biopsy evidence of vasculitis (21 with NSVN; and six with SVN, of whom four had Churg–Strauss syndrome [CSS], one had rheumatoid arthritis [RA] and one had Sjogren’s syndrome). In all patients with SVN, peripheral neuropathy was the presenting feature. The clinical and laboratory features are summarized in Table 1. The remaining 16 patients were diagnosed with chronic inflammatory demyelinating polyneuropathy or its variants (n = 7), pure neuritic leprosy (n = 6), amyloidosis (n = 1), multiple myeloma (n = 1), and acute inflammatory demyelinating polyneuropathy with asymmetric presentation (n = 1). Clinically, multiple mononeuropathy or asymmetric polyneuropathy was the most common presentation (23/27, 85.1%) followed by distal symmetric polyneuropathy (four of 27, 14.8%). Three patients had cranial neuropathy. A slight male predominance (M:F = 16:11) was noted (Table 1). 3.1. Pathological features The pathological features are described in Table 2. 3.1.1. Definite vasculitis (n = 13) Thirteen patients (48.1%) had definitive evidence of vasculitis in the biopsy (two with necrotizing vasculitis, and 11 with microvasculitis). Both patients with necrotizing vasculitis in muscle and nerve biopsy had systemic vasculitis (one each were diagnosed with RA and CSS) (Supplementary Fig. 1A–C). Evidence of microvasculitis was observed in 11 patients (Supplementary Fig. 1D, E), all of whom had NSVN (nerve alone – six of 11, muscle alone – three of 11, both nerve and muscle, two of 11).
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Table 1 Clinical and laboratory parameters of 27 patients with vasculitic neuropathy Clinical parameter
Entire series (n = 27)
NSVN (n = 21)
Male: female Age in years Duration of symptoms in months Constitutional symptomsa Clinical courseb Pain Paresthesias Cranial nerve involvementc
16:11 53.9 ± 13.43 9.7 ± 13.19 10 (37%) AM-4, SAP-13, CP-9, SM-1 9 (33.3%) 18 (66.6%) 3 (11.1%)
12:9 54.2 ± 13.75 12.0 ± 14.2 4 (19%) AM-1, SAP-10, CP-9, SM-1 6 (28.5%) 15 (71.4%) 2 (9.5%)
Clinical pattern of nerve involvement True MM Extensive overlapping MM DSP
15 (55.5%) 8 (29.6%) 4 (14.8%)
12(57.1%) 5 (23.8%) 4 (19.0%)
Common nerves involved (electrophysiology) Peroneal Sural Posterior tibial Ulnar Median
92.5% 70.3% 22.4% 51.8% 29.8%
95.2% 66.6% 19% 52.3% 53.3%
Electrophysiology Mixed sensory and motor Predominantly sensory
21 (77.7%) 6 (22.2%)
15 (71.4%) 6 (28.5%)
Laboratory parameters Raised ESR Leucocytosis CRP positive Eosinophiliad ANA RA Anemia ANCAe Anti-Rof
12 (44.4%) 8 (29.6%) 3 (11.1%) 4 (14.8%) 4 (14.8%) 5 (18.5%) 3 (11.1%) 2 (7.4%) 1(4.7%)
7 (33.3%) 4 (19.8%) 2 (9.5%) 0 1 (4.7%) 2 (9.5%) 1(4.7%) 0 0
AM = acute monophasic, ANA = anti-nuclear antibody, ANCA = anti-neutrophil cytoplasmic antibodies, CP = chronic progressive, CRP = c-reactive protein, CSS = Churg-Strauss, DSP = distal symmetric polyneuropathy, ESR = erythrocyte sedimentation ratio, MM = multiple mononeuropathy, NSVN = non-systemic vasculitic neuropathy, RA = rheumatoid arthritis, SM = subacute monophasic, SP = subacute progressive. a Constitutional symptoms in our series were fever, weight loss, fatigue, loss of appetite. b Clinical course was characterized using two nominal scales2: 1 = acute – at least one attack of sudden onset symptoms over one to two days or progression of symptoms to plateau in less than four weeks, subacute-progression over four to eight weeks, chronic- progression over more than eight weeks. 2 = monophasic, relapsing, progressive. C sssss in two and hypoglossal in one. d Four patients with CSS had eosinophilia. e ANCA was positive in two patients with CSS (one with p-ANCA; one with c-ANCA). f One patient with anti-Ro antibody was later diagnosed with Sjogren’s syndrome.
In the nerve biopsies, vasculitis involved exclusively the epineurial vessels sparing endoneurial vessels. The infiltrates were predominantly lymphohistiocytic admixed with neutrophils in the presence of necrotizing vasculitis (Supplementary Fig. 1). Axonal pathology predominated with asymmetric fiber loss (12/13) or Wallerian degeneration (seven of 13). Vascular thickening and sclerosis causing luminal narrowing was frequent (12/13). Epineurial capillary proliferation was infrequent (five of 13). Perls’ Prussian blue stain highlighted hemosiderin deposits in eight of 13 (61.5%) surrounding inflamed vessels in the epineurium and rarely in the perineurium. The PBM biopsy revealed vasculitis involving mostly epimyseal vessels in addition to evidence of neurogenic atrophy in seven of 13 patients (53.8%) (two of two with SVN, five of 11 in NSVN). Perls’-positive hemosiderin deposits were seen in all seven patients showing vasculitis (SVN two of two, NSVN five of 11) as fine punctate positivity in the endomysium between myofibres as well as larger clumps surrounding the inflamed large epimyseal or perimyseal vessels (Supplementary Fig. 1F). Perls’ Prussian blue stain was positive in nine of 13 (69.2%) biopsies of patients with definite vasculitis. Of these it was seen in both nerve and muscle in six patients (five with NSVN and one with SVN with RA), nerve alone in two (both with NSVN) and muscle alone in one patient (SVN with CSS).
The overall diagnostic yield of SPN biopsy in patients with definite vasculitis was 76.9% (10/13), and 53.8% (seven of 13) for PBM biopsy. In the NSVN cohort, the diagnostic yield of SPN biopsy was higher [72.7% (eight of 11), than PBM [45.4% (five of 11)]. But in three patients, microvasculitis was detected in muscle biopsy alone. Thus, the addition of muscle biopsy enhanced the diagnostic yield by 23%. In patients with SVN, the nerve and muscle biopsies were equally diagnostic, notwithstanding that the number of patients is small.
Table 2 Biopsy findings in superficial peroneal nerve/peroneus brevis biopsies
Definite Necrotizing vasculitis Microvasculitis Suspicious Possible
Series (n = 27), n (%)
NSVN (n = 21), n (%)
SVN (n = 6), n (%)
2 (7.4) 11 (40.7) 10 (37) 4 (14.8)
0 11 (52.3) 7 (33.3) 3 (14.2)
2 (33.3) 0 3 (66.6) 1 (1.2)
NSVN = non-systemic vasculitic neuropathy, SVN = systemic vasculitic neuropathy.
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3.1.2. Probable vasculitis (n = 14) Features suspicious of vasculitis on histological grounds was noted in 10/27 patients (37%) (three with SVN, seven with NSVN) (F Supplementary Fig. 2). Perivascular inflammation was detected in the nerve biopsy alone in five, both nerve and muscle biopsies in four, and isolated muscle involvement was noted in one patient (Supplementary Fig. 2A, B). In the nerve biopsies, asymmetric nerve fiber loss was noted in all patients (100%), followed in decreasing order of frequency by: vascular sclerosis with luminal obliteration or narrowing (four of 10, 40%), epineurial capillary proliferation (three of 10, 30%) and Wallerian degeneration (two of 10, 20%) (Supplementary Fig. 2C–E). Perls’ Prussian blue stain detected hemosiderin in three of 10 (30%) patients (nerve and muscle in two, and muscle alone in one) (Supplementary Fig. 2F). Features of possible vasculitis on histological grounds was observed in four patients (SVN – one with CSS, and three with NSVN). All patients had supportive features in nerve biopsy (asymmetric nerve fibre loss in three, vascular sclerosis and luminal narrowing/ neovascularization in two) in the absence of perivascular inflammation, in addition to changes in muscle biopsy in two patients. Perls’ Prussian blue stain detected hemosiderin in two instances, both in the muscle biopsy. All patients had clinical and laboratory features favoring a diagnosis of vasculitis and were therefore categorized as having probable (clinical presentation with mononeuritis multiplex, response to steroids, positive RA factor in one, raised erythrocyte sedimentation ratio in two). One patient with eosinophilia was later diagnosed as with CSS. Overall Perls’ Prussian blue stain was useful in detecting perivascular hemosiderin deposits in 14/27 (51.8%) patients (nerve and muscle biopsy – eight; muscle biopsy alone – four; nerve biopsy alone – two). Interestingly, the muscle biopsy more frequently revealed positivity for Perl’s Prussian blue stain (12/14, 85.7%) compared to nerve biopsy (10/14, 71%). In three patients, hemosiderin deposition was evident on routine H&E stain itself, but in 11 instances, Perl’s Prussian blue stain was required for demonstration of hemosiderin, thus enhancing the sensitivity of detection by 17.3%. 3.1.3. Sensitivity and specificity The sensitivity of SPN/PBM biopsy in diagnosing vasculitic neuropathy based on definitive criteria was 76.4% with 100% specificity. By including patients who were suspicious for vasculitis (Table 2), the sensitivity increased to 85.1% but the specificity dropped to 87.5%. For the group with NSVN, the sensitivity increased from 78.5% to 85.7% in comparison to 83.3% from 33.3% in patients with SVN.
Statistical significance of parameters used to diagnose probable vasculitis (vascular and non-vascular) was assessed by the odds ratio. Asymmetric nerve fiber loss, Wallerian degeneration and Perls’ stain for hemosiderin as markers of vasculitis were found to be statistically significant (p < 0.05) as markers of vasculitis, whereas vessel wall thickening with sclerosis and epineurial neovascularization did not attain statistical significance (p > 0.05).
4. Discussion As treatment of vasculitic neuropathies involves long-term, potentially cytotoxic medications, histologic confirmation is essential. Peripheral nerve biopsies commonly employed for diagnosis include the sural, SPN, and superficial radial nerve, depending on clinical/electrophysiological involvement. Of these, the sural nerve is most commonly biopsied for diagnosis.1,7,26–28 Its sensitivity in suspected cases of vasculitis varies from 20%9,10 to 50% in different studies.6,10,14,19 Although a randomized control study has not been undertaken, several studies have recommended using combined SPN/PBM biopsy (Table 3). These studies have mostly assessed the diagnostic yield of SPN/PBM rather than its sensitivity and specificity. The criteria used for pathological diagnosis is variable in these studies and the distinction of SVN from NSVN is not uniform, which makes comparisons difficult (Table 3). In the present study, we assessed the sensitivity and specificity of SPN/PBM biopsy and the usefulness of supportive criteria in the diagnosis of vasculitis. Clinical and electrophysiological evidence of frequent involvement of the superficial peroneal nerve in cases of vasculitic neuropathy and experimental evidence demonstrating that the sciatic bifurcation is a water shed zone and hence susceptible to ischemia supports the utility of SPN/PBM biopsies in the diagnosis of vasculitis.21 Interest in combined nerve and muscle biopsy followed the demonstration of vasculitis in muscle biopsies in 40 to 50% of patients with polyarteritis nodosa.12 In the series of Said et al., 37/83 patients demonstrated necrotizing vasculitis in the muscle biopsy and the overall diagnostic yield of muscle biopsy was significantly higher compared to nerve biopsies (80% compared to 55%),6 similar to other studies.17,19,20,29 In the present study however, the overall diagnostic yield of the PBM biopsy was 55.5% compared to 76.9% in SPN, the combined muscle and nerve biopsy enhancing the diagnostic yield by 23%. Collins et al. reported a diagnostic yield of 30% for combined SPN/PBM biopsy11 in contrast to 20% for sural nerve biopsies alone using definitive criteria for diagnosis. 8–10 Sural nerve biopsy along with tibialis anterior, gastrocnemius,8 or vastus lateralis biopsy,13 did not significantly improve the diag-
Table 3 Studies of the diagnostic yield of combined nerve/muscle biopsies Yearref
Biopsy
No. of patients
Muscle biopsy alone
Nerve biopsy alone
Overall nerve + muscle biopsy
Pathologic criteria used
19886 200520 200019
SPN/PBM SPN/PBM SPN/PBM
83 425 70
Necrotizing vasculitis Necrotizing vasculitis Necrotizing vasculitis
19 30 2 88 7 5 202
Definite, probable, possible (all NSVN)
65%
78%
58% 47% 50% 43%
200617
SPN/PBM Sural Superficial radial SPN/PBM Sural Superficial radial SPN/PBM
20% 45% 90% (100% – NSVN, 86% SVN) NA
30% 31.5% 30%
200314
45% 28% 50% (14%– NSVN, 67% – SVN) NA
Present study
SPN/PBM
31.6% 16% 53.8%
26.6% 76% 76.9%
41.6% 8% 48.1%
Definite, microvasculitis Definite, probable, possible (SVN and NSVN)
199618
27
Definite
NA = not available, NSVN = non systemic vasculitis, PBM = peroneus brevis muscle, SPN = superfician peroneal nerve, SVN = systemic vasculitis.
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nostic yield, probably due to the fact that proximal muscles are more frequently involved in vasculitic pathology. The sensitivity of nerve biopsy in the diagnosis of vasculitis depends on several factors, including patient selection, timing of the biopsy in relation to symptoms, empirical steroid therapy, the nerve biopsied, the length of the biopsy and the histologic criteria used for diagnosis.23 If transmural inflammation and vessel wall destruction are considered mandatory for diagnosis, the sensitivity reaches 30% for SPN/PBM biopsy6,19 but increases to 86% if patients considered suspicious for vasculitis are also included, with a corresponding specificity of 85%.19 In our study, the sensitivity increased from 76.4% to 85.1% with specificity of 87.5%. Other studies reported sensitivity ranging from 55% to 70%.13,14 The pooled sensitivity of SPN/PBM biopsy is 64% as against 56% for sural nerve alone11 supporting the view that SPN/PBM biopsy has a higher diagnostic yield and higher sensitivity compared to sural nerve biopsy in diagnosing vasculitic neuropathy.2,3,14,17,19,30 Prospective randomized studies with a uniform set of diagnostic criteria comparing sural and SPN/PBM nerve biopsies are essential for a definite conclusion. Following introduction of the term ‘‘NSVN’’ by Dyck et al. in 1987 for patients with vasculitis confined to the peripheral nervous system,3 several reports of NSVN have been published.13,14,17,19,31,32 In the present cohort, 21 patients were diagnosed with NSVN in accordance with criteria proposed by Collins et al.14 NSVN should be confined to involvement of nerve alone; however, the presence of vasculitis in the muscle was noted in seven of 21 patients (33.3%) in our study, similar to other published studies.3,14,17 Necrotizing vasculitis or active vascular destruction are uncommon in NSVN, though sequelae of vascular pathology such as vessel wall sclerosis, luminal occlusion, and hemosiderin deposition, are similar to that seen in systemic vasculitis. The possibility that NSVN may be a forme fruste of systemic vasculitis needs to be substantiated.
5. Conclusion The combined SPN/PBM biopsy offers better diagnostic yield, with the aid of supportive features of vascular damage. Routine use of Perl’s Prussian blue stain in muscle and nerve biopsies enhances the chances of diagnosing patients with systemic and non-systemic vasculitis with non-diagnostic features in the biopsy.
Conflict of interest/disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
Acknowledgements We wish to acknowledge the help of Dr. B. G. Tilak, and Dr. Shankarappa, Department of Plastic Surgery, Bowring and Lady Curzon Hospital, Bangalore, for their assistance in performing the combined nerve and muscle biopsies. The authors also acknowledge the technical help of Mrs.Rajyasakti V, Mr. Prasanna Kumar and Mr. Shivaji Rao and assistance with photography of Mr. Manjunath K, Human Brain Tissue Repository, Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2011.11.034. References 1. Midroni G, Bilbao JM. Vasculitic neuropathy. In: Midroni G, Bilbao JM, editors. Biopsy Diagnosis of Peripheral Neuropathy. Boston: Butterworth-Heinemann; 1995. p. 241–62. 2. Gabriel CM, Howard R, Kinsella N, et al. Prospective study of the usefulness of sural nerve biopsy. J Neurol Neurosurg Psychiatry 2000;69:442–6. 3. Dyck PJ, Benstead TJ, Conn Dl, et al. Nonsystemic vasculitic neuropathy. Brain 1987;110:843–53. 4. Davidson JR, Sundstrom WR. Sural nerve biopsy in systemic necrotizing vasculitis. Arthritis Rheum 1988;31:149–50. 5. Kissel JT, Slivka AP, Warmolts JR, et al. The clinical spectrum of necrotizing angiopathy of the peripheral nervous system. Ann Neurol 1985;18:251–7. 6. Said G, Lacroix-Ciaudo C, Fujimura H, et al. The peripheral neuropathy of necrotizing arteritis: a clinicopathological study. Ann Neurol 1988;23:461–5. 7. Wees SJ, Sunwoo IN, Oh SJ. Sural nerve biopsy in systemic necrotizing vasculitis. Am J Med 1981;71:525–32. 8. Claussen GC, Thomas TD, Goyne C, et al. Diagnostic value of nerve and muscle biopsy in suspected vasculitis cases. J Clin Neuromuscul Dis 2000;1:117–23. 9. Hellmann DB, Laing TJ, Petri M, et al. Mononeuritis multiplex: the yield of evaluations for occult rheumatic diseases. Medicine (Baltimore) 1988;67:145–53. 10. Rappaport WD, Valente J, Hunter GC, et al. Clinical utilization and complications of sural nerve biopsy. Am J Surg 1993;166:252–6. 11. Collins MP, Kissel JT. Neuropathies with systemic vasculitis. In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy, 4th ed., Vol. 2. Philadelphia: WB Saunders, 2005: 2335-2404. 12. Fort JG, Griffin R, Tahmoush A, et al. Muscle involvement in polyarteritis nodosa: report of a patient presenting clinically as polymyositis and review of the literature. J Rheumatol 1994;21:945–8. 13. Bennett DL, Groves M, Blake J, et al. The use of nerve and muscle biopsy in the diagnosis of vasculitis: a 5 year retrospective study. J Neurol Neurosurg Psychiatry 2008;79:1376–81. 14. Collins MP, Periquet MI, Mendell JR, et al. Nonsystemic vasculitic neuropathy: insights from a clinical cohort. Neurology 2003;61:623–30. 15. Hurley ME, Davids JR, Mubarak SJ. Single-incision combination biopsy (muscle and nerve) in the diagnosis of neuromuscular disease in children. J Pediatr Orthop 1994;14:740–4. 16. Prayson RA. Skeletal muscle vasculitis exclusive of inflammatory myopathic conditions: a clinicopathologic study of 40 patients. Hum Pathol 2002;33:989–95. 17. Vital C, Vital A, Canron MH, et al. Combined nerve and muscle biopsy in the diagnosis of vasculitic neuropathy. A 16-year retrospective study of 202 cases. J Periph Nerv Sys 2006;11:20–9. 18. Chia L, Fernandez A, Lacroix C, et al. Contribution of nerve biopsy findings to the diagnosis of disabling neuropathy in the elderly. A retrospective review of 100 consecutive patients. Brain 1996;119:1091–8. 19. Collins MP, Mendell JR, Periquet MI, et al. Superficial peroneal nerve/peroneus brevis muscle biopsy in vasculitic neuropathy. Neurology 2000;55:636–43. 20. Said G, Lacroix C. Primary and secondary vasculitic neuropathy. J Neurol 2005;252:633–41. 21. Low PA, Lagerlund TD, Mcmanis PG. Nerve blood flow and oxygen delivery in normal, diabetic, and ischemic neuropathy. Int Rev Neurobiol 1989;31:355–438. 22. Kissel JT, Mendell JR. Vasculitic neuropathy. Neurol Clin 1992;10:761–81. 23. Burns TM, Schaublin GA, Dyck PJ. Vasculitic neuropathies. Neurol Clin 2007;25:89–113. 24. Jennette JC, Falk RJ. Small-vessel vasculitis. N Engl J Med 1997;337:1512–23. 25. Dyck PJB, Engelstad J, Dyck PJ. Microvasculitis. In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy, 4th ed., Vol. 2. Philadelphia: WB Saunders, 2005: 2405– 414. 26. Barohn RJ, Kissel JT, Warmolts JR, et al. Chronic inflammatory demyelinating polyradiculoneuropathy. Clinical characteristics, course, and recommendations for diagnostic criteria. Arch Neurol 1989;46:878–84. 27. Hawke SH, Davies L, Pamphlett R, et al. Vasculitic neuropathy. A clinical and pathological study. Brain 1991;114:2175–90. 28. Nicolai A, Bonetti B, Lazzarino LG, et al. Peripheral nerve vasculitis: a clinicopathological study. Clin Neuropath 1995;14:137–41. 29. Deprez M, De Groote CC, Gollogly L, et al. Clinical and neuropathological parameters affecting the diagnostic yield of nerve biopsy. Neuromusc Dis 2000;10:92–8. 30. Said G. Necrotizing peripheral nerve vasculitis. Neurol Clin 1997;15:835–48. 31. Davies L, Spies JM, Pollard JD, et al. Vasculitis confined to peripheral nerves. Brain 1996;119:1441–8. 32. Sugiura M, Koike H, Iijima M, et al. Clinicopathologic features of nonsystemic vasculitic neuropathy and microscopic polyangiitis-associated neuropathy: a comparative study. J Neurol Sci 2006;241:31–7.
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Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Clinical Study
Usefulness of superficial peroneal nerve/peroneus brevis muscle biopsy in the diagnosis of vasculitic neuropathy J.B. Agadi a,⇑, Govindarajan Raghav a, A. Mahadevan b, S.K. Shankar b a b
Department of Neurology, Bowring and Lady Curzon Hospital, Bangalore 560001, India Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India
a r t i c l e
i n f o
Article history: Received 17 March 2011 Accepted 6 November 2011
Keywords: Diagnostic yield Perls’ stain Peroneus brevis biopsy Sensitivity Specificity Superficial peroneal nerve Vasculitic neuropathy
a b s t r a c t Sensitivity, specificity, and diagnostic yield of the superficial peroneal nerve (SPN)/peroneus brevis muscle (PBM) biopsy in 43 patients with clinically suspected vasculitic neuropathy was studied. Biopsies were classified as ‘‘definite’’, ‘‘suspicious’’ or ‘‘possible’’ in accordance with established criteria. Vasculitis was detected in 27 patients (21 with non-systemic vasculitis, and six with systemic vasculitis). In patients with ‘‘definite’’ vasculitis (n = 13), the sensitivity of SPN/PBM biopsy was 76.4% with 100% specificity. By including patients suspicious for vasculitis (n = 10), sensitivity increased to 85.1% but the specificity dropped to 87.5%. The overall diagnostic yield of SPN biopsy in those patients with definite vasculitis was 76.9% (10/13), and 53.8% (7/13) for muscle biopsy. The addition of muscle biopsy increased the diagnostic yield by 23%. Asymmetric nerve fiber loss, Wallerian degeneration and presence of hemosiderin were statistically significant markers of probable vasculitis. Muscle tissue was more likely to show hemosiderin (85.7%) than a nerve biopsy (71%). A combined SPN/PBM biopsy offers excellent diagnostic yield in the diagnosis of vasculitic neuropathy. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Vasculitides are a heterogenous group of disorders with systemic or localized involvement of various organ systems which is dictated by the caliber of vessels involved. Several forms of vasculitis involve the peripheral nervous system, either as an isolated process (non-systemic vasculitic neuropathy) without other organ involvement, or more commonly, as part of a systemic disease. Despite the development of newer, non-invasive serological tests for a wide range of auto-antibodies, diagnosis of vasculitis neuropathy often relies on pathological demonstration of vasculitis in involved peripheral nerves suitable for biopsy.1–3 Histological evaluation of the sural nerve is most often used for diagnosis. Several studies have examined the sensitivity of sural nerve biopsy in the diagnosis of vasculitic neuropathy.4–7 Mandatory pathologic criteria for a ‘‘definitive’’ diagnosis require demonstration of transmural inflammatory infiltrates in vessel walls, and evidence of vascular destruction with fibrinoid necrosis, endothelial disruption or hemorrhage. These criteria are, however, met in only 20% of sural nerve biopsies, leaving as many as 80% undiagnosed.8–10 To enhance the diagnostic sensitivity, several workers have used alternative features that reflect ischemic injury (multifocal nerve fibre loss and secondary vascular changes without
⇑ Corresponding author. Tel.: +91 80 25592813; fax: +91 80 25591325. E-mail address: [email protected] (J.B. Agadi). 0967-5868/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2011.11.034
distinct vessel wall destruction) in the presence of perivascular inflammation to define ‘‘suspicious’’, ‘‘probable’’ or ‘‘healed’’ vasculitis. Applying these criteria, the pooled diagnostic sensitivity of sural nerve biopsies in different series reaches 56%.11 Said and Lacroix6 and Fort et al.12 documented the presence of vasculitis in muscle biopsy in 45% to 50% of patients with vasculitic neuropathy, and advocated a combined nerve and muscle biopsy to enhance the diagnostic yield. Subsequent studies have evaluated the usefulness of combined sural nerve and quadriceps, gastrocnemius/tibialis anterior or soleus muscle biopsies.8,13–16 Only a few studies have assessed the diagnostic yield of superficial peroneal nerve (SPN)/peroneus brevis muscle (PBM) biopsy in diagnosing vasculitic neuropathy.6,11,17–20 These studies, however, do not address the sensitivity and specificity of SPN/PBM biopsies in diagnosis of vasculitis. Logically SPN/PBM biopsy would be ideal, first on account of technical ease of obtaining a combined nerve and muscle biopsy through a single incision, and second, the peroneal nerve is clinically the most commonly involved nerve in the lower limbs in vasculitis.21 At our Institute, which is primarily a referral centre for neurological disorders, this procedure has been practiced consistently for over a decade. As an audit, this study was undertaken to determine the sensitivity, specificity, and the diagnostic yield of a combined SPN/PBM biopsy in patients with clinically suspected vasculitic neuropathy. The study also aimed to assess the significance of supportive pathologic features in patients who were ‘‘suspicious’’ for vasculitis.
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2. Material and methods 2.1. Participants and clinical data acquisition This study was undertaken as an audit and was approved by the Institutional Scientific Ethics Committee. The muscle and nerve biopsies were performed as diagnostic procedures following the informed consent of patients. All patients with clinically suspected vasculitis who underwent combined SPN/PBM biopsies between 1998 and 2008 at the neurological services of Bowring and Lady Curzon Hospital, a primary and secondary referral centre in South India, were reviewed. The patient clinical and demographic details as well as laboratory findings were retrieved from the medical records. All patients were evaluated clinically by the neurologist (J.B.A.). Electrophysiologic, biochemical and serological testing was performed to exclude other causes of peripheral neuropathy.
2.2. Biopsy and histopathology A full thickness biopsy of the SPN (1.5–2 cm in length) and PBM were obtained by a single incision in the lower third of the leg.22 The histopathologic analysis was carried out at the Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore. The nerve biopsy was fixed in 2.5% buffered gluteraldehyde and processed for paraffin embedding. Sections (2 lm thick) were stained with hematoxylin and eosin (H&E), Masson trichrome stain for collagen, Kulchitsky-Palstain for myelin, and Perls’ Prussian blue for extravasated hemosiderin (iron) from damaged and leaky vessels. In selected sections, immunohistochemistry was performed by the indirect immunoperoxidase technique to label macrophages (CD68, monoclonal, 1:50, Biogenex, Fremont, CA, USA) and Leukocyte Common Antigen (CD45, polyclonal, 1:50, Biogenex) to detect inflammatory infiltrates. Tissue from the peroneus brevis was divided into two: one part was flash frozen in isopentane-cooled liquid nitrogen. Cryosections (8 lm thick) were stained with H&E, modified Gomori trichrome, oxidative enzymes (succinate dehydrogenase [SDH], nicotinamide adenine dinucleotide-teterazolium reductase [NADH-TR]) and adenosine triphosphatase (ATPase) (pH 9.5 and pH 4.6) stains. The other portion was fixed in 10% buffered formalin and processed for paraffin embedding. Deparaffinised sections were stained with H&E, Masson trichrome stain for collagen and Perls’ Prussian blue to demonstrate hemosiderin deposits.
2.3. Biopsy criteria The histological diagnosis of the nerve biopsies were classified as ‘‘definite’’, ‘‘suspicious’’ or ‘‘possible’’ vasculitis in accordance with previously established criteria.11,19,23 Vasculitis was considered definite if at least one blood vessel had transmural infiltration by inflammatory infiltrates in addition to signs of vascular injury such as fibrinoid necrosis, fragmentation of internal elastic lamina or hemorrhage into the vessel wall24 or in the presence of microvasculitis when inflammatory infiltration involved small arterioles (<30–40 microns) that lacked internal elastic lamina as well as venules.25 The biopsy was considered suspicious of vasculitis if, in the absence of transmural vessel infiltration, at least one vessel in the biopsied nerve was rimmed by inflammatory cells in the presence of one of the following supportive pathologic features: vascular wall thickening and sclerosis, narrowing or obliteration of the lumen, thrombosis with or without recanalization, epineurial capillary proliferation/neovascularization, periadventitial hemosiderin
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deposits, asymmetric nerve fiber loss or active Wallerian-like degeneration.19 Sections that showed only supportive features in the absence of inflammation were classified as possible vasculitis.19 Both the suspicious and possible histologic categories required the presence of clinical, laboratory and electrophysiological criteria to be considered as clinically probable vasculitic neuropathy.19 Nerve biopsies with none of the above features or isolated perivascular lymphohistiocytic infiltrates of variable degrees were considered negative for vasculitis.19 In the muscle biopsies, vasculitis was considered definite in presence of necrotizing vasculitis and probable when transmural or perivascular inflammation was not accompanied by necrosis but demonstrated supporting evidence of vascular injury.19 2.4. Clinical criteria Patients were classified as having systemic vasculitic neuropathy (SVN) in the presence of positive auto-antibodies or clinical, radiologic or biopsy evidence of other systemic organ involvement. Patients were classified as having non-systemic vasculitic neuropathy (NSVN) in the absence of the above, according to the criteria of Collins et al.11 2.5. Statistical analysis Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software (version 12) (SPSS, Chicago, IL, USA). A bivariate analysis using Fisher’s exact test was performed to determine the odds ratio and statistical significance of the supportive histological features in the suspicious and possible categories of vasculitis biopsies. 3. Results A total of 43 patients were analyzed in the study: 27 revealed biopsy evidence of vasculitis (21 with NSVN; and six with SVN, of whom four had Churg–Strauss syndrome [CSS], one had rheumatoid arthritis [RA] and one had Sjogren’s syndrome). In all patients with SVN, peripheral neuropathy was the presenting feature. The clinical and laboratory features are summarized in Table 1. The remaining 16 patients were diagnosed with chronic inflammatory demyelinating polyneuropathy or its variants (n = 7), pure neuritic leprosy (n = 6), amyloidosis (n = 1), multiple myeloma (n = 1), and acute inflammatory demyelinating polyneuropathy with asymmetric presentation (n = 1). Clinically, multiple mononeuropathy or asymmetric polyneuropathy was the most common presentation (23/27, 85.1%) followed by distal symmetric polyneuropathy (four of 27, 14.8%). Three patients had cranial neuropathy. A slight male predominance (M:F = 16:11) was noted (Table 1). 3.1. Pathological features The pathological features are described in Table 2. 3.1.1. Definite vasculitis (n = 13) Thirteen patients (48.1%) had definitive evidence of vasculitis in the biopsy (two with necrotizing vasculitis, and 11 with microvasculitis). Both patients with necrotizing vasculitis in muscle and nerve biopsy had systemic vasculitis (one each were diagnosed with RA and CSS) (Supplementary Fig. 1A–C). Evidence of microvasculitis was observed in 11 patients (Supplementary Fig. 1D, E), all of whom had NSVN (nerve alone – six of 11, muscle alone – three of 11, both nerve and muscle, two of 11).
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Table 1 Clinical and laboratory parameters of 27 patients with vasculitic neuropathy Clinical parameter
Entire series (n = 27)
NSVN (n = 21)
Male: female Age in years Duration of symptoms in months Constitutional symptomsa Clinical courseb Pain Paresthesias Cranial nerve involvementc
16:11 53.9 ± 13.43 9.7 ± 13.19 10 (37%) AM-4, SAP-13, CP-9, SM-1 9 (33.3%) 18 (66.6%) 3 (11.1%)
12:9 54.2 ± 13.75 12.0 ± 14.2 4 (19%) AM-1, SAP-10, CP-9, SM-1 6 (28.5%) 15 (71.4%) 2 (9.5%)
Clinical pattern of nerve involvement True MM Extensive overlapping MM DSP
15 (55.5%) 8 (29.6%) 4 (14.8%)
12(57.1%) 5 (23.8%) 4 (19.0%)
Common nerves involved (electrophysiology) Peroneal Sural Posterior tibial Ulnar Median
92.5% 70.3% 22.4% 51.8% 29.8%
95.2% 66.6% 19% 52.3% 53.3%
Electrophysiology Mixed sensory and motor Predominantly sensory
21 (77.7%) 6 (22.2%)
15 (71.4%) 6 (28.5%)
Laboratory parameters Raised ESR Leucocytosis CRP positive Eosinophiliad ANA RA Anemia ANCAe Anti-Rof
12 (44.4%) 8 (29.6%) 3 (11.1%) 4 (14.8%) 4 (14.8%) 5 (18.5%) 3 (11.1%) 2 (7.4%) 1(4.7%)
7 (33.3%) 4 (19.8%) 2 (9.5%) 0 1 (4.7%) 2 (9.5%) 1(4.7%) 0 0
AM = acute monophasic, ANA = anti-nuclear antibody, ANCA = anti-neutrophil cytoplasmic antibodies, CP = chronic progressive, CRP = c-reactive protein, CSS = Churg-Strauss, DSP = distal symmetric polyneuropathy, ESR = erythrocyte sedimentation ratio, MM = multiple mononeuropathy, NSVN = non-systemic vasculitic neuropathy, RA = rheumatoid arthritis, SM = subacute monophasic, SP = subacute progressive. a Constitutional symptoms in our series were fever, weight loss, fatigue, loss of appetite. b Clinical course was characterized using two nominal scales2: 1 = acute – at least one attack of sudden onset symptoms over one to two days or progression of symptoms to plateau in less than four weeks, subacute-progression over four to eight weeks, chronic- progression over more than eight weeks. 2 = monophasic, relapsing, progressive. C sssss in two and hypoglossal in one. d Four patients with CSS had eosinophilia. e ANCA was positive in two patients with CSS (one with p-ANCA; one with c-ANCA). f One patient with anti-Ro antibody was later diagnosed with Sjogren’s syndrome.
In the nerve biopsies, vasculitis involved exclusively the epineurial vessels sparing endoneurial vessels. The infiltrates were predominantly lymphohistiocytic admixed with neutrophils in the presence of necrotizing vasculitis (Supplementary Fig. 1). Axonal pathology predominated with asymmetric fiber loss (12/13) or Wallerian degeneration (seven of 13). Vascular thickening and sclerosis causing luminal narrowing was frequent (12/13). Epineurial capillary proliferation was infrequent (five of 13). Perls’ Prussian blue stain highlighted hemosiderin deposits in eight of 13 (61.5%) surrounding inflamed vessels in the epineurium and rarely in the perineurium. The PBM biopsy revealed vasculitis involving mostly epimyseal vessels in addition to evidence of neurogenic atrophy in seven of 13 patients (53.8%) (two of two with SVN, five of 11 in NSVN). Perls’-positive hemosiderin deposits were seen in all seven patients showing vasculitis (SVN two of two, NSVN five of 11) as fine punctate positivity in the endomysium between myofibres as well as larger clumps surrounding the inflamed large epimyseal or perimyseal vessels (Supplementary Fig. 1F). Perls’ Prussian blue stain was positive in nine of 13 (69.2%) biopsies of patients with definite vasculitis. Of these it was seen in both nerve and muscle in six patients (five with NSVN and one with SVN with RA), nerve alone in two (both with NSVN) and muscle alone in one patient (SVN with CSS).
The overall diagnostic yield of SPN biopsy in patients with definite vasculitis was 76.9% (10/13), and 53.8% (seven of 13) for PBM biopsy. In the NSVN cohort, the diagnostic yield of SPN biopsy was higher [72.7% (eight of 11), than PBM [45.4% (five of 11)]. But in three patients, microvasculitis was detected in muscle biopsy alone. Thus, the addition of muscle biopsy enhanced the diagnostic yield by 23%. In patients with SVN, the nerve and muscle biopsies were equally diagnostic, notwithstanding that the number of patients is small.
Table 2 Biopsy findings in superficial peroneal nerve/peroneus brevis biopsies
Definite Necrotizing vasculitis Microvasculitis Suspicious Possible
Series (n = 27), n (%)
NSVN (n = 21), n (%)
SVN (n = 6), n (%)
2 (7.4) 11 (40.7) 10 (37) 4 (14.8)
0 11 (52.3) 7 (33.3) 3 (14.2)
2 (33.3) 0 3 (66.6) 1 (1.2)
NSVN = non-systemic vasculitic neuropathy, SVN = systemic vasculitic neuropathy.
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3.1.2. Probable vasculitis (n = 14) Features suspicious of vasculitis on histological grounds was noted in 10/27 patients (37%) (three with SVN, seven with NSVN) (F Supplementary Fig. 2). Perivascular inflammation was detected in the nerve biopsy alone in five, both nerve and muscle biopsies in four, and isolated muscle involvement was noted in one patient (Supplementary Fig. 2A, B). In the nerve biopsies, asymmetric nerve fiber loss was noted in all patients (100%), followed in decreasing order of frequency by: vascular sclerosis with luminal obliteration or narrowing (four of 10, 40%), epineurial capillary proliferation (three of 10, 30%) and Wallerian degeneration (two of 10, 20%) (Supplementary Fig. 2C–E). Perls’ Prussian blue stain detected hemosiderin in three of 10 (30%) patients (nerve and muscle in two, and muscle alone in one) (Supplementary Fig. 2F). Features of possible vasculitis on histological grounds was observed in four patients (SVN – one with CSS, and three with NSVN). All patients had supportive features in nerve biopsy (asymmetric nerve fibre loss in three, vascular sclerosis and luminal narrowing/ neovascularization in two) in the absence of perivascular inflammation, in addition to changes in muscle biopsy in two patients. Perls’ Prussian blue stain detected hemosiderin in two instances, both in the muscle biopsy. All patients had clinical and laboratory features favoring a diagnosis of vasculitis and were therefore categorized as having probable (clinical presentation with mononeuritis multiplex, response to steroids, positive RA factor in one, raised erythrocyte sedimentation ratio in two). One patient with eosinophilia was later diagnosed as with CSS. Overall Perls’ Prussian blue stain was useful in detecting perivascular hemosiderin deposits in 14/27 (51.8%) patients (nerve and muscle biopsy – eight; muscle biopsy alone – four; nerve biopsy alone – two). Interestingly, the muscle biopsy more frequently revealed positivity for Perl’s Prussian blue stain (12/14, 85.7%) compared to nerve biopsy (10/14, 71%). In three patients, hemosiderin deposition was evident on routine H&E stain itself, but in 11 instances, Perl’s Prussian blue stain was required for demonstration of hemosiderin, thus enhancing the sensitivity of detection by 17.3%. 3.1.3. Sensitivity and specificity The sensitivity of SPN/PBM biopsy in diagnosing vasculitic neuropathy based on definitive criteria was 76.4% with 100% specificity. By including patients who were suspicious for vasculitis (Table 2), the sensitivity increased to 85.1% but the specificity dropped to 87.5%. For the group with NSVN, the sensitivity increased from 78.5% to 85.7% in comparison to 83.3% from 33.3% in patients with SVN.
Statistical significance of parameters used to diagnose probable vasculitis (vascular and non-vascular) was assessed by the odds ratio. Asymmetric nerve fiber loss, Wallerian degeneration and Perls’ stain for hemosiderin as markers of vasculitis were found to be statistically significant (p < 0.05) as markers of vasculitis, whereas vessel wall thickening with sclerosis and epineurial neovascularization did not attain statistical significance (p > 0.05).
4. Discussion As treatment of vasculitic neuropathies involves long-term, potentially cytotoxic medications, histologic confirmation is essential. Peripheral nerve biopsies commonly employed for diagnosis include the sural, SPN, and superficial radial nerve, depending on clinical/electrophysiological involvement. Of these, the sural nerve is most commonly biopsied for diagnosis.1,7,26–28 Its sensitivity in suspected cases of vasculitis varies from 20%9,10 to 50% in different studies.6,10,14,19 Although a randomized control study has not been undertaken, several studies have recommended using combined SPN/PBM biopsy (Table 3). These studies have mostly assessed the diagnostic yield of SPN/PBM rather than its sensitivity and specificity. The criteria used for pathological diagnosis is variable in these studies and the distinction of SVN from NSVN is not uniform, which makes comparisons difficult (Table 3). In the present study, we assessed the sensitivity and specificity of SPN/PBM biopsy and the usefulness of supportive criteria in the diagnosis of vasculitis. Clinical and electrophysiological evidence of frequent involvement of the superficial peroneal nerve in cases of vasculitic neuropathy and experimental evidence demonstrating that the sciatic bifurcation is a water shed zone and hence susceptible to ischemia supports the utility of SPN/PBM biopsies in the diagnosis of vasculitis.21 Interest in combined nerve and muscle biopsy followed the demonstration of vasculitis in muscle biopsies in 40 to 50% of patients with polyarteritis nodosa.12 In the series of Said et al., 37/83 patients demonstrated necrotizing vasculitis in the muscle biopsy and the overall diagnostic yield of muscle biopsy was significantly higher compared to nerve biopsies (80% compared to 55%),6 similar to other studies.17,19,20,29 In the present study however, the overall diagnostic yield of the PBM biopsy was 55.5% compared to 76.9% in SPN, the combined muscle and nerve biopsy enhancing the diagnostic yield by 23%. Collins et al. reported a diagnostic yield of 30% for combined SPN/PBM biopsy11 in contrast to 20% for sural nerve biopsies alone using definitive criteria for diagnosis. 8–10 Sural nerve biopsy along with tibialis anterior, gastrocnemius,8 or vastus lateralis biopsy,13 did not significantly improve the diag-
Table 3 Studies of the diagnostic yield of combined nerve/muscle biopsies Yearref
Biopsy
No. of patients
Muscle biopsy alone
Nerve biopsy alone
Overall nerve + muscle biopsy
Pathologic criteria used
19886 200520 200019
SPN/PBM SPN/PBM SPN/PBM
83 425 70
Necrotizing vasculitis Necrotizing vasculitis Necrotizing vasculitis
19 30 2 88 7 5 202
Definite, probable, possible (all NSVN)
65%
78%
58% 47% 50% 43%
200617
SPN/PBM Sural Superficial radial SPN/PBM Sural Superficial radial SPN/PBM
20% 45% 90% (100% – NSVN, 86% SVN) NA
30% 31.5% 30%
200314
45% 28% 50% (14%– NSVN, 67% – SVN) NA
Present study
SPN/PBM
31.6% 16% 53.8%
26.6% 76% 76.9%
41.6% 8% 48.1%
Definite, microvasculitis Definite, probable, possible (SVN and NSVN)
199618
27
Definite
NA = not available, NSVN = non systemic vasculitis, PBM = peroneus brevis muscle, SPN = superfician peroneal nerve, SVN = systemic vasculitis.
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nostic yield, probably due to the fact that proximal muscles are more frequently involved in vasculitic pathology. The sensitivity of nerve biopsy in the diagnosis of vasculitis depends on several factors, including patient selection, timing of the biopsy in relation to symptoms, empirical steroid therapy, the nerve biopsied, the length of the biopsy and the histologic criteria used for diagnosis.23 If transmural inflammation and vessel wall destruction are considered mandatory for diagnosis, the sensitivity reaches 30% for SPN/PBM biopsy6,19 but increases to 86% if patients considered suspicious for vasculitis are also included, with a corresponding specificity of 85%.19 In our study, the sensitivity increased from 76.4% to 85.1% with specificity of 87.5%. Other studies reported sensitivity ranging from 55% to 70%.13,14 The pooled sensitivity of SPN/PBM biopsy is 64% as against 56% for sural nerve alone11 supporting the view that SPN/PBM biopsy has a higher diagnostic yield and higher sensitivity compared to sural nerve biopsy in diagnosing vasculitic neuropathy.2,3,14,17,19,30 Prospective randomized studies with a uniform set of diagnostic criteria comparing sural and SPN/PBM nerve biopsies are essential for a definite conclusion. Following introduction of the term ‘‘NSVN’’ by Dyck et al. in 1987 for patients with vasculitis confined to the peripheral nervous system,3 several reports of NSVN have been published.13,14,17,19,31,32 In the present cohort, 21 patients were diagnosed with NSVN in accordance with criteria proposed by Collins et al.14 NSVN should be confined to involvement of nerve alone; however, the presence of vasculitis in the muscle was noted in seven of 21 patients (33.3%) in our study, similar to other published studies.3,14,17 Necrotizing vasculitis or active vascular destruction are uncommon in NSVN, though sequelae of vascular pathology such as vessel wall sclerosis, luminal occlusion, and hemosiderin deposition, are similar to that seen in systemic vasculitis. The possibility that NSVN may be a forme fruste of systemic vasculitis needs to be substantiated.
5. Conclusion The combined SPN/PBM biopsy offers better diagnostic yield, with the aid of supportive features of vascular damage. Routine use of Perl’s Prussian blue stain in muscle and nerve biopsies enhances the chances of diagnosing patients with systemic and non-systemic vasculitis with non-diagnostic features in the biopsy.
Conflict of interest/disclosures The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.
Acknowledgements We wish to acknowledge the help of Dr. B. G. Tilak, and Dr. Shankarappa, Department of Plastic Surgery, Bowring and Lady Curzon Hospital, Bangalore, for their assistance in performing the combined nerve and muscle biopsies. The authors also acknowledge the technical help of Mrs.Rajyasakti V, Mr. Prasanna Kumar and Mr. Shivaji Rao and assistance with photography of Mr. Manjunath K, Human Brain Tissue Repository, Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jocn.2011.11.034. References 1. Midroni G, Bilbao JM. Vasculitic neuropathy. In: Midroni G, Bilbao JM, editors. Biopsy Diagnosis of Peripheral Neuropathy. Boston: Butterworth-Heinemann; 1995. p. 241–62. 2. Gabriel CM, Howard R, Kinsella N, et al. Prospective study of the usefulness of sural nerve biopsy. J Neurol Neurosurg Psychiatry 2000;69:442–6. 3. Dyck PJ, Benstead TJ, Conn Dl, et al. Nonsystemic vasculitic neuropathy. Brain 1987;110:843–53. 4. Davidson JR, Sundstrom WR. Sural nerve biopsy in systemic necrotizing vasculitis. Arthritis Rheum 1988;31:149–50. 5. Kissel JT, Slivka AP, Warmolts JR, et al. The clinical spectrum of necrotizing angiopathy of the peripheral nervous system. Ann Neurol 1985;18:251–7. 6. Said G, Lacroix-Ciaudo C, Fujimura H, et al. The peripheral neuropathy of necrotizing arteritis: a clinicopathological study. Ann Neurol 1988;23:461–5. 7. Wees SJ, Sunwoo IN, Oh SJ. Sural nerve biopsy in systemic necrotizing vasculitis. Am J Med 1981;71:525–32. 8. Claussen GC, Thomas TD, Goyne C, et al. Diagnostic value of nerve and muscle biopsy in suspected vasculitis cases. J Clin Neuromuscul Dis 2000;1:117–23. 9. Hellmann DB, Laing TJ, Petri M, et al. Mononeuritis multiplex: the yield of evaluations for occult rheumatic diseases. Medicine (Baltimore) 1988;67:145–53. 10. Rappaport WD, Valente J, Hunter GC, et al. Clinical utilization and complications of sural nerve biopsy. Am J Surg 1993;166:252–6. 11. Collins MP, Kissel JT. Neuropathies with systemic vasculitis. In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy, 4th ed., Vol. 2. Philadelphia: WB Saunders, 2005: 2335-2404. 12. Fort JG, Griffin R, Tahmoush A, et al. Muscle involvement in polyarteritis nodosa: report of a patient presenting clinically as polymyositis and review of the literature. J Rheumatol 1994;21:945–8. 13. Bennett DL, Groves M, Blake J, et al. The use of nerve and muscle biopsy in the diagnosis of vasculitis: a 5 year retrospective study. J Neurol Neurosurg Psychiatry 2008;79:1376–81. 14. Collins MP, Periquet MI, Mendell JR, et al. Nonsystemic vasculitic neuropathy: insights from a clinical cohort. Neurology 2003;61:623–30. 15. Hurley ME, Davids JR, Mubarak SJ. Single-incision combination biopsy (muscle and nerve) in the diagnosis of neuromuscular disease in children. J Pediatr Orthop 1994;14:740–4. 16. Prayson RA. Skeletal muscle vasculitis exclusive of inflammatory myopathic conditions: a clinicopathologic study of 40 patients. Hum Pathol 2002;33:989–95. 17. Vital C, Vital A, Canron MH, et al. Combined nerve and muscle biopsy in the diagnosis of vasculitic neuropathy. A 16-year retrospective study of 202 cases. J Periph Nerv Sys 2006;11:20–9. 18. Chia L, Fernandez A, Lacroix C, et al. Contribution of nerve biopsy findings to the diagnosis of disabling neuropathy in the elderly. A retrospective review of 100 consecutive patients. Brain 1996;119:1091–8. 19. Collins MP, Mendell JR, Periquet MI, et al. Superficial peroneal nerve/peroneus brevis muscle biopsy in vasculitic neuropathy. Neurology 2000;55:636–43. 20. Said G, Lacroix C. Primary and secondary vasculitic neuropathy. J Neurol 2005;252:633–41. 21. Low PA, Lagerlund TD, Mcmanis PG. Nerve blood flow and oxygen delivery in normal, diabetic, and ischemic neuropathy. Int Rev Neurobiol 1989;31:355–438. 22. Kissel JT, Mendell JR. Vasculitic neuropathy. Neurol Clin 1992;10:761–81. 23. Burns TM, Schaublin GA, Dyck PJ. Vasculitic neuropathies. Neurol Clin 2007;25:89–113. 24. Jennette JC, Falk RJ. Small-vessel vasculitis. N Engl J Med 1997;337:1512–23. 25. Dyck PJB, Engelstad J, Dyck PJ. Microvasculitis. In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy, 4th ed., Vol. 2. Philadelphia: WB Saunders, 2005: 2405– 414. 26. Barohn RJ, Kissel JT, Warmolts JR, et al. Chronic inflammatory demyelinating polyradiculoneuropathy. Clinical characteristics, course, and recommendations for diagnostic criteria. Arch Neurol 1989;46:878–84. 27. Hawke SH, Davies L, Pamphlett R, et al. Vasculitic neuropathy. A clinical and pathological study. Brain 1991;114:2175–90. 28. Nicolai A, Bonetti B, Lazzarino LG, et al. Peripheral nerve vasculitis: a clinicopathological study. Clin Neuropath 1995;14:137–41. 29. Deprez M, De Groote CC, Gollogly L, et al. Clinical and neuropathological parameters affecting the diagnostic yield of nerve biopsy. Neuromusc Dis 2000;10:92–8. 30. Said G. Necrotizing peripheral nerve vasculitis. Neurol Clin 1997;15:835–48. 31. Davies L, Spies JM, Pollard JD, et al. Vasculitis confined to peripheral nerves. Brain 1996;119:1441–8. 32. Sugiura M, Koike H, Iijima M, et al. Clinicopathologic features of nonsystemic vasculitic neuropathy and microscopic polyangiitis-associated neuropathy: a comparative study. J Neurol Sci 2006;241:31–7.