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Is Perls Prussian Blue Stain for Hemosiderin a Useful Adjunct in the Diagnosis of Vasculitic Neuropathies?
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.310064
Keywords: Hemosiderin, nerve biopsy, Perls Prussian blue stain, sensitivity, specificity, vasculitic neuropathy
Nerve biopsies are often indicated to determine the etiology of peripheral neuropathies and aid in the management.[1],[2] Availability of genetics, autoantibody tests, and imaging studies has reduced the need for invasive biopsies. However, ~20% still remain undiagnosed, requiring nerve biopsy for determining etiology. The most frequent indication for nerve biopsy is for suspected vasculitic neuropathy and differentiate from other immune-mediated neuropathies that closely mimic vasculitis. Nerve biopsy in suspected vasculitic neuropathy is “diagnostic” only in 8 to 33% of biopsies.[3],[4],[5],[10],[11] In the absence of transmural inflammation/fibrinoid necrosis diagnostic of “definite vasculitis,” supportive features of vascular injury such as Perls Prussian Blue stain (PPB) to detect hemosiderin serve as a useful adjunct.[9] Inflammation in nerve biopsies is also seen in immune-mediated neuropathies such as CIDP, diabetes, and paraproteinemic neuropathies. Distinction is critical for determining treatment and prognosis. Presence of hemosiderin in the biopsy is often used as a pointer towards vasculitis. However, vascular injury is seen in several other infectious and immune-mediated disorders, such as inflammatory demyelinating neuropathies (AIDP/CIDP), leprosy, paraproteinemic neuropathies particularly POEMS, diabetes-related neuropathies, and neuropathy of aging and could theoretically produce hemosiderin deposits in the nerve. Hence, we sought to determine the frequency of PPB-positive hemosiderin deposits in inflammatory and noninflammatory neuropathies (such as hereditary and nutritional neuropathies) and thereby evaluate its utility in diagnosis of vasculitic neuropathy. There is only a single published study that has evaluated the frequency of Perls positivity in vasculitic neuropathies.[2]
Patient selection Between the years 2000 to 2013, 209 nerve biopsies performed for diagnosis of peripheral neuropathy were retrieved from the archives of the Department of Neuropathology, NIMHANS. The demographic, clinical, and pathologic features were reviewed and diagnoses categorized using recommended standard criteria for diagnosis [Table 1].
The patients' clinical details, neurological examination, electrophysiological findings, CSF analysis, routine hematology, blood glucose, other biochemical parameters, serological tests for HIV, HBsAg, systemic vasculitis and collagen vascular work-up, paraprotien (M-protein), cryoglobulins, and other ancillary investigations like bone scans, chest X ray, ultrasound abdomen, CT chest/abdomen for excluding neoplastic etiology as indicated based on clinical diagnoses were reviewed from charts. Nerve biopsies Peripheral nerve biopsies [sural nerve (n = 200), superficial peroneal nerve (n = 5), dorsal cutaneous branch of ulnar (n = 4)] were fixed in 2.5% glutaraldehyde and processed for paraffin embedding. Sections (2-3 micron thick) were stained with hematoxylin and eosin (H and E), Masson's trichrome for collagen, and Kulchitsky Pal (K Pal) stain for myelin. Serial sections were stained with PPB reaction for ferric iron (Perls 1867).[15] In brief, the deparaffinized sections were treated with freshly prepared mixture of ferrocyanide and hydrochloric acid for 10 mins, washed well in distilled water for 5 minutes, and counterstained with filtered Neutral red stain for 1 minute. Sections were then dehydrated in absolute alcohol, cleared, and mounted. A known positive control section (lung/spleen) was included in each run for quality assurance. Where indicated, additional stains for lepra bacilli (Fite-Faraco) and amyloid (Congo red) were performed. Slides were reviewed independently by four pathologists (SB, AM, AU, and YTC). Statistical analysis Mean and range were derived and significance was calculated using Chi-square test for categorical variables. Sensitivity, specificity, PPV, and NPV were calculated. Probability level (P value) <0.05 was considered significant.
Vasculitic neuropathy (n = 78) Vasculitis was diagnosed in 37.5% of nerve biopsies (78/208). [Nonsystemic vasculitis (NSVN) [65.4% (51/78)] and systemic vasculitides (SVN) [34.6% (27/78)]. Of systemic vasculitides, rheumatoid arthritis was most common (n = 9), followed by SLE (n = 4), undifferentiated systemic vasculitis (n = 4), Sjogren's (n = 3), HIV (n = 3), Scleroderma (n = 2), and Churg-Strauss syndrome (n = 2). Based on pathological criteria,[9] 46 were “definite” vasculitis with transmural infiltration and fibrinoid necrosis [Figure 1]a, 29 were “probable,” and 3 were “possible” vasculitis.
PPB stain detected hemosiderin in 48.7% of vasculitides [58.69% (16/27) in SVN and 43.1% (22/51) in NSVN] [Table 2].
PPB-positive deposits were frequent in “definite” (28/46; 60.86%), compared to “probable” vasculitis category (10/29; 34.48%). In all three cases of “possible” vasculitis, no hemosiderin deposits were seen as perivascular inflammatory infiltrates were minimal despite prominent vascular proliferation. Hemosiderin deposits were localized to epineurium in all cases (38/38), surrounding neovascularized/recanalized/inflamed vessels. Perineurial deposits were detected in approximately half (17/38, 44.73%), while endoneurial deposits were infrequent (12/38, 31.57%). In nine cases (23.68%), all three compartments revealed PPB-positive hemosiderin deposits. In cases of rheumatoid arthritis and Churg-Strauss syndrome, necrotizing vasculitis involved nutrient vessels [Figure 1]a and smaller epineurial venules and arterioles. In Churg-Strauss syndrome, eosinophils were prominent with plasma cells in rheumatoid arthritis. In SLE, Sjogren's syndrome and HIV, small caliber epineurial and endoneurial arterioles, and venules were inflamed. Vessels showing active fibrinoid necrosis and inflammation curiously lacked presence of hemosiderin [Figure 1]b. Instead, PPB-positive hemosiderin was seen to surround newly formed vessels, suggesting that chronic rather than acute vascular damage leads to leakage of blood and hemosiderin formation. In “probable” vasculitis, transmural inflammation or fibrinoid necrosis was absent but vascular sclerosis, luminal narrowing, recanalization, and neovascular proliferation were seen with variable inflammation [Figure 1]c and [Figure 1]e. Hemosiderin deposits surrounded sclerotic and recanalized vessels [Figure 1]d and [Figure 1]f and clusters of newly formed vessels in epineurium, in proximity to the inflamed vessels. In probable vasculitis, PPB positivity was detected in 34.4%, enhancing the sensitivity of diagnosis. Deposits were most frequently epineurial (10/29; 34.4%) with only rare deposits in the perineurium (2/29, 6.8%) and endoneurium (1/29, 3.4%). Leprous neuritis (n = 32) Thirty two cases diagnosed as leprous neuritis and classified histologically according to Ridley & Jopling criteria.[1] Most frequent was chronic resolving BT (10 cases) followed by borderline tuberculoid (BT, 9 cases), polar tuberculoid form (TT, 5 cases), borderline (BB, 4 cases), and borderline lepromatous (BL, 4 cases). Hemosiderin deposits were detected in 18 (56.2%) in endoneurium and perineurial compartments where inflammation was maximal. This was in contrast to vasculitic neuropathy with epineurial deposits. In BT and TT, epithelioid granulomas and dense lymphoid infiltrates expanded fascicles [Figure 2]a and [Figure 2]c. Perineurium was inflamed, with lymphocytes spilling into the epineurium to enclose the small epineurial vessels. Multinucleate Langhan's giant cells with central foci of caseous necrosis were seen in the nerve abscesses (two cases) [Figure 2]a. PPB test detected presence of hemosiderin in all five cases of TT and four of nine cases of BT encircling the epithelioid granulomas in peri- and endoneurial compartments [Figure 2]d, with the largest deposits in nerve abscesses [Figure 2]b. Despite dense epineurial perivascular inflammation, hemosiderin deposits were curiously not seen. In BB type with diffuse epithelioid histiocytes without granulomas, hemosiderin deposits were detected in all three compartments.
In lepromatous group with endoneurial foamy macrophages and lymphocytic infiltrates without vasculitis, hemosiderin deposits were scant, but involved all three compartments [Figure 2]e and [Figure 2]f. In 10 cases, the biopsy showed completely fibrosed fascicles enclosed and thickened perineurium. Numerous proliferating hyalinized vascular vessels were seen in perineurium and endoneurium without any inflammation. Hemosiderin deposits were rare (3/10). Two of the cases with lepra reaction revealed epineurial vasculitis in nerve biopsy and hemosiderin was detectable in one case. Diabetic neuropathy (n = 37) Nerve biopsies were performed in diabetics with distal symmetric sensory neuropathy (n = 18) CIDP (n = 11), plexopathy (n = 6), and mononeuritis multiplex (n = 2). Microangiopathic changes were seen in the endoneurial and epineurial compartments [Figure 3]a with loss of small myelinated fibers and inconspicuous regenerative activity characteristic of diabetic neuropathy. The perineurium was thickened and vascularized.
Endoneurial inflammation was seen in diabetics with CIDP (n = 11), in contrast to epineurial vasculitis in diabetics with mononeuritis multiplex (n = 2), and necrotizing vasculitis in diabetic plexopathy (n = 1) [Figure 3]c. Inflammation was absent in diabetic sensory neuropathies. Hemosiderin deposits were detected in 7 (18.9%), with CIDP/vasculitis/plexopathy as fine punctuate labeling in the perineurium [Figure 3]b and [Figure 3]d and surrounding clusters of small proliferating newly formed vessels in the epineurium with punctate deposits extending along the septa surrounding the adipose tissue. Hemosiderin was absent in endoneurium around hyalinized microvasculature probably due to hyaline thickening of the basement membrane preventing leakage of blood products. Overall the hemosiderin deposits did not correspond to degree or location of inflammation. CIDP (n = 15) Nerve biopsies in cases of CIDP revealed evidence of demyelination with inflammatory infiltrates surrounding predominantly endoneurial rather than epineurial vessels. PPB stain failed to detect hemosiderin in any of these cases. POEMS (n = 12) Nerve biopsies in cases of POEMS syndrome revealed variable axonopathy with demyelination. Inflammation was infrequent. However, a striking feature seen in the biopsies was the presence of prominent proliferating blood vessels forming a linear row around the perineurium [Figure 4]. In 4 out of 12 cases (33.3%), and hemosiderin deposits were seen exclusively in the perineurium, though not surrounding the proliferating vessels.
Hereditary neuropathy/Charcot-Marie-Tooth disease (n = 15) Fifteen cases of biopsy confirmed hereditary motor sensory neuropathy (CMT) were reviewed. These cases had no inflammation in the biopsy except in four cases with epineurial sparse sprinkling of lymphocytes. None of the cases revealed PPB-positive hemosiderin deposits. Age-related ischemic neuropathy (n = 12) Nerve biopsies revealed age-related arteriolosclerotic changes and vascular with nonuniform loss of myelinated fibers in sectoral pockets due to ischemic injury. Inflammation was absent and no hemosiderin deposits were detected despite prominent vascular changes. Vitamin B12 deficiency neuropathy (n = 7) All the seven cases revealed chronic axonopathy with secondary demyelination. No inflammation or hemosiderin deposits were detected. Statistical analysis Sensitivity of PPB positivity was highest in vasculitic neuropathy (48.7%) compared with all other types of peripheral neuropathy (22.3%), including CIDP, POEMS, leprous neuropathy, diabetic neuropathy, CMT, and age-related neuropathies, reaching statistical significance (χ2 = 15.7, P < 0.001). When compared with other infectious/immune-mediated neuropathies (POEMS, CIDP, leprous neuritis, and diabetic neuropathy), sensitivity was lower (48.7% versus 30.2%), but statistically significant (χ2 = 6.0, P = 0.015) compared to diabetes (18.9%, χ2 = 7.6, P = 0.002), and age-related ischemic neuropathy (0%, χ2 = 8.0, P = 0.005)]. PPB positivity in POEMS was 33.3% but the was not statistically significant. In disease negative controls (noninflammatory neuropathies like CMT, age-related ischemic neuropathy, and Vitamin B12 neuropathy), no PPB positivity was seen. The calculated specificity of PPB stain was 77.69% with PPV of 56.71% and NPV of 71.63%. The specificity increased to 89% if leprous neuropathy was excluded (as leprosy and vasculitis are easily distinguishable histologically). The PPV increased to 77.55% while NPV dropped to 68.5%.
PPB stain is most often used in suspected cases of vasculitic neuropathies that fail to demonstrate hallmark features of transmural inflammation/necrosis (Collins et al. 2007).[9] In the peripheral nerve, vessel wall injury, leads to local hemorrhage, and deposition of hemosiderin, visible as golden yellow pigment in H and E stain. Hemosiderin pigment contains iron in the form of ferric hydroxide that is complexed to protein but can be unmasked by various chemicals. The ferrous form of iron (transport form) is unstable and gets oxidized (Fenton reaction) to ferric state, which is the stable storage form and demonstrable in tissues using PPB stain. In this stain, ferric iron, if present in tissues reacts with ferrocyanide, to form insoluble ferric ferrocyanide, which stains as bluish or purple deposits. The intensity of staining gives rough indication of quantity of free ferric iron in the tissue. Demonstration of hemosiderin in peripheral nerve biopsies is considered one of the criteria for diagnosis of “probable” vasculitis in the absence of transmural inflammation and fibrinoid necrosis.[8] However only a single study in published literature has assessed the utility of PPB for iron in the diagnosis of vasculitic neuropathy.[11] This study evaluated 16 cases of vasculitis and 12 cases of AIDP/CIDP. They reported that PPB stain is a highly sensitive and specific marker for systemic/PNS vasculitis (81%). Ten of 12 cases of vasculitis (PNS vasculitis – 5, polyarteritis nodosa and Wegener's granulomatosis – 5), 2 of 3 cases of SLE, and 1 case of rheumatoid arthritis were positive but none of AIDP/CIDP (n = 12). The study however did not include other inflammatory neuropathies such as leprosy, diabetic neuropathies with mononeuritis multiplex/plexopathy, and paraproteinemic neuropathies in which microvascular injury is central to the pathogenesis. In our study, we evaluated 78 cases, and PPB positivity was seen in only 47.4% cases of vasculitis in contrast to the study by Adams et al.[2] and was more frequent in systemic than nonsystemic vasculitis (55.5% vs 43.1%). The lower frequency in our study is possibly due to inclusion of “probable” and “possible” category of vasculitis. Intriguingly, some of the cases with transmural inflammation, fibrinoid necrosis, and endothelial injury did not reveal PPB-positive hemosiderin deposits. In this acute stage of injury, presence of nonferric forms or soluble ferrous forms of iron may not readily be detected by the PPB staining. In chronic vascular injury evidenced by neovascularization and luminal obliteration/reacanalization, PPB stain readily detected the presence of hemosiderin deposits. PPB stain enhanced the diagnostic yield of clinically suspected vasculitis, particularly in absence of definite features. In the group of “probable” vasculitis, detection of hemosiderin in 10 out of 29 cases (34.4%), enhanced the diagnostic yield with therapeutic implications. In the category of “possible vasculitis” however, PPB stain did not help in resolving the diagnosis probably as the degree of vascular injury was presumably insufficient to cause vascular damage and leakage. In leprous neuritis, inflammation involved endoneurium and perineurial infiltrates and vasculitis is frequent in lepra reactions such as erythema nodosum leprosum (ENL). There are no studies that have evaluated the occurrence of hemosiderin in nerve biopsies of leprosy. In our study, hemosiderin deposits were very frequent, detected in 56% of biopsies but was localized within the inflamed endoneurial and perineurial compartments in contrast to epineurial location in vasculitis. In diabetic neuropathy, the presence of iron deposits in nerve biopsies from patients with lumbosacral radiculoplexus neuropathy was reported by Dyck et al.[13] Said et al. (2007) studied 22 diabetic patients with multifocal sensory-motor deficits, and 6 had necrotizing vasculitis of perineurial and endoneurial blood vessels.[12] Endoneurial red blood cells were present in 11 and endoneurial hemorrhage in 5. Ferric iron deposits that characterize old bleed was seen in seven patients. We studied 37 cases with various forms of diabetic neuropathy, including symmetric sensory neuropathy, mononeuritis multiplex, CIDP, and plexopathy. Iron deposits were most frequent in diabetic neuropathy with CIDP, preferentially localized to perineurium, even in absence of inflammation. In diabetes with mononeuritis multiplex and plexopathy, epineurial and perineurial PPB-positive deposits were detected. Interestingly, no hemosiderin deposits were seen around endoneurial microangiopathic changes. The hyaline thickening of the basement membrane of microvasculature probably prevented leakage of blood/blood products. This is in contrast to the study by Said et al. (2007),[12] who reported endoneurial PPB-positive deposits. POEMS syndrome is characterized by polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes. The pathogenesis and the clinical syndrome is attributed to overproduction of vascular endothelial growth factor (VEGF) levels inducing a rapid and reversible increase in vascular permeability and neovascularization.[7] Endoneurial microvascular endothelial injury secondary to high VEGF levels is presumed to cause hyperpermeability. In our study, 4 of 12 cases (33.3%) of POEMS syndrome revealed hemosiderin deposits exclusively in the perineurium. AIDP results from perivascular epineurial and endoneurial T-cell rich infiltrate along with macrophage-mediated stripping of layers of otherwise normal appearing myelin.[4] Circulating antibodies against endoneurial targets access through a damaged blood nerve barrier. CIDP is characterized by symmetric progressive or relapsing/remitting sensory and motor polyradiculopathy involves particularly proximal nerves with endoneurial inflammation and onion bulb formation in chronic cases. In the study by Adams and colleagues, hemosiderin deposits were seen in 12 cases of CIDP/AIDP similar to our study. Ischemic neuropathy is less common, and diagnosis can be challenging, with patients having a mononeuritis multiplex or sensory-motor, axonal-demyelinating neuropathy. These are however noninflammatory and accordingly, no hemosiderin deposits were detectable in any of our cases. In hereditary and B12-deficient neuropathies also, no hemosiderin deposits were detectable in the biopsies.
Hemosiderin deposits were most common in cases of leprous neuropathy (56%) and vasculitic neuropathies (48.72%) [59.25% in systemic and 43.13% in nonsystemic vasculitides (χ2 = 1.5, P = 0.227)]. PPB staining increased sensitivity of diagnosis in probable vasculitis (34.4%), wherein transmural inflammation is not seen. However, a considerable number of other inflammatory/immune-mediated neuropathies were also positive for hemosiderin with overall 11.22% positivity (POEMS-33.3%, diabetes-18.9%, CIDP-6.3%), if leprosy is excluded. The overall sensitivity was higher in vasculitic neuropathy (48.72%) compared to other inflammatory neuropathies (11.22%) and was statistically significant (P < 0.05). No hemosiderin was detectable in nonimmune neuropathies (hereditary, B12 deficiency, and age-related ischemic neuropathies) suggesting high sensitivity and specificity for inflammatory neuropathies. The location of hemosiderin deposits varied, being more common in epineurial compartment in vasculitis, in contrast to to endoneurial and perineurial location in leprosy, and exclusively perineurial in POEMS syndrome and diabetic neuropathy. Acknowledgements The author wishes to acknowledge Mr. Prasanna Kumar and Mrs. Rajasakthi V from Human Brain Tissue Repository (Brain Bank) for technical assistance and Mr. Manjunath K, Department of Neuropathology, NIMHANS, Bangalore, for assistance with montages of microphotographs. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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