Muscle histopathology in today's era of molecular genetics: Role and limitations.

INTRODUCTION: Past few decades have seen an increasing application of techniques like electron microscopy, western blotting, and molecular genetics in the evaluation of muscle diseases. However, due to their limited availability, histopathological interpretation of muscle biopsies still remains an important component of diagnostic approach to muscle diseases. A systematic methodology is required in the evaluation and interpretation of muscle biopsies. This study was undertaken to analyze the histopathological spectrum of 164 muscle biopsies and to assess the diagnostic yield of basic histopathological procedures in the work up of muscle biopsy.
MATERIALS AND METHODS: Retrospective analysis was done for 164 cases of muscle biopsies. Step-wise approach was adopted to assess the efficacy of routine stains, enzyme histochemistry, and immunohistochemistry. Based on hematoxylin and rosin (H and E) appearance, biopsies were broadly categorized into destructive, nondestructive but myopathic, and inflammatory morphology. Role of special stains, enzyme, and immunohistochemical stains in each category was then evaluated.
RESULTS: On the basis of histopathological features, 164 muscle biopsies were broadly categorized into biopsies with abnormal histopathological features (115) and biopsies with normal histopathology (49). Abnormal muscle biopsies were further categorized into destructive morphology (56.5%), nondestructive but myopathic morphology (30.5%), and inflammatory pathology (13%). A near definitive diagnosis could be made in 115 cases out of 164 muscle biopsies on the basis of routine histopathology, enzyme histochemistry, and immunohistochemistry.
CONCLUSION: Though advanced techniques like electron microscopy, western blotting, and molecular genetics are essential for confirmatory diagnosis, a substantive diagnostic yield can be offered with the basic armamentarium of routine (frozen) stains, enzyme histochemistry, and immunohistochemistry.

Introduction

Muscle disorders are known to be phenotypically and genetically heterogeneous. Muscle biopsy in these disorders is an important and at times an indispensible diagnostic tool for diagnosing or corroborating the clinical impressions.[123] Rapid advances have been seen in the techniques applied for diagnosing muscle diseases. Application of molecular genetic testing and electron microscopy offer the prospect of an accurate diagnosis which forms the basis of patient management and, also, family counseling. However, not all centers are equipped with these advanced diagnostic modalities and not all patients can afford these. Mainstay of diagnosis, in such instances, continues to remain histopathology, enzyme, and immunohistochemistry. There is a paucity of literature, however, that specifically assesses the diagnostic yield of the basic armamentarium of routine and special stains, enzyme histochemistry, and immunohistochemistry.[4]

This study was undertaken to analyze the histopathological spectrum of 164 muscle biopsies received in our department over the last 5 years. The aim was to observe the histopathological spectrum of changes seen in muscle diseases and assess the diagnostic yield of basic procedures like routine stains on frozen sections, enzyme histochemistry, and immunohistochemistry in the work up of muscle biopsy.

Materials and Methods

A retrospective analysis of the histopathology records and clinical case files was done in 164 cases of muscle biopsies received over a period of 5 years in our department. Following histopathological features were analyzed in detail: Fascicular architecture, variation in fiber size, nuclear features, fibers showing abnormal features like necrosis, splitting, basophilia, phagocytosis, cellular reactions, and endomysial as well as perimysial fibrosis. In addition, findings pertaining enzyme histochemistry and immunohistochemistry were also analyzed.

Step-wise approach was adopted to assess the efficacy of routine stains (on frozen sections), enzyme histochemistry, and immunohistochemistry. Based on hematoxylin and eosin (H and E) appearance, biopsies were broadly categorized into: Muscle biopsies with destructive morphology, muscle biopsies with nondestructive morphology, muscle biopsies with inflammatory pathology, and normal muscle biopsies.

Role of special stains, enzyme histochemistry, and immunohistochemistry was further evaluated in each category.

Results

Based on the histopathology, 164 muscle biopsies [results summarized in Figure 1] were broadly categorized into abnormal (115) and normal (49) biopsies. After analyzing the histopathological features on hematoxylin and eosin stain, the abnormal biopsies were further categorized into biopsies showing destructive morphology, biopsies showing nondestructive but myopathic features, and biopsies showing inflammatory morphology [Table 1]. Biopsies with destructive morphology predominantly showed effaced architecture, marked variation in fiber size, excessive internal nuclei and nuclear clumps, fibers showing necrosis, myophagocytosis, splitting, and basophilia. Endomysial as well as perimysial fibrosis was variable [Figure 2]. Biopsies with nondestructive but myopathic morphology predominantly showed relatively preserved architecture. Myopathic features like mild-to-moderate variation in fiber size, angulated atrophic fibers, and internalization of nuclei in few/fair number of fibers. Centrally placed nuclei were observed in a single case. Endomysial as well as perimysial fibrosis was mild [Figure 3]. Biopsies with inflammatory morphology predominantly showed relatively preserved architecture, mild variation in fiber size, internalization of nuclei in few fibers, marked inflammatory infiltrate, necrotic fibers, prominent myophagocytosis, fiber splitting, and basophilic fibers. Perifascicular atrophy was evident in one case [Figure 4 and Table 2].

Figure 1

Diagnostic algorithm for the muscle biopsies. H and E = Hematoxylin and eosin, MGT = modified Gomori trichrome, NADH = nicotinamide adenine dinucleotide reductase, SDH = succinate dehydrogenase, ATPase = adenosine triphosphatase, IHC = immunohistochemistry

Table 1

Table showing broad categorization of muscle biopsies

Figure 2

H and E staining showing (a) totally effaced architecture with marked endomysial and perimysial fibrosis, (b) marked variation in fiber size, (c) internalization of nuclei with nuclear clumps, (d) necrotic fibers undergoing myophagocytosis, (e) fiber splitting, and (f) basophilic fibers

Figure 3

H and E staining showing (a) relatively preserved architecture, (b) mild-to-moderate variation in fiber size, (c) angulated fibers, and (d) mild endomysial and perimysial fibrosis

Figure 4

H and E staining showing (a) marked inflammatory infiltrate, (b) myophagocytosis, and (c and d) perifascicular atrophy

Table 2

Table showing histopathological features for various categories of muscle biopsies on H and E stain

Modified Gomori trichrome (MGT) stain did not add much to the biopsies with destructive pattern. In contrast amongst nondestructive but myopathic muscle biopsies, MGT showed ragged red fibers (RRFs) in four cases [Figure 5a] suggesting mitochondrial myopathy, red staining rods in a single case [Figure 5b] suggesting nemaline myopathy, and darkly stained areas with red stained cytoplasmic bodies in one case suggesting myofibrillar myopathy [Figure 5c]. Biopsies with inflammatory pathology showed fibers with vacuoles rimmed by red granular material in three cases that suggested inclusion body myopathy (IBM) [Figure 5d and Table 3].

Figure 5

MGT showing (a) ragged red fibers, (b) red staining rods, (c) red staining cytoplasmic bodies, and (d) vacuoles rimmed by red granular material

Table 3

Table showing histopathological features for various categories of muscle biopsies on MGT stain, enzyme histochemistry and immunohistochemistry

Muscle biopsies with destructive morphology with enzyme histochemistry revealed lobulated fibers in 15 cases suggesting calpainopathy (limb girdle muscular dystrophy (LGMD) 2A) [Figure 6]; moth eaten [Figure 7a] and whorled fibers, small type 1 fibers in one case [Figure 7b] suggesting facioscapulohumeral dystrophy (FSHD), type 1 fiber predominance in a single case [Figure 7c] suggesting congenital muscular dystrophy (CMD); and type 1 fiber atrophy, and ring fibers, moth eaten fibers in one case that suggested myotonic dystrophy. Biopsies showing nondestructive but myopathic morphology with enzyme histochemistry revealed large group of atrophic fibers [Figure 8a], fiber type grouping [Figure 8b], large type 1 fibers with ATPase (pH 9.4) in 11 cases suggesting neurogenic disorders; small type 1 fibers with dark centers, and pale peripheral halos with NADH in one case suggesting centronuclear myopathy; and centrally or peripherally placed cores with NADH in three cases suggesting central core disease [Figure 9a], accumulation of oxidative enzyme stain in the center of fibers and pale peripheral halos with nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) in two cases suggesting myotubular myopathy, type 1 fibers smaller than type 2 fibers with ATPase (pH 9.4) in 12 cases suggesting congenital fiber type disproportion [Figure 8c]. Biopsies in the category of inflammatory morphology revealed intense and aggregated NADH-TR activity in perifascicular fibers in three cases that suggested the diagnosis of dermatomyositis [Figure 9b].

Figure 6

Nicotinamide adenine dinucleotide tetrazolium reductase showing (a and b) large number of lobulated fibers, and SDH showing (c and d) extensive lobulation in fair number of muscle fibers

Figure 7

NADH showing (a) moth eaten fibers, (b) small type 1 fibers, and (c) type 1 fiber predominance

Figure 8

ATPase at pH9.4 showing (a) large group of atrophic fibers, (b) fiber type grouping, and (c) smaller type 1 fibers

Figure 9

NADH showing (a) centrally and peripherally placed cores, (b) intense perifascicular NADH activity

A definitive diagnosis was rendered by immunohistochemistry among biopsies showing destructive morphology and biopsies with inflammatory pathology. Dystrophin 1, 2, and 3 showed complete membranous immunonegativity in most of the fibers in 18 cases [Figure 10c], thus providing a definite diagnosis of Duchenne muscular dystrophy (DMD). Uneven, patchy labeling with reduced intensity on most of the fibers in eight cases confirmed these cases as Becker muscular dystrophy (BMD) [Figure 10b]. Complete membranous immunonegativity for dysferlin in 13 cases confirmed the diagnosis of dysferlinopathy (LGMD 2B). A definitive diagnosis of sarcoglycanopathies (LGMD 2C, 2D, 2E, 2F) was made in all eight cases where complete membranous immunonegativity for α, β, γ, and δ sarcoglycans was observed. Inflammatory cells showing immunopositivity for LCA and CD68 among the biopsies showing inflammatory morphology confirmed all the nine cases as polymyositis.

Figure 10

Immunohistochemistry showing complete membranous immunopositivity for (a) dystrophin 1, 2, and 3; (b) patchy membranous positivity for dystrophin 1, 2, and 3; and (c) complete membranous immunonegativity for dystrophin 1, 2, and 3

Diagnostic yield (based on H and E, MGT, enzyme histochemistry, and IHC) was variable among the three categories. The broad categorization was based on H and E features dividing the muscle biopsies (n = 164) into normal (49), destructive (65), nondestructive but myopathic (35), and inflammatory (15) morphology. For the biopsies showing destructive and inflammatory pattern IHC was the most specific diagnostic tool through which among the destructive morphology we could provide a definite diagnosis in 47 (72.3%) cases; whereas with enzyme histochemistry, a probable diagnosis was provided in 18 (27.7%) cases; however, for inflammatory morphology a definite diagnosis was provided in nine (60%) cases, while MGT provided a probable diagnosis in three cases (20%) and similarly a probable diagnosis was provided by enzyme histochemistry in three (20%) cases. In contrast, among the biopsies showing nondestructive, but myopathic pattern a probable diagnosis was given in 29 cases (82.8%) by enzyme histochemistry, while with MGT a probable diagnosis was given in six (17.2%) cases [Table 4].

Table 4

Table showing diagnostic yield with H and E, MGT, enzyme histochemistry and immunohistochemistry

Among the muscle biopsies showing abnormal histopathology, histopathological diagnosis showed concordance with the clinical diagnosis in 72.2% of cases (83/115). Discordance was observed in 25.2% of cases (29/115). In three (2.6%) cases no provisional diagnosis was mentioned in the requisition forms.

Discussion

Tremendous advances in our understanding of the molecular basis of muscle diseases over the past few years have led to several conceptual shifts in our approach to clinicopathologic diagnosis of muscle biopsy specimens. However, one cannot get far in the discussion of muscle diseases before entering the somewhat arcane world of specimen processing, histochemical staining, immunohistochemical staining, and electron microscopy.[5] Unfortunately though, availability of these diagnostic tools is still limited in majority of the laboratory settings in our country. Under such circumstances, interpretation of muscle biopsy histopathology in the perspective of clinical details can serve to narrow down to probable diagnoses. An algorithmic approach to muscle biopsy was adopted in our department to maximize diagnostic yield. The techniques available were routine histopathology and some special stains on frozen and paraffin embedded sections, enzyme histochemistry (NADH, SDH, and ATPase), and immunohistochemistry (dystrophin 1, 2, and 3; merosin; dysferlin; and α, β, γ, and δ sarcoglycans). With this basic and essential panel for muscle biopsy workup, we could offer a reasonable possible diagnosis in 47/65 (72.3%) muscle biopsy cases showing destructive morphology and in 9/15 (60%) muscle biopsies showing inflammatory morphology. However, among the biopsies showing nondestructive but myopathic morphology with this basic panel, a probable diagnosis was provided in all 35 cases with the help of MGT and enzyme histochemistry. Definite diagnosis could not be rendered in this category as molecular genetic analysis and electron microscopy (EM) is required for providing a definite diagnosis as described by Rollins et al., in their study assessing the diagnostic yield of muscle biopsy in patients with clinical evidence of mitochondrial cytopathy.[6]

The limited diagnostic yield within the category of muscle biopsies showing destructive morphology could be attributed to the lack of diagnostic modalities like immunoblot and molecular genetic analysis at our center as LGMD 2A require immunoblot,[7] while LGMD2B and FSHD need molecular genetic analysis for definite diagnosis[8] as described by Upadhyaya and Cooper[9] in their study elaborating molecular genetics of FSHD. Similarly the unsatisfactory diagnostic yield within the category of muscle biopsies showing nondestructive but myopathic morphology could be attributed to the lacking diagnostic modalities including molecular analysis and EM as molecular analysis is required for confirmation of neurogenic disorders, diminishing the role of muscle biopsy in neurogenic disorders as emphasized by Echaniz-Laguna et al.[10] Similarly in all congenital myopathies, EM is essential for the diagnosis and for directing molecular analysis. To maximize the diagnostic yield for the third category of muscle biopsies showing inflammatory pathology again molecular genetic analysis is required specifically for definitive diagnosis in cases of IBM as described by Nonaka et al.[11]

Conclusion

To conclude, the authors would highlight that though tools like electron microscopy, western blotting, and molecular genetics are available at apex centers, at most places pathology diagnosis still rests on routine stains on frozen sections, enzyme histochemistry, and immunohistochemistry. Though advanced techniques like electron microscopy, western blotting, and molecular genetics are essential for confirmatory diagnosis; a substantive diagnostic yield can be offered with the basic armamentarium of routine and special stains, enzyme histochemistry, and immunohistochemistry. In our experience, this basic laboratory support helps the clinicians in planning further management and guidance to the patient and family in majority of the cases.