Just as every pathologist has particular preferences for routine stains, so muscle histochemists have tended to develop preferences for particular reactions, especially in the interpretation of fibre types.
In the early days of the application of histochemical techniques to the study of muscle, large batteries of enzymes were routinely studied in muscle biopsies (see ). While these many enzyme reactions were of special interest and value in a research context, it became apparent that much of the information required in the assessment of diseased muscle could be obtained from a smaller number of procedures, and additional methods were only necessary in specific circumstances.
In this chapter, we discuss the histological and histochemical methods which we routinely use on biopsies (see Table 2.1 ). General application and illustration of the techniques are discussed in Chapter 3, Chapter 4 . The theoretical background of the techniques and variations to the staining techniques can be found in standard textbooks ( ) or earlier editions of this book.
|Haematoxylin and eosin (H&E)||General structure of the sample: fibre size and contours, position of nuclei, fibrosis, inflammation, nerves, blood vessels|
|Gomori trichrome||Mitochondria red (type 1 fibres darker); fibres with abnormal mitochondria ragged red|
|Nemaline rods, cytoplasmic bodies, reducing bodies, tubular aggregates red|
|Membranous whorls or rimmed vacuoles red|
|Verhoeff–van Gieson||Highlights connective tissue (red) and elastin and myelin (black)|
|Oil red O||Intracellular lipid seen as red dots|
|Lipid of adipose tissue red (may spread over the section)|
|Sudan black||As for oil red O but a black end product (phospholipids also stain black)|
|Periodic acid-Schiff||Fibre type pattern; fibres with excess glycogen heavily stained; fibres with loss of glycogen white; diastase-resistant polyglucosan material dark pink|
|Reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR)||Fibre type pattern; distribution of mitochondria; abnormal mitochondria, myofibrillar disruption, cores|
|Succinate dehydrogenase (SDH)||Fibre type pattern; fibres with abnormal mitochondria, fibres devoid of activity, cores|
|Cytochrome c oxidase (COX)||Fibre type pattern; fibres with abnormal mitochondria, fibres devoid of activity, cores|
|Combined cytochrome c oxidase and succinic dehydrogenase||Fibres devoid of cytochrome c oxidase activity appear blue|
|ATPase or myosin isoforms||Distribution and involvement of fibre types and their subtypes|
|OPTIONAL TECHNIQUES, DEPENDING ON THE RESULTS OF THE ABOVE AND PHENOTYPE|
|Phosphorylase||Absent in type V glycogenosis (McArdle disease) and when glycogen absent; fibre type pattern|
|Phosphofructokinase||Absent in type VII glycogenosis; fibre type pattern|
|Myoadenylate deaminase||Absent/deficient in exertional myalgia (significance uncertain); fibre type pattern; tubular aggregates very darkly stained|
|Acid phosphatase||High in lysosomal storage disorders and rimmed vacuolar myopathies; high in necrotic fibres; highlights lipofuscin|
|Alkaline phosphatase||High in blood vessels and perimysium in some inflammatory myopathies|
|Acetylcholinesterase||Neuromuscular junctions, myotendinous junctions, vacuoles in XMEA, denervated/non-innervated fibres positive|
|Non-specific esterase||Neuromuscular junctions, myotendinous junctions, phagocytic areas, small angulated denervated fibres positive|
|Menadione-linked α-glycerophosphate dehydrogenase||Reducing bodies and granular inclusions in acid maltase deficiency positive; fibre type pattern if substrate used|
|Congo red||Shows presence of amyloid|
The most important stain used routinely is haematoxylin and eosin (H&E), which clearly shows the overall structure of the tissue in relation to the fibres, nuclei, fibrous and adipose tissue, the presence of inflammatory cells and vacuoles, and vascular and neural components. In addition, the distribution of mitochondria may be distinguished, depending on the specific haematoxylin used. With the H&E stain, nuclei stain blue, the muscle fibres pink and the connective tissue a lighter pink. Basophilic fibres may be recognized by their blue stain. If Harris’ haematoxylin is used, the mitochondria can be seen as small dots. Cross-striations are not easily visible in unfixed frozen material. Prominent red, eosinophilic areas may be visible within fibres and may correspond to abnormal accumulations of myofibrillar material or to cytoplasmic bodies (see Ch. 4 ).
It is sometimes easier to observe subtle increases in endomysial connective with the modified Gomori trichrome technique ( ), in which the muscle fibres stain a greenish-blue colour, and the collagen is a lighter but clearly distinguishable blue–green colour. Nuclei stain red with the Gomori stain, and the myelin of the nerve stains a foamy red colour. Nerves may appear poorly stained in the absence of myelin. Abnormal accumulations of myofibrillar material may appear to be a darker green–blue colour. A major application of the modified Gomori technique is the identification of red-staining structures such as rods, cytoplasmic bodies, tubular aggregates, reducing bodies, abnormal mitochondria and the membranous myelin-like whorls of rimmed vacuoles. Mitochondrial accumulations appear as red aggregates of stain and the intermyofibrillar mitochondria appear as a series of fine dots throughout the fibre. Normal muscle fibres frequently show peripheral aggregates of mitochondria, and care is needed not to overinterpret their significance. Connective tissue can also readily be revealed with stains such as van Gieson or picrosirius, both of which stain collagen bright red in contrast to the yellow–green of the fibres. As excess connective tissue is visible with H&E and the Gomori trichrome, an additional stain for connective tissue is a matter of personal choice. There is some advantage, however, in using the Verhoeff–van Gieson combination, as it also demonstrates the presence of myelin (black) in the peripheral nerves and elastin (black) in the blood vessels. Mitochondria and the intermyofibrillar network are also visible in cross-sections of the fibres as fine dark dots. These histological techniques therefore also reveal a difference between fibre types (see later), with the higher mitochondrial content of type 1 fibres giving a darker colour to the fibre. Details of all staining techniques are given at the end of this chapter.
Additional stains which may prove helpful in particular instances are various techniques for nucleic acids (DNA and RNA), cresyl fast violet or toluidine blue for metachromatic material, alizarin red for calcium, phosphotungstic acid haematoxylin (PTAH), which may demonstrate such structures as the rods in nemaline myopathy, and Congo red to show amyloid in inclusion body myositis or other disorders, but these are not necessary as part of a routine panel.
Histochemical techniques are essential for the study of muscle biopsies for four main reasons. First, they demonstrate the non-uniform nature of the tissue by revealing the different biochemical properties of specific fibre types and their selective involvement in certain disease processes. Secondly, they may show an absence of a particular enzyme (e.g. phosphorylase in McArdle disease or cytochrome c oxidase in some mitochondrial disorders). Thirdly, an excess of a particular substrate can be demonstrated (e.g. glycogen in glycogen storage diseases or lipid in carnitine deficiency or some mitochondrial-related disorders). Fourthly, they may show structural changes in the muscle which would not be apparent with routine histological stains, such as the enzyme-deficient cores in core myopathies, ‘moth-eaten’ fibres, and abnormalities in the distribution of mitochondria.
The number of histochemical techniques for routine use has diminished over the years; the most important are summarized in Table 2.1 . Enzyme histochemistry has become firmly established as a link between the morphology and biochemistry of tissues. The indispensable value of enzyme histochemistry to the study of muscle highlights the need to freeze a biopsy as fixation destroys the activity of many enzymes. With histochemical and immunohistochemical techniques, it is now possible to demonstrate many enzymes. It is clearly beyond the scope of this book to cover the whole range, but the following section highlights the application of those of particular importance to the diagnostic pathology of muscle. Minimal biochemical background of the enzyme reactions is given here, but further reference can be made to the excellent manual on enzyme histochemistry of , to the textbooks on histochemistry cited previously and to standard biochemistry textbooks.
The most useful oxidative enzymes studied in muscle are reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH) and cytochrome c oxidase (COX).
The principle of the histochemical technique for NADH-TR and SDH is to employ a colourless, soluble tetrazolium salt as an electron acceptor which is reduced to a deeply coloured, insoluble formazan product at the site of the enzyme activity. The commonly used tetrazolium salt is nitroblue tetrazolium (NBT) [2,2′-di- p -nitrophenyl-5,5′-diphenyl-3,3′-(3,3′-dimethoxy-4,4′-biphenylene) ditetrazolium chloride], which gives a bluish final end product. Thus the intensity of the formazan reaction product is a reflection of the number of mitochondria within a fibre and reveals the characteristic checkerboard pattern of fibre types. Some caution in interpretation, however, is needed with regard to specificity with the techniques for NADH-TR and SDH because tetrazolium salts have a strong affinity for phospholipids and with the reaction for NADH-TR the sarcoplasmic reticulum is also revealed. This can, however, be advantageous as the technique for NADH-TR is useful for showing disruption and distortion of myofibrils and the internal structure of whorled fibres (see Ch. 4 ). The technique for SDH, in contrast, is specific for mitochondria, as is the technique for COX.
COX is very sensitive to fixation and is inhibited by cyanide and azide. Even brief fixation in formaldehyde, glutaraldehyde or alcohol can produce negative results in the histochemical reaction, emphasizing the need for frozen sections. It is an integral component of the mitochondrial membrane and is encoded by mitochondrial DNA. Succinate dehydrogenase, in contrast, is encoded by nuclear DNA. The method commonly used to demonstrate COX activity uses diaminobenzidine as an electron donor and produces a brown end product that can be enhanced by osmium. The reaction for COX reveals differences in mitochondrial number and their distribution in different fibre types (see Ch. 3 ). It is also an important method for demonstrating fibres devoid of activity caused by certain mutations in mitochondrial DNA. A combination of the technique for COX and SDH provides a clear method for identifying fibres that are deficient in COX but retain SDH activity, as they appear blue in contrast to the brownish-blue/grey of normal fibres.
In vivo, phosphorylase is a cytoplasmic enzyme concerned with the degradation of glycogen by destruction of α-1,4′-glycosidic linkages. The histochemical method (see ) relies on the conversion of the inactive b form of the enzyme to the active a form, followed by staining of the polysaccharide that is formed by iodine. The purple colour is unstable and fades but can be made permanent using Schiff reagent. Dehydration in alcohol and mounting in synthetic resin also preserves the end product but the colour may be slightly altered. Phosphorylase activity varies with fibre type and is another technique that shows the checkerboard pattern of fibre types. Absence of phosphorylase from muscle fibres occurs in McArdle disease, and it is therefore questionable if this technique needs to be performed routinely if there is no clinical indication of a glycogenosis, but it should always be checked in a patient with a history of cramps. Absence of phosphorylase staining is also seen if there is a defect in glycogen synthesis as the method of demonstration relies on endogenous glycogen. Fibres, or focal areas such as cores, that are devoid of glycogen also therefore show an absence of phosphorylase.
Adenosine Triphosphatase (ATPase)
Myosin ATPase, which is calcium activated, is the most important enzyme for revealing fibre types. The method for its localization relies on the release of phosphate, the capture of this by calcium and the substitution of the calcium by cobalt. The cobalt is then replaced by sulphide, and the end product is a black precipitate of cobalt sulphide. The reaction is carried out at a non-physiological pH of 9.4, and preincubation at different acid pHs of 4.3 and 4.6 is used to demonstrate the reciprocal pattern and subdivision of fibre types (see section on fibre types in Ch. 3 ).
In considering the validity of this reaction, it should be borne in mind that it takes place at a very alkaline pH which may not occur in vivo. Furthermore, there is a physical alteration of the tissue at some stage during the reaction. When muscle tissue is air dried and exposed to calcium, the intermyofibrillar network is in some way altered so that later in the reaction it disintegrates. Thus with the reaction for ATPase at pH 9.4, the intermyofibrillar network is dissolved out of the section and no ATPase can be demonstrated in this location even though the enzyme may be present there. The reaction thus becomes essentially a myosin ATPase reaction.
The ATPase method has historically been accepted as the standard method for demonstrating fibre types but the advent of immunohistochemistry and the application of antibodies to myosin is equally reliable and has certain advantages ( ). This is discussed in more detail in subsequent chapters. A considerable amount of data has accumulated over the years from ATPase-stained sections, particularly with regard to morphometric analysis (see Ch. 4 ), and it may be some time before myosin immunolabelling completely replaces the ATPase method, although the use of antibodies is increasing and they are better for identifying hybrid fibres co-expressing more than one isoform of myosin. The ATPase can be a difficult method with which to get consistently good results, and several laboratories now rely on myosin immunolabelling for routine assessment of the main fibre types. Identification of the subtypes of type 2 fibres is more difficult with antibodies, but when assessment of 2A and 2B fibres is required this can be obtained from sections stained for oxidative enzymes.
Additional Enzyme Studies
Additional methods that may be useful in association with certain clinical features are included in the list of methods. Although several of these formed part of a routine set of procedures in the early years of muscle pathology, they only add additional diagnostic information in certain situations. With increasing awareness of subtle changes associated with some genetic defects, however, some are of value.
Acid phosphatase is localized mainly in lysosomes and may thus be used to indicate foci of degeneration and necrosis within muscle fibres. Very little is apparent in normal muscle fibres, except in perinuclear regions where it is seen as focal deposits associated with lipofuscin. Lipofuscin is more abundant in muscle from adults than from children and there may therefore appear to be more perinuclear acid phosphatase activity in adults. In type II glycogenosis and lysosomal disorders, acid phosphatase is useful as it demonstrates subtle increases and the activity associated with the vacuoles. It also highlights the presence of macrophages. Acid phosphatase activity is also abundant in vitamin E deficiency and Batten disease, and the deposits are autofluorescent. The colour of the autofluorescence can be used to distinguish the two types of deposit as in vitamin E deficiency they are orange–yellow but yellow in Batten disease.
Alkaline phosphatase is found primarily in cell membranes where active transport processes occur, such as the endothelium of arterioles and the arterial part of capillaries, and also in endoplasmic reticulum, Golgi apparatus and pinocytotic vesicles. The reaction is usually negative in muscle fibres but may be positive in focal necrotic fibres in various disease situations, and in some regenerating or non-innervated fibres. Its major use is in the assessment of inflammatory myopathies when perimysial areas may be intensely stained.
Phosphofructokinase may be useful to study if a glycogenosis is suspected, but only a result of total absence can be relied on. A deficiency is difficult to access histochemically and requires biochemical analysis.
Menadione-linked α -glycerophosphate dehydrogenase reveals a fibre type pattern with type 2 fibres more intensely stained than type 1, but is of particular diagnostic value in distinguishing reducing bodies. These, and the abnormal accumulation of myofibrillar material in some myofibrillar myopathies (see Ch. 16 ), as well as some unusual granular structures observed in acid maltase deficiency ( ), are the only abnormal structures to stain with this technique, even without substrate. Tubular aggregates can also show a slight degree of staining but are more easily identified by other stains. As the occurrence of these structures is very rare, this technique is often not included in a routine panel. In this technique the menadione is reduced by sulphydryl-containing structures.
Staining for myoadenylate deaminase is favoured by some, as a deficiency may be the only feature of note in some patients. Interpretation of the significance of a deficiency is hampered, however, by the presence of a common polymorphism in the normal population that obliterates the enzyme. A secondary reduction in enzyme activity may also occur for unknown reasons. Abundant tubular aggregates are also revealed by the reaction, even without substrate.
Acetylcholinesterase highlights areas with high cholinesterase activity, such as neuromuscular junctions. Myotendinous junctions also stain, but the reason is not known. It is also useful for studies of the vacuoles in the X-linked myopathy with excess autophagy vacuoles (XMEA).
Non-specific esterase also stains neuromuscular and myotendinous junctions and, similar to acid phosphatase, highlights phagocytic areas. It also stains small denervated fibres. A two-fibre pattern may also sometimes be seen.
The periodic acid-Schiff (PAS) stain, which has a very long history in histochemistry, is frequently used to demonstrate glycogen in muscle. It is worth bearing in mind, however, that not only glycogen but also other polysaccharides, as well as neutral mucopolysaccharides, muco- and glycoproteins, glycolipids and some unsaturated lipids and phospholipids, are stained with this reaction. The glycogen is demonstrated with Schiff reagent (fuchsin-sulphurous acid), which produces a reddish-purple stain and shows a fibre type pattern. The specificity of the PAS reaction for glycogen may be checked by using α-amylase digestion, and the use of celloidin helps to retain the glycogen. Although glycogen storage may be rare, the PAS technique is also useful in revealing damaged and some denervated fibres in several disorders as these may be devoid of glycogen and appear white.
Neutral lipid can be demonstrated in normal muscle and takes the form of small droplets with a distribution similar to that of mitochondria. It can be demonstrated with the Sudan black or oil red O technique. Nile red is also favoured by some workers and is fluorescent in the presence of high lipid ( ). The concentration and size of the droplets vary with the fibre type and this must be taken into consideration in interpretation. Membranous areas high in phospholipids, such as those with high numbers of mitochondria, are also highlighted by Sudan black and Nile red but are not apparent with oil red O. In disorders affecting lipid metabolism, the excessive accumulation of lipid shows up as larger and more extensive droplets. Assessing the number and size of lipid droplets is also useful in mitochondrial disorders. The routine inclusion of a stain for lipid is a matter of choice which can be driven by clinical information and the patient population referred.
Proliferation of adipose tissue is a common feature of muscular dystrophies but also occurs in spinal muscular atrophies and other disorders. The unstained content of fat cells is readily apparent on routine histological stains but it can also be strikingly demonstrated with lipid stains. Stains for lipid in the presence of adipose tissue may, however, lead to diffuse spread of reaction product over large areas of the section.
It has been found useful to look for the deposition of amyloid in inclusion body myositis ( ). In addition, amyloid can be pathologically deposited in muscle. In sporadic forms of inclusion body myositis and some hereditary myopathies with vacuoles many of the characteristic rimmed vacuolated fibres contain amyloid. These myopathies are often referred to as hereditary inclusion body myopathies and have several pathological features in common with sporadic inclusion body myositis but rarely show lymphocytic inflammation. Amlyoid is composed of protein in a β-pleated sheet conformation. Ultrastructurally, it appears as tangled masses of unbranched double filaments of variable length. Each filament is 2.5–3.5 nm in diameter and separated by a 2.5 nm space, giving a total diameter of 8–10 nm. The most common method for demonstrating amyloid uses Congo red, and one at high alkaline pH was recommended by Mendell ( ). Amyloid stained with Congo red is visible as a red deposit with normal bright field optics but also shows ‘apple-green’ birefringence with polarized light and is most easily seen using fluorescence with an excitation filter suitable for fluorochromes such as Texas red ( ). More recently, the luminescent conjugated oligothiophenes (LCOs), in particular pentamer formyl thiophene acetic acid (p-FTAA), have successfully been shown to bind protein inclusions in muscle biopsies from patients with inclusion body myositis ( ).
Histological and Histochemical Methods
In this section we list the methods of the techniques that form our routine panel of tests and that we consider to be the minimum for diagnosis. We also include additional methods used when clinical features are indicative. We have not attempted to produce a fully comprehensive list of techniques, nor included a wide selection of other methods which are available for some of the stains or enzymes. For such further information, reference should be made to one of the standard histochemical texts ( , ).
All histological and histochemical techniques are performed on frozen sections (10 μm) mounted on coverslips or slides, as described in Chapter 1 . Sections can be stored frozen until required and should be thoroughly air dried before use. If sections are stained flat, a circle around each section, drawn with a hydrophobic pen, prevents the spread of solutions. Several histological stains are now commercially available as ready-made solutions (e.g. haematoxylin). Methods for making them from the individual constituents are given here for those who may prefer this. The synthetic mountants that we routinely use are DPX or Pertex (Histolab Products AB, Gothenburg, Sweden), and when an aqueous mountant is required we use Aquamount (National Diagnostics) or VectaMount AQ (Vector Laboratories). Others are commercially available. Glycerin jelly can also be used and sections rarely dry out when mounted in this (which can occur with some aqueous mountants). DNA can be extracted from such sections, as coverslips can easily be removed with warm water.
Place sections in Harris’ haematoxylin for 3 minutes.
Blue in Scott’s tap water substitute or Tris buffer (pH 10.5) if tap water is acid. Otherwise, run in tap water for 2 minutes.
Differentiate in 0.2% acid alcohol (HCl–alcohol) until pink – if needed.
Re-blue as appropriate (step 2).
Place in 1% eosin for 15–20 seconds (or longer).
Wash quickly in distilled water.
Dehydrate rapidly in ascending alcohol series.
Clear, and mount in synthetic resin (DPX).
|Harris’ haematoxylin powder||21.5 g|
|Absolute alcohol||10 mL|
|Distilled water||200 mL|
5 g eosin/100 mL distilled water
Dilute to 1% for use.
Alkaline Solution (Scott’s Tap Water)
|Potassium bicarbonate||2 g|
|Magnesium sulphate||20 g|
|Distilled water||1 L|
Nuclei blue; fibres red with mitochondria as dark dots; connective tissue pink. If staining in haematoxylin is too long the fibres may appear too basophilic. Mayer’s haematoxylin is a good alternative if less basophilia is preferred but the mitochondria will not be visible.
Stain in Verhoeff’s stain for 20 minutes (until black).
Wash in distilled water.
Differentiate in 2% ferric chloride for a few seconds.
Wash in three changes of distilled water.
Rinse in 70% alcohol for 1 minute.
Wash in three changes of distilled water.
Counterstain with van Gieson mixture for 2 minutes.
Dehydrate in ascending alcohol series, clear and mount in synthetic resin.
Dissolve 1 g haematoxylin in hot 100% ethyl alcohol – 20 mL.
Add 8 mL Lugol’s solution containing 2% iodine and 4% potassium iodide.
Add 8 mL of 10% ferric chloride solution.
NB. This solution is good for 4–6 weeks at 4°C.