Introduction

The Verhoeff stain, also known as the Verhoeff-van Gieson stain, is a histological staining procedure developed by Frederick Herman Verhoeff in 1908. The Verhoeff stain is one of the most commonly-used stains to visualize elastic tissue, as found in blood vessel walls, elastic cartilage, lungs, skin, bladder, and some ligaments.

Function

Elastic fibers are components of the extracellular matrix of connective tissue produced by fibroblasts. Each elastic fiber is composed of an elastic microfibril containing glycoproteins and fibrillin-1 that serves as a scaffold for the deposition of elastin proteins. Elastin fibers are crosslinked by the action of lysyl oxidase, generating a branching, three-dimensional elastin matrix and contributing to the substance’s pliability.

Elastin is also produced by perivascular smooth muscle cells, which forms the elastic lamellae found within the tunica media. The elastic tissue of blood vessel walls contains elastin only, without microfibrillar support. The elastin is organized into fenestrated lamellae in concentric rings between layers of myocytes. A unique feature of these fibers is the ability to stretch up to 50% of their length before recoiling to their original length after deformation. This characteristic enables elastin within the tunica media of arteries to facilitate pressure wave propagation of blood flow, particularly in large elastic blood vessels such as the aorta and pulmonary arteries. This property, known as the Windkessel effect, maintains relatively uniform arterial blood pressure, despite pulsatile blood flow. [1]

Tissue Preparation

Verhoeff staining is effective on tissues preserved in any fixative, although formalin-based fixatives tend to be most successful. The Verhoeff staining process occurs in two steps. The initial stage is the Verhoeff stain component, using hematoxylin, iron (III) chloride and an iodine solution. Iron (III) chloride and iodine act as mordants and aid in the oxidation of hematoxylin to hematein, which is responsible for staining elastic elements. Elastin possesses a strong affinity for the hematoxylin-iron complex and thus, retains the stain longer than other tissue elements following decolorization. Excess iron (III) chloride is used to differentiate the tissue; then sodium thiosulfate is used to remove excess iodine. The subsequent van Gieson counterstain utilizes picric acid and acid fuchsin to stain collagen and muscle fibers, producing contrast against the hematoxylin stain. Under-differentiation is preferred in the Verhoeff stain, as picric acid used in the van Gieson counterstain serves to differentiate the elastic fibers further. Each slide must be differentiated individually as the appropriate duration of differentiation varies according to the amount of elastic tissue in each specimen, which may make preparation difficult. [2]

Several modifications of the standard Verhoeff staining procedure have been developed to enhance specificity for elastic components or to improve staining of specific tissues. An adaptation of the Verhoeff procedure that advocates no decolorizing step and counterstaining with Bismarck Brown Y dye may improve staining of elastic fibers and lamellae. Another alteration of the standard Verhoeff staining procedure involving a unique tissue fixation process and counterstaining with a modified light green solution has been employed to visualize the elastic laminae of small-caliber blood vessels. [3]

Microscopy, Light

Elastic fibers are not well-differentiated on traditional hematoxylin and eosin (H and E) stains of tissue sections. They may appear somewhat refractile on H and E staining, but often cannot be clearly distinguished from collagen fibers and smooth muscle. [2] The Verhoeff stain enables visualization of these fine structures under traditional light microscopy. The procedure results in elastic fibers and nuclei being stained black, collagen stained red and cytoplasmic elements stained yellow.

A variation of the elastin stain is the Masson trichrome-Verhoeff stain. The elastic tissue remains darkly stained as in the traditional Verhoeff stain, although the Masson stain results in a contrasting stain of muscle fibers and keratin red, while collagen and bone are stained green or blue. The use of this combination staining technique is most applicable for the study of vascular pathologies and can be used to differentiate arterioles, which have two or more elastic lamellae, from veins, which have a singular lamella. [4], [5], [6] 

A similar combination technique, known as the Verhoeff-Martius-scarlet-blue trichrome stain, can differentiate between new, mature and aged fibrin by staining them yellow-orange, red and blue, respectively. As with the standard Verhoeff stain, elastic tissue is stained darkly. This procedure is most appropriate for evaluation of connective tissue and vascular disorders, particularly necrotizing vasculitis. [7]

Pathophysiology

Many disorders are known to have detrimental effects on both visceral and cutaneous elastic tissue. Marfan syndrome, caused by autosomal dominant mutations in the fibrillin-1 gene (FBN1), results in a defective scaffold for elastin deposition. Stains of aortic and cutaneous tissue samples from animal models of Marfan syndrome demonstrated decreased density and increased fragmentation of elastic lamellae and fibers, with tangled microfibrils. [8], [9], [10], [11] These abnormalities predispose Marfan patients to joint hypermobility and thoracic aorta aneurysms and dissections.

Pseudoxanthoma elasticum (PXE), also known as Grönblad-Strandberg syndrome, is a progressive, genodermatosis typically inherited in an autosomal recessive manner. Mutations in the ATP-binding cassette subfamily C member 6 (ABCC6) gene result in ectopic calcification of elastic fibers in connective tissue throughout the body. [12] Manifestations of PXE include yellow papules along flexural surfaces, cutaneous laxity, retinal complications, premature atherosclerosis and valvular cardiac sequelae. Diagnosis of PXE is best accomplished through skin biopsy displaying elastorrhexis, characterized by fragmentation and mineralization of elastic fibers within the mid-reticular dermis on Verhoeff stain or stains for calcium. [13] Additionally, cardiac biopsy specimens from patients with PXE stained with the Verhoeff stain demonstrate coarse, curled, fragmented, disorganized and palisaded elastic fibers with substantial degeneration within the endocardium. [14]

Verhoeff’s stain has also been used in the visualization of elastic fibers within elastofibromas. Elastofibromas are rare, ill-defined tumor-like growths composed of irregular and enlarged elastic fibers. They present as a firm, deep, rubbery, slow-growing mass, with a characteristic periscapular location deep to the skeletal musculature. [15], [16] Elastofibromas may be bilateral, although are uncommonly painful. The etiology of elastofibromas is unclear but may be secondary to an inherited predisposition, enzymatic defect causing abnormal elastic fiber formation or repeated friction. Microscopic evaluation reveals dense bands of collagen fibers, adipose tissue, and abnormal elastic fibers. [17] As viewed following Verhoeff stain, the elastic fibers are coarse, thick and globular, resulting in a beaded or “string-of-pearls” appearance with a serrated edge. [17], [18], [19] 

The Verhoeff stain is particularly useful in the evaluation of pathologies of the elastic lamina found within elastic arteries, such as the aorta. The degenerative changes of the valvular leaflets seen in senile aortic stenosis may be visualized, in part, using the Verhoeff stain. One defining characteristic of early stenotic lesions is aortic-side subendothelial thickening with displacement and fragmentation of the internal elastic lamina, visualized on Verhoeff staining. [20], [21] Arteriosclerosis may appear as atrophy and fragmentation of elastin fibers within the lamina. [22], [23] Potential pathogenic mechanisms include lipid deposition, intimal fibrosis and increased local concentrations of elastases within the arterial elastic tissue. [23], [24], [22]

The Verhoeff stain has also been commonly utilized in dermatopathology. Normal hair follicles are typically embedded within a sheath of elastic fibers. Histological alterations of the presence and distribution of follicular elastin can be used to differentiate various forms of alopecia, particularly those with a scarring or fibrosing etiology. [25], [26], [27]

Unique distributions of elastic fibers can be identified by Verhoeff staining of several cutaneous neoplasms and similar lesions. [2] In melanoma, there is a lack of elastic fibers between malignant melanocytes; the elastic fibers appear forced downwards and crushed at the base of the tumor. [28], [29] In a case of melanoma arising within a nevus, elastic fibers may be noted to be absent from the melanoma component and retained within the nevus aspect of the lesion. [2] Melanocytic nevi present with elastic fibers dispersed throughout the benign melanocytes and in the papillary dermis, are oriented perpendicularly to the epidermis and have a forked appearance. [28] In keratoacanthomas, elastic fibers are limited to the basal aspects of the lesion (“elastic trapping”), differentiating them from hypertrophic lichen planus and squamous cell carcinoma. [30] In dermatofibromas, thickened and fragmented elastic fibers are arranged in parallel within the reticular dermis, sparing the pilosebaceous units. Scars typically demonstrate many fine elastic fibers on electron microscopy, although these are not well-identified by Verhoeff stains. [27], [31] Scars appear anelastotic on light microscopy until at least three months following the inciting trauma, although elastic fibers may be apparent on electron microscopy. [27], [31], [32] This discrepancy may be explained by the inability of the Verhoeff stain to identify thin, newly-formed elastic fibers.

Hereditary and acquired disorders of cutaneous elastic tissue are commonly initially evaluated by skin biopsy, for which the Verhoeff-van Gieson stain may be utilized to visualize elastic fibers. Disorders of increased elastic tissue include focal elastosis and elastomas, while disorders of diminished elastic fibers include anetoderma, nevus anelasticus, cutis laxa and perifollicular elastolysis, among others. [33] Additionally, structural abnormalities of elastin fibers can be observed in cutis laxa and penicillamine-induced elastosis perforans serpiginosa. [34], [2]

Clinical Significance

Elastic fibers comprise approximately 3% of the dry weight of the dermis. Although elastin is a major component of the skin, it fails to stain discernably on standard hematoxylin and eosin preparations. Thus, the Verhoeff stain is often mandatory in the investigation of the organization of dermal elastic fibers and related pathologies, including pseudoxanthoma elasticum and elastosis perforans serpiginous.

In addition to its ubiquity in dermatopathology, the Verhoeff stain is the most widely employed histological stain to evaluate vascular elastic fibers. The Verhoeff stain highlights the elastic laminae of blood vessels and can be utilized to differentiate between arteries, with multiple elastic laminae, and veins, with a single lamina. Fragmentation and atrophy of the elastic lamina may occur in arteriosclerosis and can be identified through the Verhoeff staining technique. The degenerative valvular changes characteristic of aortic stenosis, including displacement and degeneration of the internal elastic lamina, may similarly be visualized. Additional modifications to the Verhoeff stain can be utilized to identify the fine elastic fibers found in small arteries, such as those implicated in the pathogenesis of pulmonary arterial hypertension. [3]