The utility of elastic Verhoeff-Van Gieson staining in dermatopathology

Authors


Dirk M. Elston, MD

Ackerman Academy of Dermatopathology, 145

East 32nd Street, New York, NY, USA

Tel: +1 800 553 6621

Fax: +1 212 889 8268

e-mail: delston@ameripath.com

Abstract

Elastic fibers are important components of the skin and are responsible for skin elasticity. Genetic defects are well-known in numerous hereditary elastic tissue disorders and skin biopsies are often the first step in the evaluation of those disorders. Verhoeff-Van Gieson elastic staining is a simple method that is used for visualizing elastic fibers. With the development of modern immunohistochemical methods, the value of routine histochemical staining is sometimes underestimated. Histochemical stains are less expensive, easy to perform and help to resolve numerous diagnostic quandaries in dermatopathology. This article focuses on the value of elastic tissue staining in dermatopathology, with a focus on primary elastic tissue disorders, alopecia, inflammatory skin disorders and neoplastic proliferations.

Connective tissue comprises of three types of fibers: collagen fibers, reticular fibers and elastic fibers. These fibers are made of proteins and composed of long peptide chains. While the main function of collagen and reticular fibers is to provide tensile strength and support respectively, the elastic fibers are mainly there for providing stretchability and flexibility. Elastic fibers can stretch up to 1.5 times their length, and snap back to their original length when relaxed. The long and branching arrangement of fibers enables them to stretch and recoil thus providing flexibility to tissues. Elastic fibers are composed of two structural components: elastin and microfibrils, and include elastin, elaunin and oxytalan.[1-4]

Detecting abnormal patterns of elastic fibers may help in diagnosis of wide range of conditions as well as genetic defects.[5-7] In such cases, special stain for evaluating elastic fibers, may be the first step, that can then lead to further more definitive studies. With the development of modern immunohistochemical methods, the value of routine histochemical staining is sometimes underestimated. Histochemical stains are less expensive, easy to perform and help to resolve numerous diagnostic quandaries in dermatopathology. This article will focus on the value of elastic tissue staining in dermatopathology, with an emphasis on primary elastic tissue disorders, alopecia, inflammatory skin disorders and neoplastic proliferations. These patterns are highlighted by sketches, which are summarized in tables.

Methods for elastic fiber identification

Various methods for staining elastic tissue exist: Weigert's, Orcein, Gomori and Verhoeff (Table 1). The main challenge in staining is to differentiate elastic fibers from collagen and smooth muscle in various tissues. This is not possible by use of just one stain, and a combination of stains is required. Van Geison stain is a commonly used counterstain, which when combined with primary elastic stains helps in better differentiation of elastic from collagen and smooth muscle.

Table 1. Common stains used for elastic fibers
Techniques/methodsStaining patternAdvantagesDrawbacksTechnical issues
  1. a

    Modified Weigert's technique (Miller's) involves boiling and is used progressively. It is fairly rapid.

  2. b

    [Correction added on 17 December 2012, after first online publication: reference citations were amended.]

Verhoeff[9, 10]b (regressive method)• Elastic fibers – blue-black to black

•Nuclei – blue/grey/black

• Collagen – red

• Muscle – orange

• RBCs and cytoplasm – yellow (with Van Geison counterstain)

• Entire elastic system (including oxytalan) of dermis is stained[10]

•Intense elastic staining (considered best elastic stain among all)

• Easy to perform

• Easy to over-differentiate

• Slides must be individually differentiated as time of differentiation is dependent on amount of elastic tissue

• Proper differentiation sometimes is difficult

• Fresh ferric chloride should be used when preparing stain

• Helpful to use duplicate sections differentiated to slightly variable ends, as precise differentiation is sometimes problematic

Gomori[10, 78]b• Elastic fibers, mast cells and mucin – deep purple

• Muscle, cytoplasm and collagen – green (with Light Green counterstain)

• Nuclei – not stained

• Easy to perform

• Stable for many weeks or months (but should be stored in refrigerator)

• Intense background staining with tissues embedded in resins and it increases after dichromate fixation• Use fresh paraldehyde and pararosanilin rich dye to prepare stain

• If aldehyde fuchsin remains red and does not change purple – paraldehyde is decomposed – use fresh paraldehyde[79]

• Large amount of purple precipitate means overaged solution – prepare fresh

Weigert's and modified Weigert's technique (Miller's elatic stain) (1898)

[79, 80]b

• Elastic fibers – blue-black to black

• Cell nuclei – red or blue

• Collagen –pink to red

• Other tissue – yellow (with Van Geison counterstain)

• Simple and reliable

• Remarkable selectivity for elastic fibers

• Differentiation not required, good for fine elastic fiber detection

• Long staining timea

• Preparation of staining solution is time consuming

• Staining in resorcin–fuchsin should be for 30–60 min Check with microscope and stain until elastic fibers black

• Alternatively may be stained overnight in Hart's resorcin–fuchsin solution

Orcein[81]b

Tencer's method

First method used

• Elatic fibers – dark brown

• Other tissues: depends on counterstain used

•Easy to perform•Less intense color than Verhoeff

• Brown staining of elastic fibers is less desirable as Van Geison cannot be used as counterstain

• Check periodically under microscope during staining to ensure proper staining
Humberstone[82]b• Elastic fibers – blue black

• Other tissue – depends on counterstain used

• Selective for elastic fibers

•No diffusion in surrounding tissue

• Stable, improves with age

• Fixation with buffered formalin yields poor staining results (use non-buffered formalin for fixation)• Treatment with potassium permanganate and oxalic acid essential for good staining

Verhoeff-Van Gieson elastic staining (EVG) is a simple method that is used for visualizing elastic fibers. This is the most commonly employed staining technique that was first described by Ira Van Gieson in 1889 as a method of evaluating collagen fibers in neural tissue.[8] Later, it was modified by Frederick H. Verhoeff, enabling the distinction between collagen and elastic fibers to become possible.[9] The basic principle of this technique involves staining the tissue with a regressive hematoxylin, consisting of ferric chloride and iodine. The tissue is overstained with a soluble lake of hematoxylin–ferric chloride–iodine. Both ferric chloride and iodine serves as mordants, but they also have an oxidizing function that assists in converting hematoxylin to hematein. The mechanism of dye binding is probably by formation of hydrogen bonds, but the exact chemical groups reacting with the hematoxylin have not been identified. This method requires that the sections are overstained and then differentiated, so it is regressive. Differentiation is accomplished by using excess mordant, or ferric chloride, to break the tissue–mordant–dye complex. Sodium thiosulfate is used to remove excess iodine. Van Gieson's solution is the most commonly used counterstain, but others may also be used.[10, 11]

Certain common technical difficulties and troubleshooting involved in EVG staining should be considered. Over-differentiation is common and results in a completely colorless background so that a clear yellow counterstain is obtained. In such cases, restaining can be done at any step provided sections have not been treated with alcohol. Counterstaining with Van Gieson's solution should not be prolonged, as picric acid present in this step differentiates the sections further. As the time of differentiation is somewhat dependent on the amount of elastic tissue present, it is better not to rely on the control for timing the differentiation of all sections, and slides must be individually differentiated to get good results, and also it is better to err on the side of underdifferentiation.[10]

Preparation of Van Gieson's solution is critical for proper differentiation of muscle and collagen. If the picric acid is not saturated, collagen will not stain red, and cytoplasm, muscle and collagen may all stain the same color. While preparing the Verhoeff's elastic stain solution, the reagents must be added in the order given, with mixing after each addition, or poor staining may result.[10]

EVG staining in primary elastic tissue disorders

Diseases of elastic tissue are classified into two main groups: diseases with diminished number of elastic fibers [i.e. anetoderma, nevus anelasticus, papular elastorrhexis, pseudoxanthoma elasticum (PXE)-like papillary dermal elastolysis, mid-dermal elastolysis, cutis laxa and perifollicular elastolysis] and disorders with increased number of elastic tissue (i.e. PXE, linear focal elastosis, late-onset focal elastosis, elastofibroma dorsi and elastoma).[12, 13] Moreover, some disorders are characterized not only by an increased amount but also structural alterations of elastic fibers like calcification of elastic fibers in PXE[14] and the ‘lumpy-bumpy’ appearance of elastic fibers in penicillamine-induced elastosis perforans serpiginosa (EPS).[15] Especially in cases of so-called ‘invisible’ dermatoses, in which no pathologic process is seen at first glance, EVG staining can be of great value.

EVG staining in primary elastic tissue disorders with diminished number of elastic fibers

Elastic tissue diseases with a diminished number of elastic fibers can grossly be classified according to the level of dermal involvement as follows: upper parts of the dermis (i.e. anetoderma,[16] nevus anelasticus, papular elastorrhexis[17] and PXE-like papillary dermis elastolysis[18]); middle part of the dermis (i.e. mid-dermal elastolysis[19]); entire dermis (i.e. cutis laxa[20]) and adventitial dermis (i.e. perifollicular elastolysis[21]). Such classification, although helpful, is not always clear cut.

Upper dermis affected

Anetoderma presents clinically as circumscribed, hypopigmented atrophic macules which are usually localized to the trunk and upper extremities.[22] Sometimes it also presents as atrophic patches. ‘Herniation phenomenon’ during palpation is a helpful sign in clinical evaluation of anetoderma. It may occur with or without preceding erythema (Jadassohn-Pellizari and Schweninger-Buzzi types). These two types of anetoderma may co-exist, and show similar histopathological features, therefore the distinction between them is only of historical interest. Anetoderma is frequently associated with autoimmune disorders such as lupus erythematosus (LE), antiphospholipid syndrome, primary hypothyroidism, etc. Rare cases of congenital and familial forms of anetoderma have been described.[23, 24] Other diseases characterized with diminished number or absence of elastic fibers (nevus anelasticus and papular elastorrhexis) are poorly described in the medical literature and have some overlap in clinical presentations.[17] Few existing reports describe nevus anelasticus as congenital or acquired lesion that usually presents as clusters of skin-colored perifollicular papules.[25] Clinical presentation of papular elastorrhexis includes presence of clustered, firm, non-follicular, skin-colored papules.[26] They tend to appear in the second decade. Some authors have proposed that papular elastorrhexis should be viewed as a variant of nevus anelasticus or Buschke-Ollendorf syndrome.[27, 28] Del Pozo et al. define it as a different clinical entity proposing that fragmentation, but not a total loss of elastic fibers, and some clinical features (late onset, non-follicular distribution, location on the skin of all trunk and extremities) should be used in differentiation of papular elastorrhexis from the other entities.[29] At present, though, the paucity of reports on both entities do not allow to make a definite conclusion about the nature of these types of lesions. PXE-like papillary dermal elastolysis is another condition characterized by loss or diminished number of elastic fibers. It occurs in postmenopausal women and is clinically indistinguishable from true PXE, but lacks systemic involvement.[18, 30]

Despite different clinical presentations, anetoderma (Fig.  1), nevus anelasticus, papular elastorrhexis and PXE-like papillary dermis elastolysis are all characterized by loss of elastic fibers mainly in the upper part of the dermis.[18, 31] In anetoderma, the process may affect the middle and rarely deeper parts of the dermis, sometimes with accompanying fragmentation and thinning of elastic fibers.[20] An inflammatory infiltrate and elastophagocytosis may be present in anetoderma and papular elastorrhexis.[13, 17] Presence of melanophages without the involvement of the basal layer was noticed in PXE-like papillary dermal elastolysis.[30] Recent study by Revelles et al. confirmed the absence of elastic fibers in PXE-like papillary dermal elastolysis by antibodies to serum amyloid P that stains microfibrils of elastic fibers.[32] Increased and thickened collagen fibers have been described in some cases of papular elastorrhexis.[26]

Figure 1.

Anetoderma, Verhoeff-Van Gieson elastic staining staining. Loss of elastic fibers in papillary and upper reticular dermis. Magnification ×40.

The mechanism of elastolysis in the diseases with diminished number of elastic fibers is not completely understood; abnormalities of the cross-linking protein desmosin and imbalance of metalloproteinases and metalloproteinase inhibitors have been proposed.[16, 33]

Mid and deep dermis affected

Mid-dermal elastolysis clinically presents as a solitary ‘silvery’ hypopigmented wrinkled patch.[34] Other clinical presentation may include symmetrical wrinkling of the skin and perifollicular protrusions. Cases of persistent reticular erythema and absence of elastic fibers in the mid dermis have been described.[35] Mid-dermal elastolysis may be associated with systemic disorders, including thyroid and other autoimmune disorders.[19] It may also appear as the ‘end-stage’ of different inflammatory conditions (psoriasis, granuloma annulare, urticaria, psoriasis, pityriasis rosea, atopic dermatitis, etc). EVG staining of mid-dermal elastolysis presents as a ‘band-like loss’ of elastic fibers in the middle part of the dermis, leaving upper and deeper elastic fibers unchanged[19] (Fig. 2). Elastic fibers around the follicle are usually preserved. Macrophages and scattered giant cell may be seen.

Figure 2.

Mid-dermal elastolysis with characteristic loss of elastic fibers in the mid-dermis (arrows). Magnification ×100.

Entire dermis affected

Cutis laxa represents a range of hereditary and acquired disorders that are characterized by degradation and loss of elastic tissue. Cutis laxa may be inherited as autosomal-dominant, autosomal-recessive or X-linked-recessive disorder with known gene defects and often is associated with internal organ involvement.[5, 36] The acquired form of cutis laxa develops after the resolution of an inflammatory insult, metabolic diseases or drugs.[37, 38] Destruction of elastic fibers as a result of neutrophilic inflammatory disease (Sweet syndrome, pyoderma gangrenosum, etc.) with the formation of cutis laxa is known as Marshall syndrome.[39] Metabolic syndromes that could be associated with cutis laxa include Menkes disease and disorders of glycosylation.[40] Cases of acquired cutis laxa after isoniazid or penicillin have been described.[41, 42] Other rare associations include malignancies (multiple myeloma), infections (Treponema and Borrelia) and connective tissue disorders.[38] Both types have a similar clinical picture of wrinkled, aged-looking skin. In the post-inflammatory type, erythema and edema precede the development of wrinkling. Hematoxylin and eosin staining fails to reveal any other pathologic changes. Total loss of elastic fibers in the upper and deeper dermis is noted in EVG staining. In the inflammatory variant, inflammation and degradation of the elastic fibers is seen before total loss develops.

Perifollicular dermis affected

Perifollicular elastolysis is characterized by loss of elastic fibers around the hair follicle. It was proposed that this phenomenon is due to the elastolytic effect of bacteria, developing after acne,[21] but later this view was challenged. No elastolytic activity of Staphylococcus epidermidis and Propionibacterium acnes was found by Dick et al. in patients with perifollicular elastolysis, who proposed that it may represent a defect of elastic fibers regenerating after the inflammatory process.[43] Perifollicular elastolysis presents as whitish papules around the hair follicle. The histopathologic features are non-specific with conventional staining, but loss or fragmentation of elastic fibers is apparent around the hair follicle with EVG staining.

EVG staining in primary elastic tissue disorders with increased number of elastic fibers

Elastic tissue disorders with an increased number of elastic fibers can be classified into diseases in which mainly the upper part of the dermis is affected (i.e. elastoderma,[44] late-onset focal dermal elastosis[45] and linear focal elastosis[46]) or the middle and deep dermis are affected (i.e. elastoma[47]).

Upper dermis affected

Only a few cases of elastoderma have been reported in the literature.[44] Clinically it resembles localized cutis laxa with an area of skin wrinkling. Late-onset focal dermal elastosis appears as skin-colored papules on the extremities, neck, axillae or groin.[45] Lesions usually have symmetrical distribution. It is predominantly seen in elderly patients. Linear focal elastosis usually develops after regeneration of striae distensae and probably represents a repair process.[48] Prolonged sun exposure has also been suggested as a possible etiology.[49] Familial cases of late-onset focal dermal elastosis and linear focal elastosis have been reported.[50, 51]

All three entities share an increased number of elastic fibers in the upper dermis. Elastic fibers can be both normal-looking or thickened.[50] Increased number of fibroblasts with prominent endoplasmic reticulum have been found in elastoderma confirming the excess synthesis of elastic fibers.[44] In each case, the diagnosis can be missed using conventional hematoxylin and eosin staining without adequate clinical information. Clinico-pathological correlation is required for the final diagnosis.

Mid and deep dermis affected

Elastoma is a variant of connective tissue nevus that usually appears in childhood and normally is localized on the trunk and extremities as firm skin-colored papules or plaques. It may present as part of Buschke-Ollendorff syndrome (elastomas and osteopoikilosis[51]) or can be also seen without osseous presentations.[47] The elastic fibers in elastomas are thickened and contain deposits of amorphous elastotic material. They are present in the middle and deep parts of the dermis.

Diseases with structural abnormalities of elastic fibers

There are disorders in which the structure of elastic fibers shows significant pathological features (i.e. PXE,[14] EPS[52] and elastofibroma dorsi[53]).

PXE is an inherited disorder characterized by neck and flexural skin lesions with yellowish papules in a pebbled appearance.[14] Other clinical findings include angioid streaks in the retina and cardiovascular disease. The disease usually is diagnosed in the second or third decade and prognosis depends on the involvement of the internal organs. The histopathologic picture of PXE is characterized by basophilic staining of elastic fibers that may be thickened, calcified, curled and frayed. Minimal changes of elastic fibers may not be detected in routinely stained slides but are clearly highlighted by EVG staining (Fig. 3). Calcification of elastic fibers is best visualized by von Kossa staining. Genetic defect of ABCC6 gene coding for transmembrane protein with two ATP domains has been found in PXE. The function of the gene is not completely understood.[54] Perforating PXE (perforating calcific elastosis) usually is localized to the periumbilical region and lacks systemic involvement. Predisposing factors for the development of perforating PXE may be obesity or multiple pregnancies.[55] The condition appears to represent a degenerative change within abdominal striae.

Figure 3.

Pseudoxanthoma elasticum. A) Hematoxylin and eosin staining, magnification ×100. B) Verhoeff-Van Gieson elastic staining staining: thickened and curled elastic fibers, magnification ×100.

Bowen et al. described the presence of PXE-like fibers in the skin of patients with certain inflammatory conditions, without evidence of PXE.[56] Associated diagnoses included lipodermatosclerosis, granuloma annulare, lichen sclerosus, morphea profunda, erythema nodosum, septal panniculitis, basal cell carcinoma and fibrosing dermatitis not otherwise specified. In contrast to classical PXE, these fibers were located focally in the deeper dermis along with the characteristic features of the associated inflammatory or neoplastic condition. All studied patients were women and 12 of 13 described cases were located on the lower extremities.[56] In two out of six patients with PXE-like elastic fibers, the defect in ABCC6 gene was found similar to the patients with classical PXE.[56] In addition to the conditions mentioned, PXE-like changes have also been reported in association with calciphylaxis and nephrogenic systemic fibrosis.[57, 58]

Several studies have shown that PXE – like presentation and similar histopathological changes in elastic fibers are seen frequently in patients with β-thalassemia.[59, 60] Long-term follow up of such patients showed that they tend to develop cardiovascular disease, angioid streaks and other vascular abnormalities similar to classical PXE.[61]

Elastofibroma dorsi is an uncommon skin lesion with unclear pathogenesis, which primarily affects the back of older individuals. Persistent physical trauma in predisposed individuals is thought to lead to degeneration of elastic tissue.[62] The diagnosis can be made in routinely stained sections, but EVG highlights the typical findings of fragmented thick elastic fibers as well as the characteristic small round deposits of elastic tissue resembling ‘strings of pearls’ (Fig. 4).

Figure 4.

Elastofibroma dorsi. Thickened collagen and elastin fibers. A) Hematoxylin and eosin staining, magnification ×400. B) Verhoeff-Van Gieson elastic staining staining: thickened elastin fibers and round deposits of collagen, magnification ×400.

The clinical features of EPS are grouped scaly papules forming arcade lesions usually located on the neck, upper extremities or face. The histopathologic picture of EPS is unique and characterized by serpiginous transepidermal channels filled with bright eosinophilic elastic fibers and debris. An increase in elastic fibers is also seen in the adjacent skin (Fig. 5).[52] Penicillamine-induced EPS is characterized by the thickened thorn-like ‘bramble bush’ appearance of elastic fibers.[15]

Figure 5.

Elastosis perforans serpiginosa. Transepidermal channel filled with thickened collagen bundles. Verhoeff-Van Gieson elastic staining staining, magnification ×100.

A summary of EVG findings in elastic tissue disorders are presented in Table 2.

Table 2. EVG findings in elastic tissue disorders
Diagnosis EVG findings
  1. EVG, Verhoeff-Van Gieson elastic staining; PXE, pseudoxanthoma elasticum.

Diseases with decreased elastic fibersPapular elastorrhexis, PXE-like papillary dermal elastolysis , anetoderma and nevus anelasticus equationLoss of elastic fibers in the upper part of the dermis.

Loss of elastic fibers in anetoderma and nevus anelasticus may also affect deeper parts of the dermis with associated fragmentation of elastic fibers.

Increased and thickened collagen fibers can be seen in papular elastorrhexis.

 Mid-dermal elastolysis equationLoss of elastic fibers in the middle part of the dermis in a band-like manner.
 Perifollicular elastolysis equationPerifollicular loss of elastic fibers.
 Cutis laxa equationTotal loss of elastic fibers in the dermis.
Diseases with increased numbers of elastic fibersLate-onset focal dermal elastosis, elastoderma and Linear focal elastosis equationIncreased number of the elastic fibers in the upper dermis.

In elastoderma and linear focal elastosis, the fibers tend to be thickened.

Late-onset focal dermal elastosis may also involve mid-dermis.

 Elastoma equationThickened elastic fibers in the middle and deep parts of the dermis.

Deposits of elastotic material may be present.

 PXE and perforating PXE equationThickened, calcified, curled and frayed collagen fibers.

Von Kossa staining is usually used to identify calcification (green dots).

 Elastofibroma dorsi equationElastic fibers with aggregated balls of elastic giving them a serrated appearance, elastic fibers resemble ‘string of pearls’.
 Elastosis perforans serpiginosa equationTransepidermal channels filled with debris and eosinophilic elastic fibers.

‘Lumpy-bumpy’ or ‘bramble bush’ appearance of elastic fibers in penicillamine-induced EPS.

EVG staining in alopecias

Diagnosis of scarring and non-scarring alopecias is often a difficult task for dermatopathologists, especially if the biopsy is inadequate or clinical information is unavailable. Although the diagnosis is made by evaluation of routinely stained sections in most cases, EVG staining represents an additional helpful diagnostic tool. EVG staining yields additional helpful histopathologic clues in fibrosing alopecias [i.e. chronic cutaneous LE, lichen planopilaris (LPP), folliculitis decalvans and the central centrifugal cicatricial alopecia variant referred to as ‘idiopathic pseudopelade’[63, 64] in which the typical ‘scar pattern’ is highlighted] and in non-scarring conditions.

EVG staining in scarring alopecia

It is important to note, that diagnostic clues appear in a time-dependent fashion and only fully evolved lesions show the characteristic patterns of loss of elastic tissue.

Both LPP and folliculitis decalvans show wedge-shaped scars in late lesions, being superficially located in LPP and more deep and extensive in folliculitis decalvans (Fig. 6).

Figure 6.

Wedge-shaped perifollicular fibrosis in lichen planopilaris. Verhoeff-Van Gieson elastic staining staining, magnification ×40.

In the acute stage, LPP is a lymphoid process, whereas folliculitis decalvans is a suppurative process characterized by successive crops of epilating pustules. Both affect the follicular infundibulum resulting in superficial wedge-shaped scars.[64] In discoid LE-associated alopecia, the inflammation usually affects the mid portions of the dermis, being centered about the follicular isthmus. With time, the scarring progresses and the EVG pattern is characterized by a scar throughout the dermis. ‘Idiopathic pseudopelade’ (central centrifugal scarring alopecia), also known as ‘hot comb alopecia’, ‘central elliptical alopecia of the black American female’ or ‘follicular degeneration syndrome’, shows dense dermal collagen with loss of the space between collagen bundles, broad fibrous tracts with an intact elastic sheath and thick recoiled elastic fibers throughout the dermis.[64] Some authors consider ‘idiopathic pseudopelade’ to be an end stage of inflammatory disorders rather than a separate entity.[65]

EVG staining in non-scarring alopecia

EVG staining is also helpful in non-scarring alopecias to accentuate the normal elastic pattern and fibrous tracts. As pattern alopecia is primarily a clinical diagnosis, sometimes biopsies are done to rule out inflammatory non-scarring conditions like diffuse alopecia areata or syphilitic alopecia. Usually non-scarring alopecias lack specific changes in EVG staining and the fibrous tracts tend to be narrow (Fig. 7).

Figure 7.

Pattern alopecia. Solar elastosis in the upper parts of the dermis and extremely narrowed fibrous tracts. Verhoeff-Van Gieson elastic staining staining, magnification ×100.

The summary of EVG staining patterns in different types of alopecia is presented in Table 3.

Table 3. Patterns of EVG staining in alopecias
DiagnosisEVG findings
Normal elastic fiber pattern equationDelicate vertical fibers in the papillary dermis and thicker parallel fibers in the reticular dermis.
Scarring alopecia 
Lichen planopilaris equationEarly: no scar or perifollicular mucinous fibrosis.

Late: superficial wedge-shaped scar, tends to be superficial without prominent adjacent inflammation in the scar.

Folliculitis decalvans equationEarly: no scar or perifollicular mucinous fibrosis.

Late: In comparison to LPP there is more extensive scarring with occasional neutrophils present in the scar and surrounding tissue.

Discoid lupus erythematosus alopecia equationEarly: no scar or perifollicular mucinous fibrosis.

Late: scar throughout dermis with complete destruction of elastic tissue.

Idiopathic pseudopelade (central centrifugal scarring alopecia) equationHyalinized dermis with broad fibrous tract with thick and ‘recoiled ’elastic fibers, because of contraction of the dermis.
Non-scarring alopecias 
Pattern alopeciaequationThe key features include miniaturization of follicular units with variability in diameter of follicles and decreased anagen/telogen ratio.

In long standing lesions, EVG highlights ‘narrow and thinned’ fibrous tract remnants as well as solar elastosis.

Traction alopecia and trichotillomania equationBoth entities are characterized by multiple catagen hairs and pigment casts in the follicular canal (marked by brown). EVG staining shows narrow fibrous tracts and no other significant changes in elastic distribution. In addition, empty anagen hair follicles are characteristic of trichotillomania.
Alopecia areata and syphilitic alopecia equationBoth entities have similar morphology in routinely stained sections, presenting with lymphoid inflammation at the bulb level in the acute stage. In older lesions, eosinophils, lymphocytes and melanin often are located in fibrous tracts remnants. EVG staining shows narrow fibrous tracts and no other significant changes in elastic distribution. It should be noted that only some cases of syphilitic alopecia have plasma cells. The rest resemble AA.
Telogen effluvium equationThe microscopic findings include an increase of telogen hairs without specific accompanying inflammatory infiltrate or miniaturization of hair follicles. EVG staining shows narrow fibrous tracts and no other significant changes in elastic distribution.

Use of EVG staining in neoplasms

Use of EVG staining in different tumors is based on the fact that tumors produce different types of stroma. We will focus on elastic fiber distribution in melanocytic lesions, keratoacanthomas vs. squamous cell carcinoma (SCC), dermatomyofibroma and scars (Table 4).

Table 4. Verhoeff-Van Gieson elastic staining staining in neoplasms
NeoplasmEVG findings
  1. EVG, Verhoeff-Van Gieson elastic staining.

Melanoma equationAbsence of elastic fibers between malignant melanocytes and nests. The elastic fibers are ‘pushed’ down and appear crushed at the bottom of the tumor.
Melanoma arising in nevus equationLoss of elastic fibers in melanoma component, retention of elastic fibers in nevus component.
Melanocytic nevi equationElastic fibers are present throughout and in between the benign melanocytes and melanocytic nests.
Keratoacanthoma equationElastic fibers are ‘trapped’ in basal parts of keratoacanthoma.
Dermatomyofibroma equationParallel arrangement of thickened elastic fibers. No involvement of the follicular units.

There are two major indications for EVG staining in melanocytic proliferations. The first is that EVG staining is helpful in the differential diagnosis between benign nevus vs. nevoid melanoma.[66] The second is the determination of exact tumor thickness (Breslow's index) in melanomas associated with benign melanocytic nevi.

In conventional melanocytic nevi, elastic fibers are retained between nests of benign melanocytes and often between individual melanocytes. In the papillary dermis, the usual fine elastic fibers are predominantly perpendicularly oriented to the plane of the epidermis and are said to have the characteristic forked appearance (Fig. 8).[66]

Figure 8.

Conventional melanocytic nevus, Verhoeff-Van Gieson elastic staining staining. Perpendicular elastic fibers throughout the melanocytic nests. Magnification ×600.

In contrast, melanomas show an absence of elastic fibers between melanocytic nests and in the tumor stroma. At the bottom of the tumor the elastic fibers look ‘crushed’ and compressed (Fig. 9). Such phenomenon also takes place in melanomas in sun-damaged skin where solar elastosis is also displaced and pushed down.[66, 67] In regressing melanomas, a band of crushed elastic fibers is retained after the melanocytic lesion has regressed.[67] Regenerating short and thin elastic fibers in the areas of melanoma regression can be seen in about 40% of melanomas with regression phenomenon.[67] The pattern of elastic fibers in melanoma with regression is different from the melanomas with scar after a surgical procedure. The newly formed scar shows absence of elastic fibers in contrast to the “crushing” pattern in under regressing melanoma.[67]

Figure 9.

Melanoma cells within the fibrous stroma, pushing down the elastic fibers, Verhoeff-Van Gieson elastic staining staining. Magnification ×100.

Two caveats should be mentioned. First, in melanocytic lesions with prior trauma or surgery, the elastic pattern is distorted by the ensuing scar and the interpretation of the elastic patterns is difficult. Second, so-called ‘Clark's or dysplastic nevi’ are characterized by marked papillary fibrosis with displacement of elastic fibers in the central part of the lesion.[68] Nevertheless, only a minority show crushing of the elastic fibers at the base of the fibrosis mimicking the pattern of melanoma.[68]

Elastic trapping is common in keratoacanthomas – the feature that is used to differentiate them from hypertrophic lichen planus and conventional SCCs (Fig. 10 A,B).[69, 70] Frequency of elastic trapping in keratoacanthomas ranges from 68% to almost 100% in different studies. In contrast, the frequency of this phenomenon in SCCs is only 0–26% (p < 0.001)[69, 70] Elastic fiber trapping is more prominent if severe sun damage is present.[70] Electron microscopy conducted by Ohashi et al. showed that basal cells of keratoacanthomas have invaginations that gradually surround elastic fibers.[71] Normal and degraded elastic fibers are seen in keratinocytes and intercellular spaces in all the layers of keratoacanthoma, and they gradually diminish toward stratum corneum, where they get eliminated off the surface (so-called transepithelial elimination).[71] Exceptions to this rule may be seen and explained by the special location of the lesions. Elastic trapping has been reported to be more frequent in keratoacanthomas of the lip, but the difference from SCCs of the lip is not significant (61 and 39%, respectively, p > 0.05).[72]

Figure 10.

A) Edge of keratoacanthoma, Verhoeff-Van Gieson elastic staining staining. Magnification ×100. B) Elastic fibers trapping, magnification ×400.

Dermatomyofibroma is a benign myofibroblastic neoplasm that presents clinically as a small, asymptomatic plaque-like tumor. The histopathologic differential diagnosis includes dermatofibroma, hypertrophic scar, dermatofibrosarcoma protuberans and piloleiomyoma. As numerous immunohistochemical stains can usually distinguish between these entities, an EVG stain characteristically shows increased, thickened and fragmented elastic fibers between the spindle-shaped cells of dermatomyofibroma in most cases[73] (Fig. 11). In contrast to dermatomyofibromas, no difference has been noticed in the density of elastic fibers in lesional and non-lesional skin in cellular dermatofibromas; however, in the majority of these cases the majority of the fibers within the lesional area show thickening and clumping in comparison to the non-lesional area. In the paucicellular variants of dermatofibromas, the elastic fibers are less dense a pattern similar to that seen in scar tissue, supporting the concept of evolutionary stages of dermatofibromas.[74] Elastic fibers are preserved in leiomyomas, but do not show thickened parallel pattern as in dermatomyofibromas.[75]

Figure 11.

Dermatomyofibroma. A) Hematoxylin and eosin staining, magnification ×100. B) Verhoeff-Van Gieson elastic staining staining: thickened and fragmented elastic fibers in the neoplasm in the parallel pattern, magnification ×20.

Elastic staining is also helpful in differentiating neoplasms from scars and keloids in ambiguous cases. Electron microscopy has shown presence of large numbers of fine elastic fibers in scars, but they are not easily identified by stains.[76] Only few newly formed elastic fibers may be identified in the scars by Weigert's and Miller's stains,[76, 77] whereas no elastic fibers are usually identified by EVG stain (Fig. 12).[64] Elastic fibers are usually not detectable until the scar has been formed for almost 3 months and never reach the density and thickness of those in normal skin.[77]

Figure 12.

Scar. Verhoeff-Van Gieson elastic staining staining: absence of elastic fibers. Magnification ×100.

Different patterns of elastic staining in neoplasms are presented in Table 4.

Conclusion

EVG staining is easily performed, cheap and available in almost every histopathology laboratory. It finds usefulness in a wide array of cutaneous disorders ranging from inflammatory to neoplastic conditions. It has the potential to narrow the differential diagnosis of inflammatory disorders as well as neoplastic processes. Valuable prognostic and staging information can be gleaned by using an EVG stain to evaluate tumor thickness in melanomas that arise in nevi. In addition, the stain is a useful tool in evaluating tricky lesions, such as distinguishing nevoid melanoma from nevi, where making the distinction is of critical importance. It contributes in the diagnosis of certain neoplasms such as keratoacanthomas and dermatomyofibromas. Finally, it is also helpful in recognizing scars, as well as in differentiating scars or keloids from neoplasms in certain situations.

Ancillary