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Year : 2019  |  Volume : 62  |  Issue : 1  |  Page : 67-72
Tetrachromic VOF/Masson's trichrome/H and E stains: Unmasking their usability in differential stromal hard tissue staining

Department of Oral Pathology and Microbiology, ITS-CDSR, Muradnagar, Ghaziabad, Uttar Pradesh, India

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Date of Web Publication31-Jan-2019


Background: Identification and differentiation of stromal hard tissue components is a challenging task. Numerous methods of demonstrating these components have been worked upon in the past. Although some of the methods have been successful, there are many drawbacks of employing them. The need of the hour, therefore is to develop and use a simple, rapid and cost-effective method of identifying stromal hard tissues as they may signify an important change in the diagnosis of the pathology. Our aim is therefore to observe the usability of tetrachromic VOF stain over Hematoxylin and Eosin and Masson's Trichrome in routinely encountered head and neck pathologies. Materials and Method: Routine cases such as Central and peripheral ossifying fibromas, osteomas, giant cell granulomas, osteomyelitis and malignancies like osteosarcomas were retrieved from the department archives and 3 sections from each block were prepared to stain with H and E, Masson's trichrome and modified tetrachromic VOF stains respectively using standard staining protocol. Results: Tetrachromic VOF takes an upper hand in stromal hard tissue differentiation irrespective of the pathology. Conclusion: Modified tetrachromic VOF is simple, cost-effective method and can be employed for diagnosis of cases with hard tissue differentiation within the stroma on routine basis.

Keywords: Eosin, hematoxylin, Masson's trichrome, tetrachromic VOF

How to cite this article:
Tandon A, Singh A, Shetty DC, Juneja S. Tetrachromic VOF/Masson's trichrome/H and E stains: Unmasking their usability in differential stromal hard tissue staining. Indian J Pathol Microbiol 2019;62:67-72

How to cite this URL:
Tandon A, Singh A, Shetty DC, Juneja S. Tetrachromic VOF/Masson's trichrome/H and E stains: Unmasking their usability in differential stromal hard tissue staining. Indian J Pathol Microbiol [serial online] 2019 [cited 2021 Oct 25];62:67-72. Available from: https://www.ijpmonline.org/text.asp?2019/62/1/67/251242

   Introduction Top

Head and neck soft tissue lesions demonstrate varying degrees of ossifications within fibrous connective tissue stroma. Naming a few, benign fibro-osseous lesions, that is, fibrous dysplasia, ossifying fibroma, and cement osseous dysplasia [1] along with soft tissue osteomas,[2] central and peripheral giant cell granulomas, osteomyelitis, and osteosarcomas [3] represent bone and cementum-like ossifications at variable degrees of maturation or destruction. The variable amounts of mature bone, immature bone, and osteoid complicate differentiation and hence the diagnosis.[3] Routine hematoxylin and eosin (H and E) staining poses problem in differentiating osteoid from calcified bone, as all the hard and soft tissue stromal components appear in varying shades of pink. Very early/early calcifications are often missed and differentiation between organic matrix of calcified tissue and soft tissue may be difficult.[3] Misdiagnosis between bone and cementum-like tissue poses yet another problem.[4]

Over the years, various connective tissue stains have been developed and practiced to differentiate stromal hard and soft tissue components. Amongst a variety of them, a few stains that retain the ability for differentiation of stromal hard tissues are Von Kossa, solochrome cyanine, Masson's Trichrome, and silver staining before decalcification.[5] Modified MacNeal's tetrachrome and Movat's pentachrome methods are also believed to give superior staining results for differentiating osteoid from mineralized bone.[6] Modified tetrachromic method and versatile mineralized bone stain (MIBS) also have the ability for differentiation of hard tissues.[3]

The tetrachrome technique exploits the principle that primary and secondary mineralization and aging of bone tissue are linked with permanent chemical and structural alterations. Changes differentiate bone matrix from the original non-mineralized osteoid and permit differential staining even after decalcification. In the tetrachrome method, differential staining has been achieved by combining phosphotungstic acid with dyes under carefully controlled conditions. Other advantage of using the technique is that they are simple and inexpensive, they require no special equipment, they can be used to stain large sections, and they can be combined with polarized light to demonstrate the underlying lamellar and woven structure of the collagenous matrix.[7] Therefore, a simple and cost-effective method was devised by Sarasquete and Gutiérrez whereby the original trichromic hematoxylin-Gutiérrez' Verde Luz-orange G-acid fuchsin (VOF) stain was modified to tetrachromic VOF stain to identify various hard tissue components with ease.[3]

The aim of this study, therefore, is to compare the effectiveness of tetrachromic VOF stain over H and E and Masson's trichrome in differentiation of various hard tissue components in the aforementioned lesions of head and neck.

   Materials and Methods Top

A retrospective study was performed in the Department of Oral Pathology and Microbiology after gaining consent from the institutional ethical committee. Formalin-fixed paraffin-embedded blocks of three cases each of fibrous dysplasia, peripheral, and central cement ossifying fibroma, osteoma, osteomyelitis, central giant cell granuloma (CGCG), and osteosarcoma were retrieved from the archives. 4-μm sections were made and stained with H and E, Masson's trichrome, and tetrachromic VOF for differentiation of hard tissues. H and E, Masson's trichrome, and tetrachromic VOF stains were prepared, and staining was performed according to the methods suggested by Suvarna et al.,[6] Culling et al.,[8] and Sarasquete and Gutiérrez,[9] respectively. The slides were examined by oral pathologists and structures were categorized as mature bone, immature bone, cementum-like calcifications, dystrophic calcifications, and osteoid based on histologic and morphologic appearance of hard tissues that were present. Immature bone has higher proportion of osteocytes than mature bone. It has two subtypes, that is, woven bone and coarsely bundled bone. In the former subtype, bundles of collagen run in various directions, and hence the name woven, whereas in the latter collagen bundles are arranged parallel to one another. In addition, the matrix of woven bone tends to be tinged with blue in H and E sections due to higher proteoglycan content. Mature bone, on the other hand, has dintictively ordered arrangement of lamellae and fewer evenly arranged and flatter osteocytes than immature bone. On the contrary, bone tissue that remains uncalcified is referred to as osteoid.[10] The criteria for identification of all the different types of hard tissue components were based on the studies done by Ralis and Watkins [7] and Belaldavar et al. (2014)[3] except for resorbing and degenerating bone where we observed our own findings due to lack of literature for the same [Table 1].
Table 1: Criteria for identification of stromal hard tissues

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   Results Top

The findings of staining procedures using H and E, Masson's trichrome, and tetrachromic VOF stains performed on all the archival cases in following categories were remarkable. The differential stains intensely stained the areas of calcifications and simplified their categorization.

Central ossifying/cement-ossifying fibroma

In cases of central ossifying fibroma, a combination of mature bone at periphery, woven bone dispersed in the stroma, and areas of dystrophic calcifications was observed. Mature bone trabeculae [Figure 1]a, [Figure 1]b, [Figure 1]c were best identified in contrasting deep reddish purple color against a pale blue background with tetrachromic VOF stain followed by H and E stain where well-defined bone trabeculae surrounded by osteoblastic rimming and osteocytes could be appreciated. In Masson's trichrome, however, bone trabeculae could be identified but they took the same shades of color as that of the surrounding stroma as also seen in H and E stain.
Figure 1: Central ossifying fibroma; mature bone (arrow head, 10×, a-c); woven bone (arrow head, 40×, d-f); dystrophic calcification (arrow head, 40×, g-i); cementum like calcifications (arrow head, 40×, j-l)

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Areas of woven bone [Figure 1]d, [Figure 1]e, [Figure 1]f in the same cases could not be identified with surety using H and E and Masson's trichrome as the newly forming bone area gave staining with the same color as that of soft tissue. However, VOF carefully demarcated the woven bone with a blue periphery in areas of suspicious calcification and could better differentiate from central more mineralized bone. However, larger osteocytic lacunae, a characteristic feature of woven bone, could also be appreciated using H and E and Masson's trichrome. Dystrophic calcification [Figure 1]g, [Figure 1]h, [Figure 1]i in relatively acellular surrounding stroma was best observed using tetrachromic stain as patchy staining over trichromic or routine staining procedures.

Central cement-ossifying fibromas also revealed areas of concentric lamellations, often referred to cementum [Figure 1]j, [Figure 1]k, [Figure 1]l. These concentric droplets were often dispersed in the stroma and their size varied from pin-point to medium to large. The larger concentric lamellations could be identified with H and E as purple in color, whereas the smaller ones were often missed. Masson's trichrome could not highlight the droplets of both small and medium size, whereas the smallest of the droplet could be appreciated using tetrachromic VOF as a dark purple ossification against a pale background.

Peripheral ossifying fibroma

Peripheral ossifying fibroma [Figure 2]a, [Figure 2]b, [Figure 2]c revealed both mature and woven bone. The findings were very much similar to that observed in central ossifying fibroma.
Figure 2: Peripheral ossifying fibroma; mature bone (arrow head, 10×, a-c)

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Fibrous dysplasia

Cases of fibrous dysplasia showed classic thin lamellar bone pattern without osteoblastic rimming [Figure 3]a, [Figure 3]b, [Figure 3]c. VOF stained these lamellar bone areas with different shades of blue, red, and purple, whereas the other two stains gave same color shades as that of the surrounding stroma.
Figure 3: Fibrous dysplasia; bone without osteoblastic rimming (arrow head, 40×, a-c) and osteoma; bone with varying degrees of calcification (arrow head, 40×, d-f)

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Osteomas, however, revealed bone formation at variable degrees of calcification [Figure 3]d, [Figure 3]e, [Figure 3]f. The degree of calcification in a bone could best be identified using tetrachromic VOF.

Central giant cell granuloma

Cases of CGCGs showed both the normal bone trabeculae and areas of osteoclastic bone resorption [Figure 4]a, [Figure 4]b, [Figure 4]c. The resorbing bone in CGCG cases appeared to show a pale blue staining with orange patches using tetrachromic VOF stain.
Figure 4: Central giant cell granuloma; resorbing bone (red arrow head) osteoclastic giant cells (yellow arrow) (40×, a-c) and osteomyelitis; degenerating bone (arrow head, 10×, d-f)

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Degenerating bone [Figure 4]d, [Figure 4]e, [Figure 4]f often referred to as sequestrum occasionally encountered in osteomyelitis revealed empty osteocytic lacunae with all stains. With VOF, the bone showed a characteristic combination of pale blue and orange which is distinct from the other stromal hard tissues.


Multiple areas of tumor osteoid were appreciated in cases of osteosarcoma [Figure 5]a, [Figure 5]b, [Figure 5]c. The distinctive color patterns in tetrachromic stain unmask the tumor osteoid most favorably followed by H and E and Masson's trichrome stain.
Figure 5: Osteosarcoma; tumor osteoid (arrow head, 40×, a-c)

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   Discussion Top

Bone formation in a variety of head and neck lesions may be of mature or immature variety.[5] Immature bone contains a relatively higher proportion of osteocytes than mature bone. Its two subtypes are known as woven bone and coarsely bundled bone.[10] Dystrophic calcifications can be identified as irregular globules with no cellularity and relatively avascular surrounding stroma,[3],[10] whereas homogenously hyalinized areas in the vicinity of bone-forming cells may be referred to as organic bone matrix/osteoid/prebone.[10],[11] Coarsely bundled bone (immature bone) differs from woven bone (immature bone) in that it contains thick collagen bundles, most of which are arranged parallel to one another with osteocytes between them. However, in H and E staining the difference might not always be appreciated.[10]

Alternatively described as lamellar bone, mature bone is characterized by a distinctive orderly arrangement that is the result of repeated addition of uniform lamellae to bony surfaces during appositional growth.[10],[12] Some microscopic features of mature bone that may be helpful in distinguishing it from immature bone are (1) the comparatively even acidophilic staining of its matrix, (2) the comparatively regular arrangement of its lamellae, and (3) the fact that its osteocytes are fewer, more evenly arranged, and present in flatter lacunae.[10]

However, such an elaborate description for differentiation of hard tissues may appear theoretical and may pose numerous practical obstructions as in some the tumor growth is so rapid that there might not be sufficient time for bone maturation, and hence the calcified components may appear in various stages of maturation making the categorization even more difficult. Hence, of much importance is the fact that bone and other hard tissue components and soft tissues are eosinophilic in routine H and E-stained sections and thus are difficult to differentiate. Combination of dyes can stain the hard and soft tissue structures differentially for easier identification. Therefore, trichrome and tetrachrome staining methods gain an upper hand in such cases.

However, a disadvantage for some stains is that they require cyanuric chloride, which is an unstable compound that alters staining balance depending on staining time and rate, while other stains require up to 90 h for staining.[3],[13],[14] Along with this, the methods also pose difficulties of time and cost-effectiveness. In addition, interpretation of staining for mature, immature bone, and tumor osteoid is challenging.[3],[6]

A variant of the original trichromic Gutiérrez' VOF technique is a one-step tetrachrome stain composed of light green SF/or fast green FCF, methyl blue, orange G, and acid fuchsin which are used simultaneously and enable the individual tissues to be selectively differentiated and stained (i.e., muscle fibres, collagen, reticulin, erythrocytes, cartilage, bone, etc.). The acid nature of orange G and the amphoteric character of light green and acid fuchsin get highlighted. These dyes are hydrophilic, and at acid pH enhance the protonization of basic groups of proteins. Tetrachromic VOF Type III G.S stain also incorporates polyacids or colorless dyes (phosphotungstic or phosphomolybdic acids), which are high-molecular-weight compounds producing displacement in chemical reactions also referred to as “differentiation.”[9]

In our study, tetrachromic VOF stained the components as varying shades of blue and red against a light blue stromal soft tissue color in comparison to H and E where all the stromal components are stained in varying shades of pink. In Masson's trichrome, the colors of calcifications though distinct over H and E often mix with soft tissue stroma as it stains in same colors of slightly different shades of blue, green, and red. We also observed marked propensity of identification of all the hard tissue components using tetrachromic VOF over the other two stains. The identification of mature bone becomes difficult in Masson's trichrome, however, if the bone is scanty as the mineralized bone takes up the same shades of color as that of the surrounding soft tissue stroma. Dystrophic calcified deposits are caused by tissue injury and necrosis. Therefore, they are irregular in shape and appear to be surrounded by acellular fibrous stroma with minimal vascularity.[3] These could be appreciated with all the stains used in our study, but the superiority of tetrachromic VOF can be proved by the fact that it identifies dystrophic calcifications as patchy staining signifying degenerative changes. Concentric lamellations often referred to as cementum-like areas were best identified using tetrachromic VOF as even the tiniest of the calcified deposit took a dark purple color against a light blue background. In the other two stains, the tiniest of concentric lamellations often got missed.

It is believed that during endochondral ossification as osteoid starts to mineralize, its staining changes from pink to black with Von Kossa' s method, and from deep blue through a narrow pale blue-pink zone (the mineralization front) to red mineralized bone with tetrachrome staining.[7] We also observed that osteoid seams as usually seen in osteosarcomas took a deep blue color in contrast to reddish purple center. Degenerating bone (sequestrum), as occasionally encountered in osteomyelitis, sometimes hard to differentiate in H and E stain can be easily recognized in shades of orange and blue and not red using tetrachromic VOF. This could be explained by the fact that only mature bone and collagen are stained red by acid fuchsin, because collagen has affinity for anionic aniline dyes of large molecular size that bind by Van der Waal forces. Type I collagen is the most common form in bone, and mature collagen undergoing mineralization is stained pink by acid fuchsin which has affinity for calcified deposits and bone.[3],[15]

Resorbing bone as seen in CGCGs due to osteoclastic activity takes up a light blue color with patches of orange showing degradation of bone collagen which could not be appreciated with either H and E or Masson's trichrome on the staining property of bone alone as there is uniformity in staining of these areas.

In addition, the maturity of calcification can be distinguished owing to the varied staining of hard and soft tissues.[3] Tetrachromic VOF produced contrasting colors of deep purple blue and red showing varied stages of bone maturation.

The contrasting interplay of colors of the bone and collagen components after VOF staining facilitated easy identification in our study as it was much easier to identify and speculate very early areas of calcification against a light background of pale blue collagen.

   Conclusion Top

Tetrachromic VOF stain requires single-step staining in even lesser time than H and E and Masson's trichrome stain and is cost-effective. It stains all the hard tissue components including bone, cementum, and dystrophic deposits distinctly and distinguishes these from stromal components. The effectiveness of the stain can be even more substantiated by the fact that even smallest of calcified deposits take up the stain brightly against the pale blue collagen background making interpretation much easier. Also, degenerating bone and tumor osteoid show a characteristic staining pattern. VOF stain appears to be reliable in terms of its use for identifying hard tissue components of stroma, and therefore we recommend its use in all the suspicious cases of normal/abnormal calcifications so that the nature of the calcified deposit could be ascertained. This would surely help in better therapeutics of the pathology.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Debta P, Debta FM, Bussari S, Acharya SS, Jeergal VA. Cancellous osteoma of maxilla: A rare case report. J Int Soc Prev Community Dent 2016;6:261-4.  Back to cited text no. 2
Belaldavar C, Hallikerimath S, Angadi PV, Kale AD. Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components. Biotech Histochem 2014;89:545-51.  Back to cited text no. 3
Fang Z, Jin S, Zhang C, Wang L, He Y. Misdiagnosis of osteosarcoma as cementoblastoma from an atypical mandibular swelling. Oncol Lett 2016;11:3761-5.  Back to cited text no. 4
Tripp EJ, MacKay EH. Silver staining of bone prior to decalcification for quantitative determination of osteoid in sections. Stain Technol 1972;47:129-36.  Back to cited text no. 5
Suvarna SK, Layton C, Bancroft JD. Bancroft's Theory and Practice of Histological techniques. 7th ed. China: Churchill Livingstone Elsevier; 2013, p. 200-4.  Back to cited text no. 6
Ralis ZA, Watkins G. Modified tetrachrome method for osteoid and defectively mineralized bone in paraffin sections. Biotech Histochem 1992;67:339-45.  Back to cited text no. 7
Culling CFA, Allison RT, Barr WT. Cellular Pathology Technique. 4th ed. Hopeland, PA: Butterworth and Co. Ltd.; 1985, p. 408.  Back to cited text no. 8
Sarasquete C, Gutiérrez M. New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues. Eur J Histochem 2005;49:211-27.  Back to cited text no. 9
Cormack DH. Ham's Histology. 9th ed. Philadelphia, PA: JB Lippincott Company; 1987. p. 279-83.  Back to cited text no. 10
Sarkar R. Pathological and clinical features of primary osseous tumours of the jaw. J Bone Oncol 2014;3:90-5.  Back to cited text no. 11
Waldron CA, Giansanti JS. Benign fibro-osseous lesions of the jaws: A clinical-radiologic-histologic review of sixty-five cases. Oral Surg Oral Med Oral Pathol 1973;35:190-201.  Back to cited text no. 12
Villanueva AR. A bone stain for osteoid seams in fresh, unembedded, mineralized bone. Stain Technol 1974;49:1-8.  Back to cited text no. 13
Villanueva AR, Lundin KD. A versatile new mineralized bone stain for simultaneous assessment of tetracycline and osteoid seams. Stain Technol 1989;64:129-38.  Back to cited text no. 14
Horobin RW, Bennison PJ. The interrelation of the size and substantivity of dyes: The role of van der walls attraction and hydrophobic bonding in biological staining. Histochemistry 1973;33:191-204.  Back to cited text no. 15

Correspondence Address:
Ankita Tandon
Department of Oral Pathology and Microbiology, ITS-CDSR, Muradnagar, Ghaziabad, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_242_18

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1]


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