| Abstract|| |
Context: Vascular anomalies present diagnostic challenges to histopathologists. Mulliken and Glowacki's classification categorizes vascular lesions into hemangiomas and vascular malformations. Aim: This study explored diagnostic clues in the histomorphology of hemangiomas and vascular malformations. Materials and Methods: A total of 120 cases of benign vascular lesions were retrieved from 12 years period. A total of 94 cases, where complete clinical details and representative paraffin sections were available, were included in this study. Hematoxylin and eosin (H and E) stain and Verhoeff's stain for elastic tissue were done on all cases and lesions were classified into hemangiomas or arterio-venous malformations (AVM). Mast cell density in all lesions was calculated from toluidine blue stained sections. Results: Ten cases of hemangiomas were reclassified as AVM on the basis of presence and absence of arteriovenous structures. Intra-lesional nerves were seen in significantly higher number of AVMs compared to hemangiomas. Medium and thick sized nerve bundles were seen in 56% of AVMs, while they were not seen in any of the hemangiomas. Mean mast cell density was significantly higher in proliferating hemangiomas (53.12 ± 27.83 cells/mm 2 ) compared to involuting hemangiomas (11.43 ± 7.9 cells/mm 2 ). Conclusions: Use of elastic tissue stains are useful ancillary tools to distinguish between AVMs and hemangiomas. The presence of arteries and arterioles are an integral part of AVMs. The presence of the intra-lesional nerves can be useful to distinguish between AVMs and hemangiomas even on H and E stained sections. The significantly higher mast cell density seen in proliferating hemangiomas compared with involuting ones, seem to suggest that mast cells play an important role in the natural history of these lesions.
Keywords: arterio-venousmalformation, elastic stain, hemangioma, mast cells
|How to cite this article:|
Pawane P, Anshu, Gangane N. Hemangiomas versus arterio-venous malformations: Role of elastic stains and mast cell density. Indian J Pathol Microbiol 2014;57:191-5
|How to cite this URL:|
Pawane P, Anshu, Gangane N. Hemangiomas versus arterio-venous malformations: Role of elastic stains and mast cell density. Indian J Pathol Microbiol [serial online] 2014 [cited 2019 Apr 24];57:191-5. Available from: http://www.ijpmonline.org/text.asp?2014/57/2/191/134661
| Introduction|| |
Vascular anomalies comprise a widely heterogeneous group of tumors and malformations.  The diagnosis and management of these lesions present diagnostic and therapeutic challenges to surgeons, radiologists, and histopathologists. This is in part due to lack of agreement regarding the nosology and classification of these lesions both for diagnostic and therapeutic purposes. 
Arteriovenous vascular malformations and hemangiomas are benign vascular lesions that are difficult to distinguish from one another clinically. Furthermore, they may be confused with each other at histopathology.  It is clinically relevant to differentiate between arteriovenous vascular malformations and hemangiomas.  In 1982, Mulliken and Glowacki  introduced a new classification based on the clinical and histological characteristics of vascular lesions. They used only two categories to classify vascular lesions: Hemangiomas and vascular malformations. Those lesions which exhibited a history of rapid neonatal growth and slow involution, characterized by hypercellularity during the proliferating phase and fibrosis and diminished cellularity during the involuting phase were hemangiomas and those present at birth which grew commensurately with the child were characterized by a normal rate of endothelial cell turnover were malformations. Mulliken and Glowacki  believed that sophisticated laboratory techniques are not necessary to assign a lesion to either of the two major categories: Hemangioma or malformation. This classification was later accepted at the 1996 biennial meeting of the International Society for the Study of Vascular Anomalies. 
Hemangiomas and arteriovenous malformations (AVMs) can be distinguished from each other histologically on the basis of absence or presence of arteriovenous structures.  In our study, hemangiomas and AVMs were distinguished from each other on the basis of the presence or absence of arteriovenous structures, using Verhoeff's stain for elastin. We sought to explore additional diagnostic clues that could help in the diagnosis and differentiation of these lesions. We also studied mast cell density in hemangiomas and AVMs to determine if they differed significantly.
| Materials and methods|| |
This study was carried out in the Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, India from June 2006 to August 2008. The study received approval from the Institutional Ethics Committee. This study includes 120 consecutive cases of benign vascular lesions reported during a 12-year period (1997-2008). Complete demographic and clinical details of the patients including name, age, sex, clinical history, site of the lesion and histologically diagnosis offered originally were retrieved from Surgical Pathology records. Cases where clinical details were incomplete, tissue blocks were unavailable or representative tissue was inadequate (n = 26) were excluded from the study group. Paraffin tissue blocks were retrieved in all the remaining 94 cases and these were included in the study.
Three sections of 4 μ thickness were cut from each block containing the representative lesion. Sections were stained by (1) Routine hematoxylin and eosin stain (2) Verhoeff's method: To look for elastic fibers for the presence of arteries and arterioles and (3) 1.0% toluidine blue: To perform mast cell count.
All cases were reclassified into hemangiomas and AVMs on the basis of the presence or absence of arteriovenous structures.  Further, lesions were evaluated for (a) presence or absence of intra-lesional nerves and (b) mast cell counts.
Mast cell counting was done in toluidine blue stained sections using Nikon YS100 model biological microscope in all the cases using 100 oil immersion objective and × 10 eye piece. With these specifications the magnification is × 1000 and field view diameter is 0.18 mm. Therefore, area of the field view using the formula for the area of a circle = πr 2 (where r is the radius of a circle) is 0.0254 sq mm. Thus, for counting in 1 mm 2 area, we have to do the cell count in 1/0.0254 = 39.37 fields, or rounded up to 40 fields. Therefore, 40 nonoverlapping fields were counted to obtain mast cell count per mm 2 area. In each representative section, 40 fields were counted to obtain the number of cells per mm 2 .
All data were entered in MS Excel and statistical analysis was performed. Chi-square test was done using STATCALC calculator in EPI-INFO software (Version 6, Centres for Disease Control and Prevention, Atlanta, 1996).
| Results|| |
The original diagnoses offered on histopathology as per surgical pathology records were hemangiomas in 60 cases and AVMs in 34 cases. Lesions which showed presence of arteries and arterioles on elastic stain reclassified as AVM [Figure 1]. The diagnoses of all the 34 cases of AVM were confirmed on review. However 10 cases originally diagnosed as hemangioma turned out to be AVM on using these criteria. The 50 hemangiomas included 46 cases of capillary hemangiomas and 4 cases of cavernous hemangiomas. Thus, this study analyzes findings seen in 44 cases of AVM and 50 cases of hemangiomas, that is, in 94 cases of benign vascular lesions.
|Figure 1: Photomicrograph of arteriovenous malformation showing prominent elastic fibers in walls of the arteries (Verhoeff's stain, ×200)|
Click here to view
Maximum cases (20; 45.45%) of AVMs were between 10 and 30 years of age, while most hemangiomas (27; 54%) were in the age group of 20-40 years. AVMs were twice as common in men than women (male:female = 2.4:1). Hemangiomas were more common in women (male:female = 1:1.38).
Most cases of benign vascular lesions were found in the face, extremities and scalp. 53.1% (50) cases were found in the head and neck region, 30.8% (29) were found on the extremities. Most AVMs (25) were found in the lower or upper extremities (17) or face (8). Most hemangiomas involved the face (15).
We evaluated the hematoxylin and eosin (H and E) stained sections for the presence or absence of intra-lesional nerves [Table 1]. Intra-lesional nerves were found in 81.8% (36 cases) of AVM [Figure 2], while they were seen in only 6% (3 cases) of hemangiomas. Thus, intra-lesional nerves were seen in significantly higher number of AVMs when compared to hemangiomas, (P = 0.0000002).
|Figure 2: Photomicrograph showing intra-lesional nerves in a case of arteriovenous malformation (H and E, ×200)|
Click here to view
We further evaluated the lesions with the presence of intra-lesional nerves; they were classified as:
- 0: where the nerve fibers were absent,
- 1+: where isolated nerve fibers were seen,
- 2+: where lesions showed the presence of medium sized nerves and,
- 3+: where thick nerve bundles were seen inside the lesion.
As can be inferred from [Table 2], 25 cases of AVM showed the presence of medium sized nerves or thick nerve bundles. These were not seen in any of the hemangiomas. Only three cases of hemangiomas showed the presence of intra-lesional nerves, and even they were in the form of isolated nerve fibers.
Mast cell density ranged from 0 to 95 cells/mm 2 in cases of AVM, while it ranged from 2 to 136 cells/mm 2 in cases of hemangiomas. The mean mast cell density in AVM was 25.31 ± 27.3 cells/mm 2 , while it was 29.36 ± 25.06 cells/mm 2 in hemangiomas. This difference was not statistically significant.
|Table 2: Distribution of cases according to pattern of intra-lesional nerves|
Click here to view
We compared mast cell density in involuting (n = 23) and proliferating (n = 16) hemangiomas. Only those cases, which showed clear cut evidence of proliferation or involution, were included in this analysis [Table 3]. Mean mast cell density was significantly higher in proliferating hemangiomas [Figure 3] compared with involuting ones showing the presence of fibrosis (P = 0.028).
|Figure 3: Photomicrograph showing large number of mast cells in a case of hemangioma (Toluidine blue, ×1000)|
Click here to view
|Table 3: Mean mast cell density in proliferating and involuting hemangiomas|
Click here to view
| Discussion|| |
Lesions were classified as AVMs if they showed the presence of arteries and arterioles, and as hemangiomas if they did not show the presence of arteries and arterioles,  on performing elastin stain. In our study, all the 34 cases of AVMs were confirmed on review. However, 10 cases of hemangiomas were reclassified as AVM on application of the histologic criteria.  AVMs are the result of errors of morphogenesis and contain complex intercommunicating arterial and venous structures. Hemangiomas on the other hand, result from derangement in angiogenesis from exuberant proliferation of vascular elements. , Therefore, arteries and arterioles are not part of the lesions.
Hemangiomas, being the result of abnormal proliferation, often respond to treatment and corticosteroids. Vascular malformations are stable cellularly and their therapeutic approaches include percutaneous catheter embolization and surgical resection.  Not only will AVMs that are misdiagnosed as hemangiomas and treated as such, fail to respond to medical treatment with pharmacologic agents; error in the surgical management of such lesions may also result in treatment failure and loss of angioaccess for proper management of the lesion in the future.  Therefore considering the therapeutic importance of accurately distinguishing between hemangiomas and AVMs, it is important that these lesions be correctly diagnosed.
Koutlas and Jessuran  used orcein stain to study the presence and distribution of elastic fibers in the vascular components from 6 cases of arteriovenous hemangiomas. Adegboyega and Qui  used Movat pentichrome stain to assess various tissue components in hemangiomas and AVMs. Because arteries and arterioles are an integral part of AVMs, the pathologists can rely on elastic stains as ancillary tools for making a definitive diagnosis of AVMs.
In our study, 45.45% of cases of AVM were between 10 and 30 years of age, while 54% of all hemangiomas were in the age group of 20-40 years. Most studies mention that hemangiomas usually appear in the 1 st year of life. They appear within a few weeks after birth and rapidly enlarge over a period of several months.  However, only 4 of our cases of hemangiomas belonged to the first decade of life. This is because most hemangiomas are followed up clinically with little or no intervention and seldom operated upon. Most of the cases in our study therefore are from adults, where the lesions were operated upon for mainly cosmetic purposes. AVMs, by definition are technically present at birth, but may not become noticeable until much later in life, with some coming to attention as late at the time immediately after puberty.  AVMs do not involute; and may sometimes increase in size because of vascular ectasia induced by conditions such as sepsis, trauma, puberty, and pregnancy. 
Intra-lesional nerves were seen in significantly higher number of AVMs as compared to hemangiomas. Medium sized nerves or thick nerve bundles were seen in 56% of the AVMs while they were not seen in any of the hemangiomas. Only three cases of hemangiomas showed the presence of intra-lesional nerves and even they were in the form of isolated nerve fibers. Only a handful of previous studies have focused on the presence of distribution of nerves in benign vascular lesions. Rydh et al.  have reported absence of nerve bundles and paucity of nerve fibers around the dilated vessels in nine cases of port-wine stains and concluded that loss of vascular tone due to the absence of adequate nerve supply may be responsible for the vascular ectasia that characterizes those lesions. Considering the absence of nerve fibers in those lesions, Adegboyega and Qui  suggested they are better classified as venous hemangiomas. Adegboyega and Qui  used S100 stain to assess the presence of nerves and nerve fibers in hemangiomas and vascular malformations. Intra-lesional nerves were identified in 91% (69/76) of their cases of AVMs. In contrast, no intra-lesional nerve was detected in any of the 91 hemangiomas. Our findings are in agreement with Adegboyega and Qui's study.  Our results showed that nerve bundles are consistently present in AVMs and absent in hemangiomas and so they can be used as a diagnostic clue to differentiate between these lesions even in H and E stained sections.
We found that the mast cell density ranged from 0 to 95 cells/mm 2 in cases of AVM while it ranged from 2 to 136 cells/mm 2 in cases of hemangiomas. The mean mast cell density in AVM was 25.31 ± 27.3 cells/mm 2 while it was 29.36 ± 25.06 cells/mm 2 in hemangiomas and this difference was not statistically significant. Literature on the occurrence of the mast cells in benign vascular lesions is sparse and contradictory. This may be accounted for by the confusion in the classification and nomenclature of cutaneous vascular lesions. Burrows et al.  found a mean of 35.9 mast cells/high power field (HPF) in four hemangiomas, while the mean cell count in seven patients with vascular malformations was 0.7/HPF. Koutlas and Jessurun  found 6-22 mast cells/HPF in the stroma and around vascular components in 6 cases of AVMs. Girard et al.  also reported increased numbers of mast cells in the stroma, but they did not further elaborate on this observation. Carapeto et al.  mentioned the presence of mast cells around vessels. Lascano  and Baroni  identified increased numbers of mast cells in several vascular tumors.
We compared the mast cell density in 16 cases of proliferating hemangiomas and 27 cases of involuting hemangiomas. The mean mast cell density in proliferating hemangiomas was 53.12 ± 27.83 cells/mm 2 , while it was 11.43 ± 7.9 cells/mm 2 in involuting hemangiomas. Mean mast cell density was significantly high in proliferating hemangiomas compared to involuting ones showing the presence of fibrosis. The wide range of values obtained from the mast cell counts proliferating hemangiomas (6-136 cells/mm 2 ) probably reflects the nature of these lesions, where there is the presence of dense proliferating nests of endothelial cells without capillary lumina at places to abundant capillaries at some places and to beginning of fibrosis and involution at others.
In their study, Glowacki and Mulliken  found that the density of mast cells was at least five times more in proliferating hemangiomas of childhood than the density of mast cells of normal skin and that the mast cell numbers fell to normal in regressing tumors. Our data raises the possibility that the mast cells may have a role in formation, maintenance and progression of these lesions.
The striking feature of proliferating hemangioma in contrast to lesions in the involuting phase is hypercellularity with high labeling of endothelial cells by ( 3 H) thymidine.  Azizkhan et al.  have shown that mast cell conditioned media stimulate the migration of microvascular endothelial cells in vitro. Heparin has been shown to be a potent stimulus of endothelial migration. Mast cells may enhance directional elongation of new capillary sprouts by sustained release of heparin. Heparin alone does not stimulate endothelial cell proliferation but is an important amplifier in angiogenesis. Hemangiomas thus may arise or be maintained by abnormal concentrations of mast cells within developing connective tissue; the endothelial cells and vascular channels may involute when stimuli from mast cells are no longer present.
Any classification is justifiable only if it has diagnostic applicability in planning therapy. Use of elastic stain helps in accurately discriminating between AVMs and hemangiomas by clearly outlining arteries and arterioles. Presence of the intra-lesional nerves can be a useful histologic feature to distinguish between AVMs and hemangiomas even on H and E stained sections. It is unlikely that mast cells alone are the direct cause of hemangiomas, yet their abundance in proliferative lesions only suggests that they may have a role in the natural history of these lesions.
| References|| |
|1.||Chang MW. Updated classification of hemangiomas and other vascular anomalies. Lymphat Res Biol 2003;1:259-65. |
|2.||Hand JL, Frieden IJ. Vascular birthmarks of infancy: Resolving nosologic confusion. Am J Med Genet 2002;108:257-64. |
|3.||Adegboyega PA, Qiu S. Hemangioma versus vascular malformation: Presence of nerve bundle is a diagnostic clue for vascular malformation. Arch Pathol Lab Med 2005;129:772-5. |
|4.||Mulliken JB, Glowacki J. Hemangiomas and vascular malformations in infants and children: A classification based on endothelial characteristics. Plast Reconstr Surg 1982;69:412-22. |
|5.||Enjolras O, Mulliken JB. Vascular tumors and vascular malformations (new issues). Adv Dermatol 1997;13:375-423. |
|6.||Girard C, Graham JH, Johnson WC. Arteriovenous hemangioma (arteriovenous shunt). A clinicopathological and histochemical study. J Cutan Pathol 1974;1:73-87. |
|7.||Folkman J, Klagsbrun M. Angiogenic factors. Science 1987;235:442-7. |
|8.||Klagsbrun M, D'Amore PA. Regulators of angiogenesis. Annu Rev Physiol 1991;53:217-39. |
|9.||Natali J, Merland JJ. Superselective arteriography and therapeutic embolisation for vascular malformations. (Angiodysplasias). J Cardiovasc Surg (Torino) 1976;17:465-72. |
|10.||Trout HH 3 rd . Management of patients with hemangiomas and arteriovenous malformations. Surg Clin North Am 1986;66:333-8. |
|11.||Koutlas IG, Jessurun J. Arteriovenous hemangioma: A clinicopathological and immunohistochemical study. J Cutan Pathol 1994;21:343-9. |
|12.||Werner JA, Dünne AA, Folz BJ, Rochels R, Bien S, Ramaswamy A, et al. Current concepts in the classification, diagnosis and treatment of hemangiomas and vascular malformations of the head and neck. Eur Arch Otorhinolaryngol 2001;258:141-9. |
|13.||Rydh M, Malm M, Jernbeck J, Dalsgaard CJ. Ectatic blood vessels in port-wine stains lack innervation: Possible role in pathogenesis. Plast Reconstr Surg 1991;87:419-22. |
|14.||Burrows PE, Mulliken JB, Fellows KE, Strand RD. Childhood hemangiomas and vascular malformations: Angiographic differentiation. AJR Am J Roentgenol 1983;141:483-8. |
|15.||Carapeto FJ, Garcia-Perez A, Winkelmann RK. Acral arteriovenous tumor. Acta Derm Venereol 1977;57:155-8. |
|16.||Lascano EF. Mast cells in human tumors. Cancer 1958;11:1110-4. |
|17.||Baroni C. On the relationship of mast cells to various soft tissue tumours. Br J Cancer 1964;18:686-91. |
|18.||Glowacki J, Mulliken JB. Mast cells in hemangiomas and vascular malformations. Pediatrics 1982;70:48-51. |
|19.||Azizkhan RG, Azizkhan JC, Zetter BR, Folkman J. Mast cell heparin stimulates migration of capillary endothelial cells in vitro. J Exp Med 1980;152:931-44. |
Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha - 442 102, Maharashtra
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]