biopsy in oral medicine and radiology , dentistry topic

Biopsy in oral medicine and Radiology

1. BIOPSY One can’t show this when it’s not there One can’t hide this when it’s there. Presented by- Arjun KR

2. TABLE OF CONTENTS Introduction History Definitions Indications & Contraindications Classification Various biopsy procedures Special considerations Essential biopsy principles 2

3. Errors Biopsy artifacts Healing of a biopsy wound Complications Conclusion References 3

4. Introduction The clinical presentation of any pathology can be the mucosal surface change (i.e., change in texture, ulceration, proliferation) or it can be submucosal structural alterations (i.e., distortion or swelling produced by a mass). The diagnosis of such pathology depends on the history, examination, laboratory studies, biopsy and other diagnostic techniques. 4

5. Among all these investigations, obtaining an accurate tissue diagnosis is the one needed in the process of forming a definitive diagnosis and treatment planning. This is referred to as the biopsy . It is essential for the purpose of records and research; to determine the prognosis in cases of malignancy and also provides an indication towards the response of the tumor to therapy (e.g., relative resistance of malignant melanoma to radiotherapy). 5

6. There are many methods used to perform this procedure. But whatever the procedure, the aim of it should be to provide a suitably representative sample for the pathologist to interpret, while minimizing perioperative discomfort for the patient. 6

7. HISTORY 7

8. In the early 16 th century, Sir Marcello Malphigi was the one who formulated the basic microscopic technique of utilizing the living tissues. He termed it Bios-living, Opsis -visualizing . Later, Sir Georianni Morgahni in the early 17 th century popularized this method through his book ‘ The site and causes of diseases ’ which laid the foundation for physiologic anatomy. From that date of Malphigi’s formulation of the technique till now, the method has spread wide with some additions and deletions to the extent that its need has become mandatory adjunct to complete history and examination of the lesion in doubt. 8

9. The term "biopsy" was introduced into medical terminology in 1879 by Ernest Besnier . "...  Not only skin changes (colloid degeneration of the dermis) were studied in a definitive manner in a series of necropsies, but we could enlighten the living many points by examining fragments of integument or diseased tissue fragments. There is in this latter mode of investigation, true biopsy (a new word that we propose to designate a new thing), regular clinical diagnostic process whose importance is considerable.”  9

10. In this quote from an article in the  weekly Gazette of Medicine and Surgery  October 10, 1879 under the Ernest Besnier signature, the word  biopsy  is first delivered.  The first diagnostic biopsy in Russia was performed in 1875 by M. M. Rudnev . 10

11. It is possible to make out three stages in more than 100 year history of the method development: an occasional use of histologic procedure, involving living organs and tissues accessible for observation and study (approximately until the late 19th century); restricted application of biopsy (until the mid-20th century); 11

12. present stage at which the method is widely adopted and its use is general and total (with respect to human organism) not only in oncology but practically in all clinical specialties 12

13. DEFINITION OF BIOPSY Biopsy is the removal of tissue for examination, microscopic analysis, chemical analysis, and bacterial analysis or a combination of all four. The term is used most frequently to indicate removal of tissue from a living subject for analysis. - Tiecke RW in 1965 13

14. Biopsy is the removal of a piece of tissue from the living body for diagnosis by microscopic examination. - Tomlins Christopher DC in 1998 Biopsy is the removal of tissue from a living subject for laboratory evaluation and analysis. - Neelima AM in 2002. 14

15. INDICATIONS FOR BIOPSY To confirm a clinical impression of a lesion. When an inflammatory lesion is not responding to conservative therapy after 10 to 14 days. For the determination of the more definitive treatment of the lesion. 15

16. To determine the nature of any intraosseous lesion which cannot be identified clinically and radiographically . To determine the nature of all abnormal tissue removed from the oral cavity, including cysts and granulomas. 16

17. Persistent hyperkeratotic changes in surface tissues. Any persistent tumescence, either visible or palpable beneath a relatively normal tissue. Lesions that interfere with local function (e.g. Fibroma). Any lesion that has the characteristics of a malignancy (e.g. Erythroplakia ). 17

18. Surface lesions: Color/texture change e.g., white, red or pigmented. Ulcerated, fissured or both. Proliferate (fibroma, papilloma). 18

19. 2. Subsurface lesions: Soft tissue- Superficial, distort surface continuity, e.g., Mucocele . Deep, detected by palpation, e.g., masses of various consistency. Hard tissue- Superficial, distort surface continuity Deep, detected by x-rays, e.g., radiopaque and radiolucent changes. 19

20. CONTRAINDICATIONS FOR BIOPSY RELATIVE For ambiguous lesions a) This should include inflammatory lesions of allergic, viral, fungal or bacterial etiology. b) Normal anatomical and racial variations, e.g., linea alba, physiological racial pigmentation, leukedema , exostoses etc. c) Lesions caused by recent trauma. 20

21. Compromised general health of the patient or a history of coagulopathy or bleeding diathesis, including patient on anticoagulant therapy. Proximity of lesion to vital anatomic, vascular, neural or ductal structures and lesions in areas of difficult surgical access. 21

22. ABSOLUTE Pulsatile lesions or those suggestive of a vascular nature should be referred for more in depth evaluation (e.g., hemangioma ). Intrabony radiolucent lesions should not be biopsied without initial aspiration. Pigmented lesions generally should not be biopsied incisionally (e.g. Spread of a melanoma, transformation of premalignant pigmented lesions to malignant ones). 22

23. Lesions that because of size or location present technically difficult surgery e.g., posterior tongue and oropharynx offer severe problems of access. Lesions that are clinically obviously malignant should be biopsied only in the facility that will assure continual care. 23

24. VARIOUS BIOPSY TECHNIQUES The type of biopsy to be performed depends on the location, size and clinical impression of the lesion. Basic types include- Incisional, excisional, exploratory, punch biopsy, needle biopsy, cytological smear, curettage biopsy, unplanned biopsy. 24

25. SURGICAL NON-SURGICAL SOFT TISSUE BONE DIAGNOSTIC CURATIVE PUNCH ELECTROCAUTERY SOFT TISSUE CURETTAGE FROZEN SECTION EXCISIONAL BIOPSY DIAGNOSTIC CURATIVE CURETTAGE TREPHINE ASPIRATION FROZEN ENUCLEATION RESECTION CURETTAGE ASPIRATION CYTOLOGY SCALPEL CAUTERY EXFOLIATIVE FNAC INCISIONAL 25

26. Tissue may be obtained as: Tissue piece: a. Incisional biopsy including geographical biopsies and vital staining b. Excisional biopsy c. Curetting d. Frozen section. 26

27. Tissue core: Fine needle cutting biopsy. Tru -cut needle biopsy. Vin Silverman needle biopsy. Cell aspirate: Fine needle aspirate biopsy. Scrapings: Exfoliative cytology. 27

28. TISSUE PIECE: INCISIONAL BIOPSY / DIAGNOSTIC BIOPSY: It is a biopsy technique that samples only a particular or representative part of the lesion. Using this technique multiple biopsy (that is, if the lesion is large or has different characteristics at different locations, more than one area of the lesion need to be sampled) can be considered. This is often done by drawing a diagram of the lesion. 28

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31. 31

32. Indication: It is chosen- when the lesion is extensive (larger than 1 cm in diameter) or potentially malignant (requiring wide excision) or to avoid an adjacent structure, e.g., nerve or artery. For central bony lesions (either cystic or solid) in determining the nature and facilitate planning for definitive removal/ reconstruction. 32

33. Other indication includes: Chronic non-healing ulcer or squamous cell carcinoma. Leukoplakia/ erythroplakia . Lichen planus . Bullous lesions (pemphigus, pemphigoid etc ). Granulomatous diseases ( crohn’s disease, orofacial granulomatosis , ulcerative colitis, tuberculosis). Minor salivary gland tumor (in palate). 33

34. Contraindications: Pulsatile/ vascular lesions. Pigmented lesions. 34

35. Disadvantages: Leaves noticeable scar. Risk of facial nerve damage. Possible spread of malignant cells (detection of cytokeratine 19 reverse transcriptase in peripheral blood drained 15 mins after incision of squamous cell carcinoma, not detected in excisional biopsy group or in controls. If at all Incisional biopsy is necessary, chemotherapy should be administered before or after biopsy thus effective in preventing secondary metastasis) 35

36. Principle: Representative areas of the lesion (the area that shows complete tissue changes) should be biopsied in wedge fashion from the edge of the lesion including some of the normal tissue. Deep narrow biopsy should be considered rather than broad, shallow one, because superficial changes may be different from those deeper in the tissues. Necrotic areas should be avoided because it may be nondiagnostic . 36

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38. GEOGRAPHICAL BIOPSY AND VITAL STAINING Lesions with areas of varying appearances and in cases of widespread dysplastic disease or field changes within the oral cavity affecting whole of palate, tongue or floor of the mouth may require several biopsies, in such situation vital staining helps to choose the appropriate area for biopsy. 38

39. Technique: 1% aqueous toludine blue dye is applied to the affected lesion for approximately 30 seconds followed by a rinse with water. Then 1% acetic acid stain is applied and rinsed with water. 39

40. Toludine blue stains the epithelial surface blue and with the application of acetic acid the blue stain from normal epithelium is lost whereas the stain is retained in premalignant and malignant erythematous lesions and are not decolorized by acetic acid. Toluidine blue is not a specific stain for cancer cells but since it is acidophilic metachromatic nuclear dye that selectively stains the acid tissue component (particularly nucleic acid DNA), the dysplastic and anaplastic cells as they contain more of DNA than normal stains, they retain toludine blue stain. 40

41. Advantages: The technique is helpful in differentiating the small dysplastic erythroplakia that requires biopsy from erythematous lesions caused by infection, inflammation or trauma. In dysplastic or malignant lesions with diffuse marginal pattern preoperative toluidine blue staining indicates the border of a lesion serving as a guide for surgical excision than does the clinical examination alone. 41

42. Disadvantage: The technique cannot show tumor that is present beneath the normal epithelium. 42

43. ELECTRO-SURGERY BIOPSY Electro-surgery refers to the cutting and coagulation of tissue using very high-frequency, low-voltage electrical currents. A blended current combines cutting and coagulation, and is useful in producing a bloodless operative field. Lesion excisions on the face are usually performed with only a cutting current to limit scarring at the wound base, which can be produced by the effects of thermal coagulation. 43

44. Electro-surgical technique The lesion is grasped with forceps through the loop electrode. The electrode is activated going under the lesion, removing the growth. 44

45. EXCISIONAL BIOPSY It is the removal of the lesion in toto at the time the surgical diagnostic procedure is performed. Not only the entire lesion is made available for pathological examination, but also complete excision may constitute definitive treatment for few lesions. 45

46. Indications: It is the usual approach for smaller lesions (less than 1 cm in diameter). It is used for clinically benign lesions, be they superficial or deep, soft or hard tissue. Includes the excision of pigmented and hyperkeratotic lesions. Fibromas and papillomas . 46

47. Mucoceles Myoblastoma Keratoacanthoma Sialoliths and smaller benign lesions of accessory salivary glands Certain cicatrial lesions 47

48. Principle: Entire lesion along with 2 to 3 mm of normal appearing surrounding tissue is excised. 48

49. 49

50. Technique: For surface excision simple elliptical approach is designed, like for the excision of pigmented and hyperkeratotic lesions, fibroma, papillomas and superficial pathology. For deep soft tissue lesions, modified elliptical that is combined with deeper dissection is chosen. 50

51. elliptical incision vertical deep 51

52. For most Incisional and excisional biopsies, ellipse technique is employed. After obtaining the anesthesia i.e., local infiltration (LA with vasoconstrictor) around the periphery of the lesion, the lesion is isolated and immobilized with a traction suture, hook or forceps. Care should be taken not to mutilate the specimen when grasping it with forceps 52

53. An elliptical area at the surface is outlined using no.15 sharp scalpel blade (to avoid tearing the tissue), incision is taken down to the connective tissue layer to form a ‘V’ at the base of the lesion, this provides a good specimen and also leaves a wound that is easy to close. The length of the incision should be three times its width to allow for tension-free primary closure. High volume suction devices should be avoided as they can aspirate small surgical specimen. 53

54. Haemostasis is achieved with resorbable suture inserted (to control bleeding and also assist in healing). Firm pressure for few minutes aid in haemostasis . The tissue is fixed immediately upon removal in 10% formalin / 70% alcohol, 20 times the volume of the surgical specimen. If the specimen is thin, then place it upon a piece of glazed paper and drop into fixative so that it prevents curling of the tissue. The biopsy specimen should be marked with a silk suture to orient the specimen to the pathologist. 54

55. CURRETTAGE BIOPSY CURRETTE is a French word ‘Curer’- meaning to clean. It is indicated for intraosseous lesions that lie in cavities such as maxillary antrum and cystic lesions within the jaws. Also used in very friable cellular lesions like sinuses and fistulae within the soft tissues when only small amounts of surface material are necessary for evaluation. 55

56. Although the sample produced is usually soft tissue but it may include bone fragment as well. These extremely small segments of tissue after fixation are centrifuged and then the sediment is placed in medium such as agar, they are then sectioned as a cellblock. 56

57. PUNCH BIOPSY: It is an alternative technique of tissue removal applicable to both incisional and excisional biopsy. 57

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59. Indications: The technique most often is used for total removal of small lesions but also finds applicability in the partial removal of superficial abnormalities. It is extremely useful when used on fixed tissue such as firmly attached palatal tissue, which heal by secondary intention regardless of the technique. 59

60. The technique is used for oral mucosal malignancies such as squamous cell carcinoma as well as leukoplakia and other mucosal abnormalities that may require multiple biopsies. It is also helpful to diagnose oral manifestations of mucocutaneous and other vesiculoulcerative diseases. 60

61. Contraindication: As a definitive surgical excision procedure of suspected malignant lesions and cases of vascular lesions.   61

62. Advantages: Technique is fast with low incidence of post surgical morbidity. Suturing is usually not required as the surgical wound heal readily by secondary intention with minimal or no scar formation and with maximum esthetic results. The need for a post operative or suture removal visit is uncommon. The technique can be used on any mucosal surface accessible to the biopsy punch. 62

63. Disadvantage: Technique is designed primarily for use with epithelial or superficial mesenchymal lesions. It is difficult to use biopsy punch to obtain adequate representative tissue deeper than the superficial lamina propria . Freely movable mucosa that cannot be well supported as with the floor of the mouth and soft palate may preclude the technique. 63

64. Punch biopsy should be used with caution when the lesion overlie significant submucosal structures such as mental foramen or nasopalatine foramen and occurs in inaccessible areas such as the maxillary posterior buccal alveolar ridge and anterior lingual aspect of the mandible. 64

65. Technique: Various types of biopsy punches are available – - Keye biopsy punch - Belt-driven. Even disposable biopsy punches are available. 65

66. After the biopsy site has been anesthetized, the site is gently blotted with sterile gauze. The edge of the blade of the biopsy punch is placed on the site and rotated back and forth using moderate pressure to an appropriate depth until the external bevel is not visible and creates a clearly defined surgical margin. 66

67. The tissue is then grasped with an atraumatic forceps and the base of the tissue core is released using a scalpel blade or fine curved scissors. Punch size varies from 2-6 mm in diameter. Suture is rarely needed, as the hemorrhage is minimal. Local pressure with sterile gauze is sufficient to induce haemostasis 67

68. Persistent hemorrhage can be treated with chemical cautery such as silver nitrate, collagen matrix, or by electrocautery . 68

69. Post- operative instructions include avoidance of inadvertent trauma to the area, either by diet or through attempts at oral hygiene for 48 hours. Warm salt mater rinses recommended for palliation. Non-steroidal anti-inflammatory agents are preferred for post-operative discomfort. 69

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71. FROZEN SECTION In surgical oncology, treatment of malignant oropharyngeal tumors involves the excision of tumor with 1 cm margin of normal tissue around the tumor- this is termed as clear margin. Failure to achieve this reduces the chance of local control (for radiotherapy) and recurrence can be expected. To overcome this problem, frozen section analysis is undertaken from the mucosal and deep surfaces of the defect intraoperatively ; and if the tumor remains then further resection is undertaken at the time of primary resection. 71

72. Indication: To make an immediate surgical therapeutic decision. To determine whether a lesion is benign, malignant or non-neoplastic. Establish the adequacy of clearance of margin after resection. Ascertain metastatic involvement of regional lymph nodes. 72

73. It reduces the time of processing from 18 hours to 5 minutes Methods of frozen section: Freezing microtome using CO 2 gas Refrigerated microtome(cryostat) 73

74. Technique: Biopsy tissue is frozen in a mixture of isopentene and solid carbon dioxide at -70 o . Sections of 5-7μm are made on a refrigerated microtome adhered to a glass slide at room temperature, fixed with formal acetic alcohol (50ml formalin, 450ml 90% alcohol and 25ml of glacial acetic acid) and stained with haematoxylin and eosin. The procedure is completed within 5-10 mins from the time of receiving specimen till it is stained. 74

75. The remainder of tissue is stored in 10% buffered formaldehyde and routinely processed; embedded in paraffin, sectioned to 3 μm and stained with haematoxylin and eosin. In this type of biopsy slides cannot be preserved for future reference. 75

76. Errors in diagnosis can be due to: Sampling by the surgeon or pathologist. Interpretation by the pathologist. Difference in communication between the two. 76

77. Disadvantages: There can be error in sampling and interpretation of frozen tissue as compared to routinely processed tissue. Differentiation between reactive epithelial changes is difficult. It has the disadvantage that only 8-16 micron thick section can be cut and finer details of tissue can not be examined. 77

78. TISSUE CORE Although FNAB is widely used technique in the diagnosis of head and neck lesions, the diagnostic accuracy is not as good as histological analysis. Many pathologists consider core tissue for the histologic study to be useful because core biopsy specimen is larger in size and thus suitable for conventional histopathological analysis than the cytologic material obtained from FNB. 78

79. Secondly the technique is simpler, easier and faster than the traditional suction method. It also eliminates the possibility of inadvertent suction of a specimen through a biopsy needle into the syringe and the fragmentation of the specimen due to the aspiration step. 79

80. Cutting needle biopsy that employed the Biopsy gun was first devised in the early 1980s. Studies assessing the clinical utility of cutting needle biopsy using Monopty biopsy instrument (18G needle), which is a newly introduced simple disposable tool for performing cutting needle biopsy in head and neck lesions like lesions of lymph nodes, salivary glands, palate and soft tissue have shown a accuracy of 88% with no significant rush artifacts or obscuring blood, both of which are problems associated with manual biopsy techniques. 80

81. Core biopsy specimen provides accurate information about cell type and tissue characteristics in head and neck lesions. It helps to determine whether a lesion is malignant or benign before performing a total resection. One of the major complications with this technique is the possible spread of tumor cells along the large-bore needle track. 81

82. TRU-CUT NEEDLE BIOPSY: It consists of wide bore 14G and consists of a long 15.2cm ) canula and trocar with a 2cm notch at the tip of the trocar . Technique: L.A. Stab incision with a scalpel Canula is inserted with the trocar fully retracted until the specimen notch is with in the tissue to be biopsied. 82

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84. VIM SILVERMAN NEEDLE BIOPSY:1938 It consists of: 1)Outer canula 16 G in size. 2)Inner trocar. 3)Inner split longitudinal needle. Technique: L.A. Small incision made with the scalpel before the canula and trocar are inserted up to the tissue to be biopsied. The trocar is then completely removed and replaced by the split cutting needle. 84

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86. Advantage: They are easy to interpret then aspiration cytology to the pathologist To distinguish between reactive changes and recurrent malignancy in possible cervical metastasis. 86

87. CELL ASPIRATE FINE NEEDLE ASPIRATION CYTOLOGY: (FNAC) Kun in 1847, gave the first description of the technique for aspiration biopsy. Greg and Gray in 1904 used needle aspiration to demonstrate the organisms in the lymph nodes. Franzem et al 1956 gave the technique of fine needle aspiration biopsy which is used today (needles of 21G or smaller). 87

88. Patient selection/Indication: 1)The disease must be localized and clearly defined by clinical examination or by any available radiological investigating technique. 2)The most commonly diagnosed malignant lesion, with this method is squamous cell carcinoma & benign lesions are Pleomorphic adenoma and relative lymph node hyperplasia. 88

89. Aspiration of soft tissue pathology is employed only when fluid is detected or suspected and in of little value in the diagnosis of solid oral lesions. Although it is not a substitute for conventional biopsy but it is valuable in producing immediate results and free of complications and even helpful in distinguishing between benign from malignant neoplasms, to initiate treatment, or even to indicate the need for further investigation. 89

90. Other advantages include – The cost savings as compared to conventional open biopsy, rapid and effective aid in diagnosis of swelling in lymph nodes and parotid tumors. Small size of the needle avoids damage to vital structure in the head and neck. Cells can be fixed, stained and examined within minutes. In cases of deep lesions ultrasound or radiological guidance may be used to ensure that the needle enters the lesion. 90

91. Treatment options including preoperative counseling can be investigated earlier without the need for further diagnostic surgery Requires little equipment Painless, no anesthesia is required Outpatient or bedside procedure Cuts down on bed occupancy for diagnostic investigations 91

92. Absence of reactive fibrosis to interfere with subsequent surgery No problems with wound healing, e.g. after radiotherapy Readily repeatable 92

93. Disadvantages: Success of FNA depends on obtaining a representative sample (if the specimen is small with few or no cells). Experience is required for interpretation. Definitive diagnosis not always possible. False negative and false positive results are possible. 93

94. Technique: Standard disposable needles (21 – 23G) are used for all palpable lesions (for children and where the eyelids are involved smaller 25 G needles are used). Thicker needles tend to cause more bleeding and are prone to blockage. The needle is attached to a standard 10-20 ml disposable syringe capable of producing good suction. The barrel of the syringe is supported with free hand and the lesion is approached, as vertically as possible 94

95. The needle is inserted into the lesion using no suction and once the needle is within the lesion the change in resilience confirms the entry of needle into the mass. Then suction or negative pressure is applied and is moved back and forth in the lesion for 10 to 15 times at different angulations making sure that the needle is within the lesion. When the mass has been adequately sampled, the negative pressure is released from the syringe and the needle is withdrawn. Then air is drawn into the syringe and the aspirate is deposited on a clean-labeled microscopic slide. 95

96. Usually 2-3 slides are prepared on each mass. One slide is immediately fixed in 95% ethanol solution and subsequently stained with Papanicolaou’s or haematoxylin and eosin stain. Another slide is allowed to dry for staining with a May- Graunwald or Wright stain. 96

97. Ideally the aspiration should have a creamy consistency with a high cell and low fluid content. The number of needle insertion necessary for a good aspirate depends on the consistency of the lesion. Even it is possible to perform FNAC using a 25 G needle alone in cases of small lesions that is just relaying on the capillary pressure which keeps the cells within the needle lumen. This technique gives a better feel for the consistency of the tissue and produces less blood in the aspiration. 97

98. After any needle biopsy, direct pressure should be applied over the site to reduce the incidence of hematoma formation. 98

99. A- needle is introduced into the mass. B – plunger is retracted after needle enters the mass C – suction is maintained while needle is moved back and forth within the mass D – suction is released and plunger returned to original position before needle is withdrawn 99

100. ASPIRATION CYTOLOGY GUN 100

101. FRANZEN’S HANDEL WITH SYRINGE & NEEDLE FITTED ON IT FOR PERFORMING FNA C 101

102. Causes of unsatisfactory yield with fine needle aspiration include: Needle missing a lesion and picking up inflammatory cells. Lack of cells in central area of necrosis, hemorrhage, cystic change. Small malignant tumour being masked by larger benign tumour . Lack of cells in dense fibro sclerotic tissue. 102

103. COMPLICATION & HAZARDS OF FNAC Haematoma : Bleeding from puncture site & haematoma formation are commonest complications of the procedure. Firm finger pressure for 2-3 minutes immediately after procedure reduces the frequency of complications. Infection : Introduction of infection is not a significant hazard. Dissemination of tumour : Local dissemination by seeding of malignant cells along the needle tract is a rare complication & reported in cancers of lung, prostate & pancreas. 103

104. LIMITATIONS OF FNAC Only a small population of cells is sampled, thus the reliability of test depends on adequacy of sample & its representative character. An inadequate sample, which is not representative of true lesion, results in false negative report. R equires clinical information or relevant investigation (e.g. x-ray finding), which further limit utility of FNAC. 104

105. USE OF FNAC: Commonest indication is to distinguish between both benign and malignant neoplasia as well as non-neoplastic conditions. Secondly to differentiate between a local recurrence or nodal metastasis 105

106. Lymph nodes – FNAB is an excellent initial diagnostic modality in the evaluation of lymphadenopathy. Many infectious processes can be diagnosed because cultures may be obtained from bacteria and fungus. Aspirates from enlarged lymph nodes can differentiate between- Reactive hyperplasia or inflammation. Malignant disease. Lymphoma. It is also used to confirm the cervical lymph node metastasis from previously treated local neoplasms. 106

107. Bone and Cartilage Aspiration using 21 or 23G needles can be undertaken if the cortical bone is thin or absent. A larger bore 18G needle can be used to gain access to the lesion prior to inserting a smaller 25G needle for the actual aspiration, or access may be gained by penetrating the cortical wall with a small bur. 107

108. Aspirated blood often indicates a vascular lesion e.g., hemangioma or aneurysmal bone cyst. Withdrawn air suggests likely entry into the maxillary sinus or a traumatic cyst. Aspirated serous fluid sometimes glistering with cholesterol crystal is indicative of a cyst. If aspiration of a central bony defect is nonproductive, the probability of a solid lesion, i.e. neoplasia or tumor, exists 108

109. Skin and soft tissue - It is usually possible to obtain tissue by incisional at excisional biopsy, but FNA is possible on all lesions larger them 5 mm. 109

110. Salivary gland swelling – Incisional or cutting biopsies are contraindicated owing to risks of tumor seeding or fistula formation. Thus FNAB is most widely used in the assessment of salivary gland masses. The primary indication for FNAB of salivary gland is to distinguish among benign, malignant and inflammatory lesions. The accuracy percentage is higher for benign as compared to malignant tumors with a sensitivity and specificity of 80% and 98%. 110

111. Terminology: Regarding the confusion over the use of the terms aspiration biopsy and aspiration cytology. If an aspirate of cells is obtained using fine needles (21-25G) the technique is called “fine needle aspiration cytology” (FNAC) or fine needle aspiration (FNA). Whereas, if a core of tissue is produced using larger bore needles (14-18G), the procedure is best referred to as fine needle cutting biopsy (FNCB) or true cut biopsy 111

112. ASPIRATION BIOPSY Aspiration biopsy is the use of a needle & syringe to penetrate a lesion for aspiration of its contents for purpose of analysis. Applicable to both intra osseous as well as soft tissue masses. 112

113. Used to rule out &/or differentiate Fluid filled cavities Vascular lesions Hematomas Empty cavities Cyst (when lesion is cystic fluid is yellow in color occasionally red due to presence of blood) 113

114. 114 INTRAOSSEOUS OR HARD TISSUE BIOPSY TECHNIQUE AND SURGICAL PRINCIPLES A lesion either on or within the osseous tissues of the jaws requires investigation. The most common intraosseous lesions we encounter are periapical granulomas and cysts of the jaws. Because these have a characteristic radiographic appearance and are usually asymptomatic, a presumptive diagnosis is frequently possible. Treatment may involve surgical removal of the cyst in the form of an Excisional biopsy. When a lesion is large, perforating into soft tissues, or suspected of malignancy, incisional biopsy is indicated.

115. 115 Before performing hard tissue biopsy, the dentist should carefully palpate the area of the jaws where the suspect lesion is located. Comparison with the opposite side is helpful. Bone that feels smooth and firm to palpation usually indicates that the lesion has not expanded or eroded the cortical plate. A spongy feel to the jaw when compressed indicates erosion or thinning of the cortical plates.

116. 116 Aspiration biopsy of radiolucent lesions Any radiolucent lesion that requires biopsy should undergo aspiration before surgical exploration. This provides valuable diagnostic information regarding the nature of the lesion. For e.g , brisk, pulsating blood may indicate a vascular lesion, which should not undergo surgical exploration by the general dentist. The return of straw colored fluid would corroborate presumptive diagnosis of a cyst, and surgical removal can then be undertaken without hesitation. The aspiration of air may indicate that then needle tip is within the maxillary sinus or a traumatic bone cavity.

117. Mucoperiosteal flaps Because of their location within or proximal to the jaws, most lesions of hard tissue must be approached through a mucoperiosteal flap. The choice of flap depends chiefly on the size and location of the lesion. The size of the lesion dictates how much access is necessary when Excisional biopsy is indicated. Access may necessitate extension of the mucoperiosteal flap.

118. 118 The location of the lesion dictates where the flap incisions are to be made. It is important to avoid major neurovascular structures when possible and to keep incisions over sound bone for closure. Optimally flap design should provide 4 to 5 mm of sound bone around the anticipated surgical margins. A central lesion that may have eroded the cortical plate of the jaw is always approached with flap elevation in an area away from the lesion and over sound bone. This technique allows establishment of the proper tissue plane for dissection.

119. 119 All mucoperiosteal flaps for biopsies in or on the jaws should be full thickness and incised through mucosa, submucosa , and periosteum . The dissection to expose bone is always performed subperiosteally

120. 120 Osseous window Lesions within the jaw (i.e., central lesion) require the use of a cortical window. If expansion of a central lesion has eroded the cortical plate to the point that an osseous void is seen once the flap has been elevated, this window can be enlarged with rongeurs or burs. If the cortical plate is intact, a rotating bur should be used to remove an osseous window.

121. The size of the window depends on the size of the lesion and the proximity of the window to normal anatomic structures such as roots and neurovascular bundles. Once the window has been created, it can be enlarged with a rongeur . The osseous window specimens should always be submitted for histopathologic examination with the primary specimen . 121

122. 122 Removal of the specimen The technique for removal of the biopsy specimen depends on the nature of the biopsy (excisional versus incisional) and the consistency of the tissue encountered. Most small lesions that have a connective tissue capsule (e.g., cysts) can be removed in their entirely. A dental curette is used to peel the c.t. wall of the specimen from surrounding bone. The concave surface of the instrument should always be kept in contact with the osseous surface of the bone cavity.

123. 123 The convex surface of the instrument is the portion that actually separates the specimen from surrounding bone. This technique is used until the specimen is free and removed. The bony cavity is inspected after irrigation with sterile saline. Any residual fragments of soft tissue within the cavity should be removed with curettes. Once the cavity is devoid of residual pathologic tissue, it is irrigated and flap is replace and sutured in its proper location.

124. SCISSORS BIOPSY Is one of the ways to remove skin tissue for a biopsy specimen. This procedure entails snipping off a growth that is attached to the skin with a stalk. Scissors biopsy is indicated for pedunculated and very superficial growth. Depending on lesion size and morphology, anesthesia may or may not be necessary. 124

125. Small forceps with teeth and a pair of sharp curved or straight iris scissors are the only surgical instruments required. The lesion to be removed is lightly grasped with forceps by gently pulling upward, traction provides a firm cutting surface and allows clear visualization of the lesion base. Bleeding after this procedure is usually minimal and can be easily controlled by application of 35% aluminium chloride solution 125

126. 126

127. SHAVE BIOPSY A scalpel or razor blade is used to scrape lesion, performed superficially or deeply. Shave excision usually extends to the level of the middle dermis, with the subcutaneous tissue left undisturbed. Skin lesions with a minimal dermal component, such as seborrheic keratoses or fibrous papules are excellent candidates for shave excision technique. 127

128. The blade is held horizontal to the skin surface and brought below the lesion. The other hand is used to stretch and stabilize the skin surrounding the lesion during the shave biopsy. Smooth, unidirectional cutting with the blade separates the lesion above from the deep (reticular) dermis below. 128

129. 129

130. Assisted guidance: Most tumours that are visible or palpable can be examined without the aid of radiologic imaging, whereas for deep lesions that cannot be palpated, as well as for small, deep mobile lesions that are difficult to palpate require radiologic control to ensure that target tissue sample is obtained and secondly this guidance reduces the number of aspirates and helpful in differentiating the tissues within a lesion. Ultrasound, computed tomography and MRI are used in percutaneous biopsy procedures 130

131. Ultrasound guided biopsy This technique has the advantages of being noninvasive , quick, and easy, and it can be performed with the patient under local anesthesia . It has an advantage over blind percutaneous biopsy because the needle can be visualized in the organ and the organ scanned after biopsy for possible complications. Another advantage is that, unlike other radiographic biopsy procedures, ionizing radiation is not used for imaging. 131

132. However, Ultrasound guided biopsy is not possible when gas/bone prevents the visualization of the biopsy region. 132

133. 133

134. CT guided biopsy A computed tomography guided biopsy, uses real-time CT images to help the doctor guide a needle to the suspect lesion to obtain a tissue sample. Occasionally, intravenous (IV) contrast is needed to help the radiologist identify and target the lesion prior to the biopsy. The CT image is immediately available on a monitor, allowing the radiologist to view the biopsy target. 134

135. Indications 1. Lymph nodes or masses that are not completely identifiable using ultrasound. 2. Lesions near the skull base: CT is optimal for localizing these lesions. 135

136. Disadvantages of CT include radiation exposure, limited possible scan plane orientations, low soft tissue contrast, and, poor vessel conspicuity without administration of intravenous contrast medium. 136

137. MRI guided Interventional MRI is a method for procedure guidance that combines the imaging benefits of magnetic resonance, including excellent tissue contrast and multiplanar imaging capability, and good vessel depiction of MRI with the increased patient access that is possible with newer magnet designs. 137

138. 138

139. Scientific interest has also focused on MRI-guided, minimally invasive thermal tumour ablation using the unique temperature sensitivity of MRI or its capability to demonstrate changes in tissue relaxation parameters (T1 and T2) that occur in the process of necrosis. The mean procedure time for MRI-guided needle insertion per pass is less than 10 minutes for aspiration as well as core biopsy. 139

140. Endoscope guided biopsy Endoscopy is defined as “the examination of the interior of a canal or hollow viscous by means of an endoscope.” Endoscopic technique may prove to be particularly important when dealing with large jaw cystic lesions that may contain neoplastic processes such as ameloblastomas or carcinomatous entities within certain regions of their lining. 140

141. Endoscopy may prove to be an important tool for the internal examination of large jaw cysts that may contain regional neoplastic processes within the cyst lining. Especially in the case in large cysts that extend into areas that are difficult to inspect and sample through a standard “bony window” technique 141

142. Endoscope positioned into the lesion. 142

143. Endoscopic view showing areas of thickened lining containing exophytic protrusions measuring up to 10 mm in diameter. 143

144. Velscope 144

145. 145

146. 146

147. 147

148. 148

149. EXPLORATION BIOPSY Used for inrtaosseous lesions of maxilla and mandible Instruments: Chisel, Bone burs, Periosteal Elevator UNEXPECTED/ UNPLANNED BIOPSY When as a result of surgical procedure (Tooth extraction) some suspicious tissue is obtained unexpectedly 149

150. TISSUE SCRAPINGS EXFOLIATIVE CYTOLOGY: Cyto – Cell -- Logos – Study Study of cells. Rudolf Virchow (1955) stated “Every cell is derived from a cell & that human disease processes were essentially disease of the cells.” 150

151. Rudolf Virchow (1821-1905) “ THE FATHER OF CELLULAR PATHOLOGY” 151

152. Dr. George N. Papanicolaou (1883-1962) 1920s “ father of exfoliative cytology” Developed PAP test for diagnosis of uterine cervix cancer 152

153. Normal oral squamous epithelium continuously sheds the most superficial cells. If malignant or other disease processes affect the area, the deeper cells lose their cohesiveness and are exfoliated at the same time as the superficial cells. 153

154. Exfoliative cytology is the study of superficial cells which have been either exfoliated or shed actually from mucous membrane, renal tubules etc. and also includes the study of those cells which have been collected being scraped or pulled off by tissue surface and may also be found in body fluids such as sputum, saliva etc 154

155. The lesion is repeatedly scraped with a moistened tongue depressor or spatula or cytobrush type instrument. The cells obtained are smeared on a glass slide and immediately fixed with a fixative spray or solution. 155

156. Indications: For quick laboratory evaluation of suspected malignant and premalignant oral lesions and multiple premalignant and extensive lesion and lesions leading to field cancerization . For sequential laboratory evaluation of post-operative or post-irradiated malignant lesions. Recurrent oral cancers after treatment. Mass screening of oral cancer. 156

157. To identify the presence of certain specific cells in non-malignant red patches or ulcerative lesions. To see malignancy associated change in buccal squamous cells in patients with malnutrition. For evaluation of vesicular lesion. For detection of sex chromosomes. For the study of buccal mucosa in various anemia. 157

158. Certain benign hereditary skin lesions having their representative oral manifestations. For the study of the change of the oral epithelial cells followed by chemotherapy. 158

159. Contradictions: Deep seated lesions (both soft and hard tissue). Fibrous lesions. Polypoid growth. Non-ulcerative lesions. Lesions do not show positive changes in the cells of the superficial layers. 159

160. Atrophic lesions. Densely keratinized lesions. Smooth surface lesions. Lesions giving inadequate specimen sampling through the adopted technique. Patients with underlying blood dyscrasias . 160

161. Merits: Exfoliative cytology implicates its importance in the field of diagnosis with the principle that any change in the superficial cells can be the reflection of the change in the immediate underlying tissue. Cytology is an adjunct to but not a substitute for the surgical biopsy. It is quick, simple, painless, bloodless, non- invasive procedure. If guards against false negative biopsies. 161

162. It helps to follow up recurrent carcinomas. It is valuable for screening of clinically non- suspected lesions. Easy and a most feasible method for detection of malignancies in oral epidemiological survey programme . It gains to evaluate the emergent concepts of proclaimed cancer, ca -in-situ, and related lessons of the oral cavity. 162

163. Demerits: Relatively limited information provided with exfoliated material when compared to a histological preparation. Positive results gives definite value whereas negative results is of considerably less value. Cytology positive with biopsy positive - definite value. Cytology positive with biopsy negative - Repeat biopsy. Cytology negative with biopsy negative - negative. Cytology negative with biopsy positive – Repeat cytology. 163

164. Exfoliative cytology is limited to tissue, which exfoliate cells into reasonably accessible sites. Majority of the lesions occur in the oral cavity are benign which do not lend themselves to cytologic smear. 164

165. Target for cytological Prediction: Squamous cell Carcinoma remains the chief target for cytological investigation. Shed cells with less cohesion or poor adhesiveness and red oral lesions usually considered as benign are the prime targets for cytological smearing. 165

166. Requisite for diagnostic cytology: Diagnosis must be based on the synthesis of entire evidence available rather than the changes in individual cell. Adequate clinical data. Uniformity of the technical method employed. Thorough knowledge of normal cells and extensive physiological variation of the sites to be diagnosed. 166

167. Technique: Instruments for removing the cells should have a 90-degree angle or straight blade. The cement spatula used in mixing crown and bridge cement at wax carver is ideal. A tongue blade slit longitudinally is a second choice, but it should be moistened to avoid dehydration damage to the cells. The cotton swab has been recommended in selected cases. 167

168. Scraping of the lesion should be done while the tissue is stretched or taut. A single stroke is preferred over many small strokes. Excess saliva should be removed by an air blast prior to removal of the cell. Smearing should be done on a standard glass slide (1x3 inches) with an etched label area atone end. The glass slide should be clean and dry. The cells should be evenly disturbed over the central one third of the slide. The slide must be labeled 168

169. Fixation must be immediate. Air dried smears are inconsistent in their staining properties. A safe and adequate fixation is 95% isopropyl alcohol, there is little danger of fire with this solution, and it is easily obtained, inexpensive, available and easy to store. The cells should be fixed for at least 1 hr before staining. If the slide is to be mailed the fixative can be discarded after fixation and the mailing container tightly sealed to prevent drying. History should be supplied to the pathologist as in done with a biopsy. 169

170. 170

171. 171

172. 172

173. 173

174. 174

175. Interpretation and Reporting: The evaluation of slide is based on the morphological features and staining quality of the cells especially the nuclei. The ratio of nucleus to cytoplasm is also a critical factor papanicolaou’s classification is often used in reporting diagnostic equation. The cytologic smear is usually reported into one of the V classes: - 175

176. Class I (Normal) indicates only normal cells. Class II (Atypical) indicates the presence of mild atypia but no evidence of malignant change. Class III (Intermediate) the cells display wider atypia , may be suggestive of cancer, but they are not clear-cut and may represent precancerous lesions, carcinoma in situ. Biopsy is recommended. Class IV (Suggestive of cancer) A few malignant cells with many cells having borderline characteristics. Biopsy is mandatory. Class V (Positive for cancer) obvious malignant cells. Biopsy is mandatory 176

177. Touch impression or Imprint cytology It is a method in which gentle grazing or sliding of glass slide over the cut surface of a resected tumor immediately after the surgery. Advantage : Feasible and economical Detect the malignancy at the tumor margin Less time consumable 177

178. A direct imprint is prepared by pressing a glass slide gently on to the freshly cut surface of the specimen, avoiding a gliding movement, which will distort the shape of the cells. The imprint slide is immediately fixed in 95 % ethyl alcohol for 5-6 seconds and then stained (rapid haematoxylin and eosin). 178

179. This is particularly useful in - In the diagnosis of certain neoplastic lesions which can simulate inflammatory lesions on frozen section In the diagnosis of certain benign inflammatory lesions which can simulate malignancy on frozen section 179

180. In the diagnosis of malignancy confined to one small area of a large specimen The amount of tissue that can be frozen for rapid intraoperative diagnosis is limited. On the other hand, the imprint technique can easily cover a larger portion of the specimen, thus reducing errors due to inadequate sampling. In the diagnosis of malignancy when the submitted specimen is limited in quantity Small fragments of tissue, which may prove to be difficult for frozen-section interpretation, are often large enough to provide sufficient cells for imprint interpretation. 180

181. Imprint of a massively necrotic carcinoma showing a cluster of viable cancer cells on a necrotic background. Diagnosis was not possible on frozen section. 181

182. Imprint of a lymph node showing malignant cells. 182

183. ARMAMENTARIUM: 183

184. Wide mouthed bottle with 10% formalin (at least 20 times the volume of the specimen should be used) For exploratory biopsy – bone burs, chisel, periosteal elevator and curette are included. Electric cautery should not be used for removal of tissue because the surgical margins get coagulated. Cautery can however be used on a postsurgical site in order to control bleeding. 184

185. Site selection and handing of the specimen- By carefully selecting the area or areas, which will produce a good diagnostic specimen, the clinician can avoid having repeat biopsy. The extent of biopsy is determined by the size and location of the lesion. Pathologists prefer the entire lesion as the most desirable specimen. 185

186. Deep sections are preferred over shallow specimens. If possible, biopsies from the mucosa should include the epithelium, lamina propria , submucosa , and a portion of any deeper tissue, such as muscle and fat, which may be affected by the clinical lesion. Sections taken from the surface or center of a lesion are often necrotic and do not represent the most typical characteristics of the lesion. The periosteum should be avoided whenever possible as it is often a barrier to growth and infections. 186

187. Sterile technique should be utilized both to prevent infection, which complicates the diagnostic procedure, and to avoid contamination of the specimen. Anesthesia in the majority of cases is local, with infiltration used more commonly than block. Infiltration must be slow and far enough from the lesion to prevent explosion of the tissue elements, and particularly the separation of epithelium from connective tissue. The anesthesia should be subjected slowly enough to provide blanching but not rapid swelling. 187

188. TISSUE STABILIZATION Soft tissue biopsies in the oral cavity are frequently performed on movable structures, such as the lips, soft palate, and tongue. Accurate surgical incisions are easiest to perform on tissues that are properly stabilized. Several methods are available to achieve tissue stabilization. An assistant’s fingers pinching the lip on the both sides of the biopsy area can immobilize the lips. 188

189. This method also aids in hemostasis by compressing the labial arteries. Instruments are available to perform this same function. Heavy retraction sutures or towel clips can be used to aid immobilization of the tongue or soft palate. When used, the sutures should be placed deeply into the substance of the tissue, away from the proposed biopsy site. They will be useful for secure stabilization without pulling through the tissue 189

190. The chalazion clamp, used by ophthalmologists, is a helpful tool for oral biopsies on the oral lips, anterior buccal mucosa, or tongue. This clamp, with a solid metal back and ring like opening anteriorly, is tightened in place around the lesion to be biopsied. It performs the two important functions of providing a firm surface to work and yields nearly complete hemostasis . Sutures can be placed in the center of the ringed opening before the clamp is loosened. 190

191. 191

192. HEMOSTASIS The use of suction device for aspiration of surgical hemorrhage during biopsy should be avoided. This is especially true of the high-volume evacuators available in most dental offices. Small surgical specimens can be easily aspirated into these devices and lost. Gauze wrapped over the tip of a low-volume suction device or simple gauze compresses are adequate in most cases, unless severe haemorrhage is encountered. 192

193. Manipulation and grasping of any lesion suspected of being a tumor should be minimal. Vigorous manipulation of malignant tumors can cause tumour cell emboli. Suturing is not often necessary when small incisional biopsies are taken, but most excisions require suturing. Black silk suture material in preferable. Orientation of the specimen is extremely important to the pathologist Multiple lesions must be adequately labeled and often oriented with a diagram of the original lesion. A black silk suture placed through each end, preferably in normal tissue will often serve to orient the lesson. 193

194. Orientation of mucosal biopsies (particularly superficial lesion) is important, especially because they are small and have limited morphologic characteristics after being immersed in formalin. Proper orientation of the surface lesion specimen assists the oral pathologist in sectioning the specimen to avoid tangential cuts. Improper orientation will lead to the sectioning of either epithelium or the connective tissue alone, but not both. 194

195. Surgeons can place sutures in the specimen to assist in orientation and provide a written description of the specimen in relation to suture. At least two adjoining margins must be clearly identified to ensure correct orientation, with the help of short suture and a long suture 195

196. 196

197. Fixation and preservation of tissue are essential to avoid distortion of histologic and cytologic details. 10% formalin (4% formaldehyde) has a relatively long shelf life, and it is the more commonly used fixative for diagnostic specimens. Other fixatives, which are used with satisfactory results, include parachlorphenol (camphorated), sodium hypochlorite, and Zephesin . When there is doubt about the action of some chemical on tissue, it is preferable to place the specimen first in cool or cold water (preferably an isotonic solution) and then in formalin or soon as possible. 197

198. Tissue should never be placed on cotton, gauze, or paper prior to fixation. There is rapid absorption of fluid from the cells, often causing extensive distortion that would not occur if the specimen is left on glass or metallic surface for several hours. Container used for fixation should have a wide mouth, so that after fixation the specimen can be easily removed. Containers that have caps that will rust should be avoided since the iron oxide will often interfere markedly with the staining reaction in the laboratory. 198

199. If culture is desired, then the tissue should not be placed in fixative until the material for bacteriological study is obtained, either by smearing the tissue on a plate or by placing it for a short time in a nutrient medium. If formalin is not available at hand, place the specimen in refrigerator at 4oC to slow down autolysis. The container should have an opening larger enough so that the tissue can be removed easily after it has hardened by fixation. 199

200. However, fresh material is needed for the following purpose: 1. Frozen section 2. Immunocytochemistry 3. Cytological examination 4. Microbiological sampling before histopathology 5. Chromosome analysis 6. Research purpose 7. Museum display 200

201. No fixative will penetrate a piece of tissue thicker than 1 cm. For dealing with specimen thicker than this, following methods are recommended: 1. Solid organ: Cut slices as necessary as but not thicker than 5 mm. 2. Hollow organ: Either open or fill with fixative or pack lightly with wool soaked in fixative. 3. Large specimen, which require dissection: Inject fixative along the vessels or bronchi as in case of lung so that it reaches all parts of the organs. 201

202. Temperature: The fixation can be carried out at room temperature. Tissue should not be frozen once it has been placed in the fixative solution, for a peculiar ice crystals distortion will result. Speed of fixation : The speed of fixation of most fixative is almost 1 mm/hour. Therefore, a fixation time of several hours is needed for most specimens. Amount of fixative fluid : This should be approximately 10-20 times the volume of the specimen. Fixative should surround the specimen on all sides. 202

203. Factor affecting fixation : 1. Size and thickness of piece of tissue. 2. Tissue covered by large amount of mucous fix slowly. 3. The same applies to tissue covered by blood or organ containing very large amount of blood. 4. Fatty and lipomatous tissue fix slowly. 5. Fixation is accelerated by agitation. 6. Fixation is accelerated by maintaining temperature around 60oc. 203

204. Labeling of the specimen: The patient’s name, the location of the specimen, and the date of the surgical procedure are all essential. If multiple biopsies are taken from the same patient, the specimens should be placed in individual containers or wrapped and labeled in individual gauze, which has been saturated in 10% formalin. If the specimen is to be mailed, it is better to place it in gauge within the specimen container so that if a rupture with loss of fluid occurs, the gauze will maintain the tissue in a moist fixed state. 204

205. Details required in pathology form Patient data • Clinical details of lesion • Any medical history with details of medication • Oral habits - all forms of tobacco and alcohol consumption • Investigations done, if any • Site and biopsy type • Clinical diagnosis with differential diagnosis • Previous biopsy done, if any, with details. 205

206. 206

207. Follow-up and Reporting of Biopsy Result to the Patient Patients should be seen 1 to 2 weeks postoperatively to ensure healing and to discuss the results of the biopsy. It is the responsibility of the clinician (not the assistant or secretary) to explain the diagnosis and any further management if necessary. If the microscopic diagnosis is inconsistent with the clinical impression, the clinician is strongly advised to discuss any concerns directly with the pathologist. 207

208. In large lesions Accessible area Characteristic portion. For multiple lesions Most representative site. Material curetted from interior of the lesion . SPECIAL CONSIDERATIONS 208

209. For red & white lesions include both red & white area 209

210. ULCERS Include margin, deep part of ulcer and site of maximal clinical activity. AVOID Superficial ulcers & necrotic tissue 210

211. For Polypoid lesions include base 211

212. For Vesiculobullous lesions Fluid is more representative. Intact vesicle or bulla should be biopsied. 212

213. For LICHEN PLANUS – representative area should be biopsied 213

214. For LEUKOPLAKIA – Most dysplastic area should be biopsied 214

215. For MUCOCELE lesions – careful excisional biopsy 215

216. For GRANULOMATOUS LESIONS – deep incisional biopsy + fresh sample to microbiology if infective agent suspected 216

217. Do not cut into pigmented and vascular lesions 217

218. 218

219. ERRORS DURING SURGICAL REMOVAL OF BIOPSY SPECIMEN 219

220. INJECTION 220

221. HANDLING THE TISSUE Handle tissue with minimum force USE OF FORCEPS Avoid use of forceps on the surface of biopsy. USE OF ELECTROCAUTERY Topic of debate Combination of cautery and scalpel should be considered . 221

222. SELECTION OF THE SPECIMEN Tissue should be representative of whole lesion. Adequate amount of tissue at least 1 x 0.5 cm in size. REMOVE WHOLE LESION 222

223. Biopsy of skin or mucosa representative sample of both epithelium and connective tissue has to be taken 223

224. Tissue Artifacts "Alterations in tissue morphology that result from various forms of mechanical, chemical or thermal insult to the tissue removed for diagnostic purposes are termed as tissue artifacts." 224

225. CAUSES OF TISSUE ARTIFACTS Clinical application of chemicals. Local injection of anaesthetics . Surgical sectioning. Excessive heat. Freezing. Surgical mishandling of the specimen. Inadequate fixation. Improper fixation medium. Faulty tissue processing. Improper staining. 225

226. 1.DURING SURGERY : Injection Artifacts Forceps Artifacts Fulguration Artifacts Laser Artifacts Suction Artifacts 226

227. 2.DURING FIXATION AND TRANSPORT: Fixation Artifacts Freezing Artifacts Artifacts during transportation 3.TISSUE PROCESSING ARTIFACTS: During Embedding During Sectioning 227

228. INJECTION ARTIFACTS Injection of large amounts of anesthetic solution into the area to be biopsied can produce following tissue changes: The needle insertion may produce hemorrhage with extravasation, which in turn masks the cellular architecture. 2) The separation of connective tissue bands with vacoulation may occur. 3) Tissue edema or distortion. 228

229. HOW TO AVOID Infiltration of an anaesthetic agent Injections directly into the lesion should be avoided. If hemeostasis is a consideration, injections deep to the lesion, or immediately after the biopsy has been performed, is effective. 229

230. FORCEPS ARTIFACTS When the teeth of the instrument penetrates the specimen, it resulted in voids or tears and compression of the surrounding tissue. The surface epithelium may be forced through the connective tissue producing small " pseudocysts ". Compression of tissues results in loss of cytological details with nuclear dimensions and relationships being especially affected. 230

231. TO AVOID Using small atraumatic or Adson forceps with or without teeth. These will produce little mechanical distortion of the tissue. The area of the lesion should never be grasped with forceps. If the use of forceps is mandatory, only the peripheral aspect should be used for holding and delivery of the specimen. 231

232. CRUSH ARTIFACT It’s a form of tissue distortion resulting from even the most minimal compression of the tissue. Crushing produces a destructive and dangerous type of artifact by rearranging tissue morphology and squeezes the chromatin out of the cell nuclei. Inflammatory and tumors cells are most susceptible. 232

233. CAUSES Mutilation of the tissues with forceps. Dull scalpel blades. HOW TO AVOID Delicate handling of specimen. A suture placed to the edge of the specimen to substitute for forceps. 233

234. FULGURATION ARTIFACTS The use of cautery for biopsies. Heat produces marked alterations in both the epithelium and connective tissue. The epithelial cells appear detached and the nuclei assume a palisading configuration. Separation of the epithelium from the basement membrane has also been reported. 234

235. The fibrous connective tissue, fat and muscle gain an amorphous appearance when subjected to such procedures. Its effect is to boil tissue fluid and precipitate protein. Microscopically such tissue shows a coagulated and torn appearance that makes histological evaluation impossible in the cauterised areas. Alternating areas of coagulation impart a " Herring Bone " appearance to the tissue if the procedure is not done properly 235

236. TO AVOID Only the cutting and not the coagulation electrode should be used while obtaining a biopsy specimen. The incision margins should be sufficiently far away from the lesion - normal tissue interface to prevent thermal changes in that significant area. Combination of electro surgery and a scalpel should always be considered 236

237. This technique involves use of the scalpel for initial incision in and around the lesion and electrosurgery to complete the removal of the specimen. This ensures superior hemostasis while reducing the amount of heat to which the tissue is exposed . 237

238. ONCOCYTOID ARTIFACT IN SALIVARY GLAND TISSUES This unusual tissue artifact occur in the serous acini of parotid gland tissue. Many of these have been misinterpreted as oncocytosis , oncocytoma and both. Serous acinar cells resemble oncocytes . 238

239. The two cells can be distinguished by the fact that the altered acinar cells possess uniform round nuclei in the basal half of the cells like their unaffected counterparts. In contrast the oncocytes have a centrally placed nucleus. It is believed that the oncocytosis artifact of salivary gland tissue subjected to electrocautery is a consequence of electrothermal discharge. 239

240. LASER ARTIFACTS Lasers are being used for excision and ablation of oral mucosal lesions with the chief advantage being minimization of intraoperative haemorrhage . DISADVANTAGE Produces the thermal artifactual changes. 240

241. The CO2, Nd : YAG systems lends to cytological artifacts in keratinocytes, with the keratinocytes showing atypical changes consisting of hyperchromatism , pleomorphism and nuclear elongation along with a wide margin of coagulative thermal changes. 241

242. ARTIFACTS INDUCED BY SUCTION TIPS Induced by the vacuum effect of the surgical suction tips. These are seen in various types of surgical specimen, most notably in the connective tissue around odontogenic cysts and dental follicles. Manifest by the formation of large often pleomorphic connective tissue vacuoles resembling traumatized adipose tissue. 242

243. In highly vascularised tissue such as tissue in periapical granulomas and in inflamed odontogenic cysts. Suctioning induces extravasation of blood and focal accumulation of erythrocytes within the connective tissue vacuoles. 243

244. IMPROPER SURGICAL REMOVAL 244

245. WRONG ORIENTATION OF SPECIMEN 245

246. CURLING AND CRUSH ARTEFACT 246

247. HEMORRHAGE ARTEFACT 247

248. SPLIT ARTEFACT 248

249. FRAGMENTATION ARTEFACT 249

250. HEALING OF BIOPSY WOUNDS The healing of a biopsy wound of the oral cavity is identical with the healing of a similar wound in any other part of the body and thus may be classified as either primary healing or secondary healing. The nature of the healing process depends upon whether the edges of the wound can be brought into apposition, often by suturing, or whether the lesion must fill in gradually with granulation tissue. 250

251. Primary healing Primary healing, healing by primary intention or healing by first intention is that type of healing which occurs after the excision of a piece of tissue with the close apposition of the edges of the wound. This is the form of healing one might expect after the excision of a lesion in an area of the oral cavity where the pliability of the tissues is such that the wound may be drawn together and sutured. 251

252. When the edges of the wound are brought into contact and held in place by sutures, the blood clots , and in a matter of hours numerous leukocytes are mobilized to the area. Connective tissue cells in the immediate vicinity undergo transformation into fibroblasts which in turn undergo mitotic division, and the new fibroblasts begin to migrate into and across the line of incision. 252

253. In time, these cells form thin, delicate collagen fibrils which intertwine and coalesce in a general direction parallel to the surface of the wound. At the same time, endothelial cells of the capillaries begin to proliferate, and small capillary buds grow out and across the wound and form new capillaries which fill with blood, and a rich network of young capillaries and capillary loops are formed. 253

254. When there is a close apposition of the edges of the wound, the surface epithelium proliferates rapidly across the line of incision and reestablishes the integrity of the surface. The delicate connective tissue fibrils eventually coalesce into denser bundles and usually contract. Shows a small linear scar which may be depressed below the surface. Wound heals rapidly. 254

255. Secondary healing: Healing by second intention, healing by granulation or healing of an open wound occurs when there is loss of tissue and the edges of wound cannot be approximated. Removal of a lesion of the palate or a large lesion of the alveolar ridge is usually followed by healing by second intention, since the edges of the wound cannot be coapted . 255

256. After the removal of the lesion, the blood filling the defect clots and the repair process begins. It is basically identical with healing by primary intention except that the fibroblast and capillaries have a greater distance to migrate; more granulation tissue must form, and the healing is slower. 256

257. Cellular proliferation begins around the periphery of the wound, and the fibroblasts and endothelial cells grow into the clot along fibrin strands. P olymorph nuclear leukocytes and, later, lymphocytes, and mononuclear phagocytes migrate into the granulation tissue from the adjacent vessels and tissues. 257

258. As the granulation tissue matures, it becomes more fibrous through condensation of collagen bundles, and the surface of the granulation tissue becomes epithelized. The lesion becomes less vascular, the only evidence of the wound may be a small depressed area of the mucosa. 258

259. Complications 259

260. 1. Haemorrahage – Immediate or subsequent haemorrhage can be serious problem following biopsy. Highly vascular tissue ( hemangioma ) In a region where venous pressure is markedly increased (e.g. a supraclavicular lymph node biopsy in the presence of superior vena caval obstruction) From a large friable tumor mass Where an adjacent blood vessel of moderate size may be severed and allowed to retract. When the wound becomes infected and late secondary haemorrhage occurs. 260

261. 2. Infection – When tumors on the various skin or mucosal surfaces are biopsied, there is always a possibility that already present bacteria, may thus gain access to the depths of the tumor and to the adjacent normal tissue. Aseptic technique must always be observed 261

262. 3. Poor wound Healing Unsatisfactory healing of the incision may be due to Ischemia of the skin overlying a tumor mass (tension) Implantation of tumor cells Previous X-ray therapy 262

263. 4. Spread of tumor cells Prominent reasons for local tumor cell contamination are the following: -Lack of awareness that the spread of tumor cells commonly occurs and is increase by prolonged and unnecessary manipulation of tissue. -Careless protection of the tissue during the Incision or Excision of malignant neoplasm. -Failure to change contaminated drapes, instruments or material when indicated 263

264. 5. Injury to adjacent organs Injury may occur to surrounding structure (blood vessels). Structures through which the biopsy is accomplished may be injured. Certain of the vital structures must be avoided in needle biopsy (blind methods) and the adequate surgical exposure is essential when the biopsy is taken with a scalpel. 264

265. Other complications Post operative pain. Paraesthesia - in the lips or the tongue, Swelling and bruising - in the tongue, lips and buccal mucosa Procedures in the floor of the mouth can lead to submandibular or sublingual duct damage. Removal of mucocoeles from the lip carries the risk of further gland damage and ‘recurrence’. 265

266. CONCLUSION For entities of uncertain significance or etiology , a biopsy provides the simplest and most speedy means of obtaining the perfect diagnosis. In the concern of patient’s welfare, correct diagnosis is of extreme importance. A carefully selected, performed and interpreted biopsy is critical in rendering an accurate diagnosis. When considering biopsy, a little forward planning and thought can greatly improve the diagnostic value obtained. 266

267. Careful handling of the tissue and prompt appropriate fixation will enable a confident histological diagnosis to be reached. Inadequate care at any stage could result in a nondiagnostic biopsy and may necessitate the patient having a repeat procedure with its ensuing physical and psychological morbidity. 267

268. R. Rajendran and B. Sivapathasundharam : Shafer’s textbook of oral pathology, 5 th edition (2006), Elsevier. Neville Brad W., Damm Douglas D., Allen Carl M. and Bouquet Jerry E.: Oral and Maxillofacial Pathology, 2 nd Edition (2004) Saunders. Martin S. Greenberg and Michael Glick: Burket’s Oral Medicine Diagnosis and Treatment, 10 th Edition (2003); BC Decker Inc. References 268

269. Marx RE. Oral and Maxillofacial Pathology. A rationale for diagnosis and treatment. 2003. Quintessence publishing co, Inc. Chicago. Cawson RA, Odell EW. Essentials of Oral Pathology and Medicine.1998. 6 th ed. Churchill Livingstone. Edinburgh. Peterson, Ellis, Hupp and Tucker: contemporary oral and maxillofacial surgery, 4 th edition. S M Balaji : Text book of oral and maxillofacial surgery, 1 st edition. Theory & practice of Histological techniques , 2 nd & 3 rd ed. , Bancroft 269

270. Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2 S I Talukder . www.talukderbd.com. Histopathology Techniques: Tissue Processing and Staining Sylvie-Louise Avon. Oral Soft-Tissue Biopsy: An Overview. J Can Dent Assoc 2012;78:c75 K. L. Kumaraswamy , M. Vidhya . Oral biopsy: Oral pathologist’s perspective. Journal of Cancer Research and Therapeutics - April-June 2012 - Volume 8 - Issue 2 270

271. Oral Brush Biopsy Technique Instruction Outcomes for Senior Dental Students David L. Hall, D.D.S. Journal of Dental Education Volume 70, Number 8 . Meta Ramirez A, Silvestre FJ.Oral biopsy in dental practice.Med Oral Patol Oral Cir Buccal 2007; 12(7):E504-10 Brian E.D. Cooke.Exfoliative cytology in evaluating oral lesions.J Dent Res 1963,42:343 . Bone Marrow Aspiration and Biopsy: Collection and Interpretation. Kathryn G. Trewhitt 271

272. Simple biopsy techniques. Christophej . R Huerter 272

273. THANK YOU 273