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Biopsy techniques in oral surgery
BIOPSY
TECHNIQUES
IN
JEROME CHESTER STOOPACK,D.D.S.,**
ORAL
SURGERY”
ST. ALBANS, LONG ISLAND, N. Y.
I
N THE last decade or so the emphasis in periodical literature and lectures on oral tumors has been on such aspects as the importance of early detection, the responsible role of the dentist in their early detection, and the necessity for biopsy. Much less, however, has been presented on the aspects to be discussed in this article; biopsy techniques and procedures. The word biopsy stems from the Greek word bias, meaning life, and apsis. meaning sight. Webster defines biopsy a~ L‘the examination of some portion of the living body, severed from the whole, for scrutiny in aid of correct diagnosis.” However, a more accurate definition would be removal of a specimen of tissue from a lesion on a living patient, for direct histological examination as an aid to correct diagnosis and treatment. It should be emphasized that biopsy is an &id and only an aid. We must not slight the other very important procedures in the “working up” of correct diagnosis and treatment planning such as case history clinical examination, radiographic examination, laboratory tests, and so forth. The role of biopsy cannot be confined to confirming or disproving a clinical impression or diagnosis. The very fact that it does h,eZp establish a diagnosis makes it a valuable aid in determining the prognosis and the treatment for a specific condition. For example, it would be very difficult clinically to differentiate between an oral squamous cell carcinoma, a basal cell carcinoma, and certain lesions of syphilis or tuberculosis. Yet microscopic examination of a biopsy specimen from the lesion would practically establish the diagnosis, and determine the prognosis and course of treatment which would, of necessity, be different in each of the four cases. It is also possible to determine microscopically the degree of malignancy of a particular lesion and to recognize whether a relatively benign lesion has malignant tendencies, as in the case of leukoplakia. Squamous cell carcinomas show a less malignant and a more,malignant type, which can be differentiated readily by a competent pathologist. When to Take the Biopsy.-We must suspect every questionable lesion, cancer being ruled out, first in all cases. Biopsy specimens should be taken on any ulceration that does not respond to conservative treatment in two weeks and on any swelling or growth that does not show signs of regression in one month’s time. If the doctor is in doubt regarding his diagnosis or his course of action, it is always wise for him to refer the patient to someone more experienced in the *The oDinions 01‘ asrertations contained in this article are the private Ones of the Writer and ape not to be construed as official OT reflecting the views of the Navy Department or the Naval Service at large. **Lieutenant Commander, Dental Corps, United Sta%es Navy: Resident, Oral Surgery Department, United State.? Naval Hospital.
1034
BIOPSY
TECHSIQITl?S
IN
OR.\I,
S[:RGERY
1oxFi
specialized field of oral diagnosis and oral surgery for confirmation or consult aCon. The important thing is to recognize the potentially dangerous l&on wrr*l (aall for c-onsultation if necessar?. For the prosthodontist, the early recognit,ion of lesions under dentrl rcs anil restorations is particularly important 1)ecanse of the difficnlty in distin52uishin~ a benign ulcerat,ion due to irritation of a faulty or ill-fitting denture. from :I malignant or potentially malignant. lesion. Immediately upon discovering an inflammatory process, ulceration or growth of any kintl lmtler or associated with dentures, the prosthodontist should take the dentures tru~a~yfrom the pnfictif for safekeeping-not merely from the patient’s mouth. The patient c*annot bcl trusted with them; thev will go back into his mouth for what, he considers 1)&f harmless periods, during so-called ’ ‘iml)ortant engagements.” With the tltAni11~‘s in safekeeping of the prosthodontist he should observe t)he lesions for the prl’wrihed periods of time under conservative treatment. If they do not c~lcar III), or show progressive improvement, during this period, biopsy shonlcl thtxn iktl performed by the doctor himself or by a consultant oral surFeon.
Partial Section or Complete Excision?-It is 01’ course llnclerstc,otl ih;it the size and the character of the lesion are at least two of the factors to bc con.sidered in deciding how much tissue will be removed for biopsy. If a tumor is a sma;l one, for esample. it is more fcasiblc to rcmov(’ tht) mass in tot0 with :I border of normal Cssue. For large massesit is better to remove a small section, including at the same time some of the adjacent normal tissue. Pigmented lesions such as nevi and melanomas should never be cut into : they should always be widely e?rcised if l)iopsy is attempted. ?jor sho~rld vascular lesions such as hemangiomas be cut, into ; rat her. the incision sho111tL he wide of the growth and through norntal tissue. Techniques of Biopsy.-There are foltr main methods of biopsy : the aspiration technique, the punch technique, the cautery or electrosurgical twhniquc, and the surgical technique. A.spircrtion Technique.-Aspiration biopsy as devised by Dr. Hayes Martin is used widely in the Head and Neck Section of Memorial Hospital for Cancer and Allied Diseases in New York City. It is indicated for tumors which lita below the surface of normal tissue where a specimen cannot be obtained except by surgical incision through normal tissue. This applies both to s&t tissue tlimors and tumors within bone. The objections to partial surgical biopsy in these deeply situated tumors arc that such a procedure modifies the clinical setting, and breaks down the natural local barriers to the spread of disease, favoring, in some cases,early metastasis. The special instruments needed for aspiration biopsy are an l&gauge needle 5 to 10 cm. in length, a 10 or a 20 C.C.syringe, glass slides for a smear, a small slice of Gelfoam or fibrin foam as a vehicle for some of the aspirated tissue, and a specimen bottle with 10 per cent formalin (Fig. I). The skin or mucous membrane at the site of puncture is painted with antiseptic solution and a small area is infiltrated with local anesthetic solution, raising a small wheal over the site selected for aspiration. With a sharp poi.nted scalpel a stab wound is made through t,he skin or mucous membrane. This
1036
JEROME
CHESTER
ST001’.4Ct(
facilitates insertion of the needle and prevents contamination of the aspirated material by the surface epithelium. (Fig. 2.) If t,he tumor is contained within bone, a small round burr is used to penetrate the cortical plate following the initial incision through the surface epithelium. The la-gauge needle is then inserted through the stab incision, through the superficial tissues, or through the opening in the bone with the syringe attached and with the piston tightly closed (Fig. 3) until the needle is felt, to enter the tumor, With the needle held stationary, a vacuum is produced in the syringe by partly withdrawing the piston. Maintaining this vacuum, the needle is then advanced a distance of 1 to 3 cm., depending upon the size of the tumor, and then withdrawn to the starting point. This manipulation is repeated a few times changing the direction of the needle, remembering to maintain the vacuum at the same time. Here the needle is again withdrawn to the starting point and the vacuum slowly released. The syringe is then detached from the needle before complete withdrawal, and the needle, which supposedly cont,ains a specimen of tumor tissue, is withdrawn separately from the tissue. Here the needle is again attached to the syringe and the specimen expelled onto the previously prepared glass slide. A portion of the tissue is placed in the small piece of Gelfoam and then immersed in formalin for fixation and section by the usual method. The remaining portion on the glass slide is gently smeared by firm flat pressure of another glass slide, for immediate staining and study. The Punch Biopsy.-There are two types of punch biopsy to be discussed and the first was again originated by Dr. Hayes Martin, Chief of the Head and Neck Section at Memorial Hospital for Cancer and Allied Diseases. The instruments consist of a series of various-sized sharp, circular, detachable punches, resembling the ordinary leather punch (Fig. 4). The desired size punch is selected, attached to the handle, and after the selected area for biopsy has been infiltrated with a local anesthetic, the instrument is applied to the tissues and rotated back and forth several times, until it has penetrated to a depth of 5 to 7 mm. (Fig. 5). This frees the specimen from all of the surrounding tissue except at the base. The punch is put aside and the specimen is removed by grasping it carefully with the tissue forceps and severing it from the base with a scalpel or dissecting shears. It is then placed in the fixing solution. Persistent bleeding can be controlled by use of a coagulating agent such as silver nitrate, by use of the cotigulating current, or by sutures properly placed. The first method is used most often and with best results in the majority of cases. The second technique in this group is performed upon lesions or growths in areas difficult to get to by ordinary methods, such as tumors of the base or back of the tongue. The instrument used is a forceps type of tissue cutter (Fig. 6). The area is anesthetized and the cutting edges of the forceps are applied to the lesion in question and tissue snipped off in such a manner that a representative specimen is obtained. It is treated in the same manner as any other specimen by placing in the formalin bottle for future study. The Electrosurgical Technique.-This third method is mentioned only because some men still persist in its use. It presents the very obvious disadvantage
Fig
Fig.
Fig.
Fig. .-Ammamentarium for aspiration biopsy. lJnw lrft to right, ~w~lpel, kge, : all piece of Gelfoam. Fig. .-Stab incision through mucous membrane of the tongltc. .-The needle inserted through the stab incision with thv piston tightly Fig.
I
1038
Fig. 4.--S& devised by Dr. Hayes Martin, Hospital for Cancer and Allied Diseases, New 74: 107, 1947.)
Chief of the Head York, N. Y. (From Fig.
Fig. 5.-The instrument it has penetrated to a depth Fig. B.-This or similar oral cavity and surrounding
and Neck Section Martin, Hayes:
at Memorial Am. J Surg.
and
times
5.
Fig. 6. applied to the tissues, of 5 to 7 mm. instrument used for structures.
rotated
back
lesions
in
forth
difficult-to-reach
several
areas
until of
the
of cooking the tissues, even with the most judicious handling of the instrument by the operator. This results in cellular dist,ortion and destruction, making microscopic diagnosis very difficult. The only atlvant,age of electrocauter!: lit\ in it,s hemostatic qualities. tt can be twecl to cwnt tvl ht~morrliage during hiops\ mrvrtiolrcvl I)t’c\-iorlsl!-. by use of the coagulating current i1S
treatment
Fig. Fig.
7.-Typical has failed 8.-Area
Fig.
7.
Fig.
8.
tongue lesion in which carcinoma to clear it up within a two-week selected for biopsy outlinc,l.
must be ~wriwl.
ruled
out
first
if conservative
Surgical Excision.-This is the method of choice on all surface or superficially located growths of soft tissue or bone. Here again we find two types: (1) partial excision; (2) total escision or therapeutic> biopsy. Partial Excision.-Partial excision is indicated in large growths or lesions located on or just beneath the surface epithelium and in some smaller growths (Fig. 7). The reason for partial excision in these cases is obvious---some lesions
1040
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XTOOPACK
of the mouth, found in systemic diseases, such as tuberculosis or syphilis, simulate neoplasms. Partial excision, correct diagnosis, and subsequent systemic treatment would eliminate the lesion readily. Total excision with the resulting large defect or deformity in cases of this type is certainly contraindicated. Fig.
Fig. Fig. Y.-Specimen the base. Fig. lO.-Suturing may aid the healing
carefully
grasped
with
9.
10. tissue
is unnecessary in most cases: process and control hemorrhage.
forceps, however,
lifted
and
one or two
dissected well-placed
away
from sutures
There is very little risk, in partial excision of surface lesions, of spreading disease or stimulating the growth of tumor cells because careful section does not break down the natural barriers in the surrounding normal tissue. Following selection of a representative region from which the specimen is to be obtained (Fig. 8)) the area should be infiltrated locally by injection deep to the lesion. A sharp, pointed scalpel should be used and a V-type of elliptical incision should be made. Thin, deep sections should be made rather than broad, shallow sections, and they should include normal tissue at the outer edge of the
BIOPSY
TECHSIQlVZS
Iii
ORSI,
Sil‘R(;ERS
1041
specimen. Following the initial incisions, the specimen should be very carefully grasped with tissue forceps at the normal tissue end, lifted and dissected away from the base (Fig. 9). It is then placed in the fixing solution. Suturing is usually unnecessary in partial excision; however, one or two well-placed sutures may aid in faster healing and controlling hemorrhage (Fig. 10).
Fig. Fig.
Il.-Total 1 %.-The
excision is initial incision
indicated should
Fig.
Il.
Fig.
12.
in lesions of this type. include a good border of normal
tissue
all
around.
Y’otnl Escision.-Total excision is indicat cd in small, surface lesions or sr1d1 growths of the superficial connective tissue layer (Fig. 11). The lesion is well outlined with an elliptical incision following local anesthesia, obtained in t,he same manner as described previously. The initial incisions should include a, good border of normal tissue all around (Fig. 12). The tissue is carefully lifted at one end with the tissue forceps and the growth carefully removed by means of a dissecting shears or scalpel, maintaining a good border of normal t,issue beneath the growth as well as at the sides. Persistent bleeders arc tied off with catgut or are coagulated.
1042
JEROME
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STOOPACK
All tension on the surface sutures is relieved and the incision is closed. This can be accomplished by undermining the surface edges of the wound and then closing or by closing in layers with subcutaneous absorbable sutures. The excised growth in toto is then treated as an ordinary biopsy section and placed in the fixing solution. As a variation in the total excision technique in areas of rich vascularity such as the tongue, it is well to consider running the sutures through before excision. The sutures are placed wide and deep to the ,lesion or growth, following local anesthesia of the area. The incisions and excision are made carefully so that the sutures are not cut and the wound immediately closed subsequent to removal of the growth.
Preparing the Specimen for Study.-To avoid drying out of the specimen, the bottle of fixing solution for receiving the section should be prepared prior to completion of the operation. The bottle should be carefully labeled with the patient’s name and also with an identifying number corresponding to the number on the tissue examination request. Ten per cent formalin is used as the fixing solution; the amount of solution should be approximately twenty times the volume of the tissue. If formalin is not available, 70 per cent alcohol can be used as a poor substitute, for shrinkage of the tissue results with its use, due to dehydration. It is desirable to have a half dozen or more prepared formalin bottles on hand. The Tissue Examination Request Form.-It is important that the tissue examination request include all the information one can give that will be of use to the pathologist. The data should be checked over before the form is passed along, with the specimen. (Fig. 13.) The request should include the following : 1. 2. 3. 4. 5. 6. 7.
Doctor submitting specimen and address. Date obtained. Patient’s age. Patient’s sex. Patient’s race. Clinical description of the lesion before excision. Description of the specimen following excision.
(The last two should be a written report of the clinical appearance of the lesion and surrounding tissues. The report should be an exact description of what one actually sees, including the size, shape, color, whether raised or not, whether soft or hard, and whether ulceration and/or hemorrhage is or ever has been in evidence. ) 8. How specimen was obtained. 9. Brief clinical history. (Includes duration of the lesion, rapidity of growth, physical condition of the patient, and one’s clinical impression or diagnosis.) 10. Patient’s name and address.
Laboratory Procedures.--&zation is accomplished by placing the specimen in the preservative, 10 per cent formalin. The length of time for fixation varies
BIOPSY TECHKIQI'ES
CLINICAL RECORD
I?; ORAl, SL!R(;ERY
TISSUE EXAMINATION I DATE OBTAlNED 4 January 1951
SPEClMEN *lJBMlTTEo BY
Dr. John Doe PATIENT'SMAE sPEC'MENConsists of a yellow round shiny 20 piece of tissue about 2 cm in diameter. It is fairly firm to touch but oan be rather
HOW
OBTIINEO
Speciumn was excised surgically incision and blunt dissection.
su lb
RACE MALE 0 FEMALE
easily
w
-- .-
depressed.
in toto by means of anellf~ticslskin
WuEF C~~~l~~~l~~~~~~~~~~~~~~~~,~a
gpyyom
lmer
----border
of
mandible, 1.5 om in diameter. Is freely movable under epidermis, fibrous No pain, discharge, ulceration or discoloration. Is (hard) in nature. irritated upon occasion by shaving. Began in 1946, grew to its present size associated rith its growth. in period of a few days. No disease of infection Es8 history of cyst removal on both cheeks and behind both ears, otherwise history is irrelevant. Impression: cyst (sebaceous) FLocC"LATIONREACTION COMPLEMENT I=,XAT,ON SlONATURE ANDTkTLE
Grossr An ellipse of skin measuring 2.0x0.6 cm. and attached below it is a large grayish yellow structure resumbling a large kidney bean. It has a smooth glistening surfaoe and is of rather soft consistency. It msa8uree 2.0x1.5x0.6 cm.Cross section of the cystic-like structure reveals that it is filled with a cheesy yellow material. Micror Sections show skin on one surface and below it is a large cyst lined by oompressed etratified aqasmaua epitheliuaL The oyst is filled with desquaamted keratin. The mall of the cyst is very thin being composed of only a few strands of fibroua connective tirsue. No aeoondary skin struotures A sebaoeoua oyst and an epidermoidal cyst are are evident in the uall. usually impossible to tell apart, Because of lack of M&&gic evidence to the contrary the diagnosis of epidenuoidal cyst is favored. No neoplasm. Diagnosis:
Fig.
Epidermoidal
1X.--Tissue
examination
cyst, neck.
request form
usecl in the United
States
Navy.
JEROME
CHESTER
STOOPACK
with the size of the tissue; however, the tissue is left in the formalin solution for from six to twenty-four hours. This coagulates the protein content of the cells, thus preventing decomposition and preserving the tissue elements in as nearly the same condition that they were in at the time of fixation. Hard tissue specimens have to be decalcified before fixing. An acid such as nitric or hydrochloric is used for this process, usually 5 per cent nitric acid. Following decalcification it is treated as a soft tissue specimen and embedded in either celloidin or paraffin. Dehydration is accomplished by placing the specimen in a series of ascending alcohols, 50, 70, 95, and then 100 per cent alcohol, over a period of twentyfour hours in an especially timed machine for the purpose. Embedding.-In order that the tissue may be sectioned by the microtome, it has to have a certain rigidity to offer sufficient resistance to the cutting edge of the knife. This may be accomplished by freezing the tissues, or, as is more commonly done, by using a material which fills the interstitial spaces of the tissue, such as paraffin or celloidin. The freezing technique is used only where immediate investigation is necessary during the course of a surgical operation, for instance, questionable deep tongue biopsies ; while the patient is on the operating table, a small section is excised, sent to the laboratory, where it is quickly frozen, sectioned, examined, If the report is negative for malignancy, the rest of the tumor and diagnosed. is removed and the wound closed. However, if the report is positive, a radical tongue dissection and a possible mandibular resection on that side are performed immediately. Embedding in paraffin is a more lengthy but a far more satisfactory process, retaining the tissue in a much more adequate form for microscopic diagnosis without the distortion that is found in the freezing technique. Since the paraffin is not soluble in alcohol, the alcohol already in the tissues as a result of the dehydration process has to be replaced by xylol or chloroform, to enable the paraffin to be taken up by all of the tissue spaces. The specimens are left in two changes of solvent for a specified length of time and then paraffin is added to the solution. They remain in this solution for a few hours and then are placed in molten paraffin for another specified time. Finally they are placed in a mold filled with molten paraffin, and this is quickly cooled into a solid block. Celloidin is used in hard tissue sections containing teeth or parts of teeth. A mixture of equal parts of ether and alcohol is used as the solvent here, to fill the tissue spaces with celloidin. This type of section requires at least six weeks and as much as three months to prepare. Sectioning is accomplished by means of the microtome. types: the sliding, the rotary, and the freezing depending tissue and the embedding medium used.
There are three upon the type of
Mounting consists of placing the cut specimens on glass slides and drying on an electrically controlled heater. Staining.-Hematoxylin and eosin are uSed universally for staining in microscopic diagnosis, and consist of an initial blue stain which is followed by
partial decolorizat,ion with acid-alcohol solution ant1 then counterstaining wit11 red rosin. Aster clearing with alcohol and sylol, ant1 thorough drying:. the sliclcs is given added protection bp means of a cover glass, itnd then is ready f~)r tllc. last step. Microscopic Eznmi~ation.-Following microscopic examination sia hp the pat,hologist, his report is returned to the tloctor submitting and thus the cyc!c is completed.
and tliagno the reqncsi (
are some precautions and conin Biopsy.-Following that should be observed to insure full diagnostic and sometime:; value of the biopsy, and to avoid unnecessary di&urement of the,
Do’s and Don’t’s siderations. therapeutic patient :
1. Acoid the use of dyes: The dyes should not he used as local antiseptics for procaine infiltration, as they prevent proper preparation of the tissue for Antiseptics used should he colorless! although solutions microscopic study. with a slight amount of color such as Metaphen are permissible. 2. Include a representatiLqe av-ea of the lesion or growth: Thin, deep sections should be obtained in partial or punch biopsy rather than broad shallow ones. Superficial tissue in cases of malignancy may only show inflammatory reactions without actually getting deep enough int,o the connective tissue layer to show tumor cell invasion. Many negative reports are made in error, because of the lack of depth of the section. 3. Include an adequate border of nom& tissue: In partial biopsies, an area of normal tissue is essential for purposes of comparison in microscopic study. Material from the center of the lesion only is frequently not of valuct because of secondary changes or infection which greatly affect the microscopic picture. In total biopsies, a good border of normal t,issue should be included all around and beneath the lesion. The pathologist should be able to report adequate excision into normal tissue in cases where microscopic diagnosis proves the lesions to be premalignant or malignant. In epithelial lesions, the normal border often gives indication of whether the lesion is malignant or not. In benign epidermoid lesions, the epithelium appears pushed up above t,he line of normal epithelium, whereas in the epidermoid malignancies, the epithelium of the lesion is invasive and appears below the line of normal epithelium and well into t,he underlying connective t,issue, 1. Plan the incisions to avoid or mininzize disfiguring the patient: If the lesion from which a biopsy is to be taken is on the face or lips, the cosmetic aftereffect has to be taken into consideration: a. Follow the natural creases (Langer’s lines of tension), if operating on the face. b. Avoid reducing the vermilion border, if operating on the lips. The use of vertical incisions should be considered here. 5. Avoid the use of the electrocautery: If, of necessity, cautery must used, extreme care should be exercised to minimize cooking the tissues.
be
1046
JEROME
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STOOPACK
6. Use care in the use of the tissue forceps: Careless handling of the tissues can crush or otherwise mutilate the specimen. This results in the appearance microscopically of large holes or of flatt,ened tissues. The specimen should be carefully held at one end with a fine forceps during excision. 7. Identify the area, if possible, in suspicious lesions: In these lesions a suture through a portion of the tissue is of great value. Placed anteriorly or posteriorly, or above or below, for that matter, a suture in a specimen that is found by the pathologist to be premalignant or malignant can often be a check on the adequacy of the excision. If the lesion has not been adequately excised, the pathologist can accurately locate for the doctor that area of the lesion by means of this previously placed suture, and the doctor can proceed accordingly without guesswork. 8. Avoid drying out of the specimen: Too frequently excised tissue is placed on the bracket table and left exposed until the operation is completed. As little as ten minutes in an area with an arid climate could cause enough cellular distortion to make microscopic diagnosis impossible. Therefore, place the specimen directly into the formalin solution following excision. 9. Use a separate bottle for each specimen: If specimens are taken from lesions in different areas on the same patient, they should be placed in separate bottles. Great confusion results if one specimen out of two or more in the same bottle proves to be premalignant, or malignant, and the pathologist cannot tell the doctor which area of the mouth is affected. 10. Accompany the specimen with a complete report: Use a comprehensive report form and fill in all pertinent, information. 11. Do not accept all negative reports: One negative report of a suspicious lesion is not necessarily diagnostic of a benign lesion. A negative report should not lull the operator into a feeling of security, when a lesion has a clinical course indicating malignant tendencies. Repeat biopsies are indicated in all There have been persistent eases in which several such suspicious lesions. negative reports on the same lesion have been followed by a positive report of malignancy. References 1. Bernier,
J. L.: An Evaluation of the Biopsy as a Diagnostic Aid, Am. J. Orthodontics and Oral Surg. (Oral Surg. Sect.) 32: 531-542, 1946. 2. Bernier, Joseph L., and Tiecke, R. W.: Biopsy, J. Oral Surg. 8: 342-348, 1950. 3. Chilton.26: N. W.: 1949.Biousv of Cancer of the Mouth, J. New Jersey I D. Sot. - ” in Diaenosis 13-15, 4. Dingman, R. 0.: Importance of Biopsy in Oral Diagnosis, J. Oral Surg. 6: 204-208, 1948. Diagnosis of Oral Tumors, J. Oral Surg. 7: 2175. Dingman, R. O., and Hayward, J. R.: 230. 1949. 6. Martin, ‘Hayes E.: Biopsy Drill Punch, Am. J. Surg. 74: 107-108, 1947. 7. Martin, Hayes E., and Ellis, E. B.: Aspiration Biopsy, Surg., Gynec. & Obst. 59: 578-589, 1934. 8. Morrey, C. W.: Responsibility of the Dentist in Mouth Cancer Detection, J. Am. Dent. A. 38: 452-453, 1949. 9. Orban, Balint: Oral Histology and Embryology, ed. 2, St. Louis, 1949, The C. V. Mosby Company. 10. Smith, J. B.: Surgical Management of Cancer of the Mouth, J. Oral Surg. 7: 95-103, 1949.
64 CLOVERFIELD
ROAD SOUTH,
VALLEY
STREAM,
N. Y.
TECHNIQUES
IN
JEROME CHESTER STOOPACK,D.D.S.,**
ORAL
SURGERY”
ST. ALBANS, LONG ISLAND, N. Y.
I
N THE last decade or so the emphasis in periodical literature and lectures on oral tumors has been on such aspects as the importance of early detection, the responsible role of the dentist in their early detection, and the necessity for biopsy. Much less, however, has been presented on the aspects to be discussed in this article; biopsy techniques and procedures. The word biopsy stems from the Greek word bias, meaning life, and apsis. meaning sight. Webster defines biopsy a~ L‘the examination of some portion of the living body, severed from the whole, for scrutiny in aid of correct diagnosis.” However, a more accurate definition would be removal of a specimen of tissue from a lesion on a living patient, for direct histological examination as an aid to correct diagnosis and treatment. It should be emphasized that biopsy is an &id and only an aid. We must not slight the other very important procedures in the “working up” of correct diagnosis and treatment planning such as case history clinical examination, radiographic examination, laboratory tests, and so forth. The role of biopsy cannot be confined to confirming or disproving a clinical impression or diagnosis. The very fact that it does h,eZp establish a diagnosis makes it a valuable aid in determining the prognosis and the treatment for a specific condition. For example, it would be very difficult clinically to differentiate between an oral squamous cell carcinoma, a basal cell carcinoma, and certain lesions of syphilis or tuberculosis. Yet microscopic examination of a biopsy specimen from the lesion would practically establish the diagnosis, and determine the prognosis and course of treatment which would, of necessity, be different in each of the four cases. It is also possible to determine microscopically the degree of malignancy of a particular lesion and to recognize whether a relatively benign lesion has malignant tendencies, as in the case of leukoplakia. Squamous cell carcinomas show a less malignant and a more,malignant type, which can be differentiated readily by a competent pathologist. When to Take the Biopsy.-We must suspect every questionable lesion, cancer being ruled out, first in all cases. Biopsy specimens should be taken on any ulceration that does not respond to conservative treatment in two weeks and on any swelling or growth that does not show signs of regression in one month’s time. If the doctor is in doubt regarding his diagnosis or his course of action, it is always wise for him to refer the patient to someone more experienced in the *The oDinions 01‘ asrertations contained in this article are the private Ones of the Writer and ape not to be construed as official OT reflecting the views of the Navy Department or the Naval Service at large. **Lieutenant Commander, Dental Corps, United Sta%es Navy: Resident, Oral Surgery Department, United State.? Naval Hospital.
1034
BIOPSY
TECHSIQITl?S
IN
OR.\I,
S[:RGERY
1oxFi
specialized field of oral diagnosis and oral surgery for confirmation or consult aCon. The important thing is to recognize the potentially dangerous l&on wrr*l (aall for c-onsultation if necessar?. For the prosthodontist, the early recognit,ion of lesions under dentrl rcs anil restorations is particularly important 1)ecanse of the difficnlty in distin52uishin~ a benign ulcerat,ion due to irritation of a faulty or ill-fitting denture. from :I malignant or potentially malignant. lesion. Immediately upon discovering an inflammatory process, ulceration or growth of any kintl lmtler or associated with dentures, the prosthodontist should take the dentures tru~a~yfrom the pnfictif for safekeeping-not merely from the patient’s mouth. The patient c*annot bcl trusted with them; thev will go back into his mouth for what, he considers 1)&f harmless periods, during so-called ’ ‘iml)ortant engagements.” With the tltAni11~‘s in safekeeping of the prosthodontist he should observe t)he lesions for the prl’wrihed periods of time under conservative treatment. If they do not c~lcar III), or show progressive improvement, during this period, biopsy shonlcl thtxn iktl performed by the doctor himself or by a consultant oral surFeon.
Partial Section or Complete Excision?-It is 01’ course llnclerstc,otl ih;it the size and the character of the lesion are at least two of the factors to bc con.sidered in deciding how much tissue will be removed for biopsy. If a tumor is a sma;l one, for esample. it is more fcasiblc to rcmov(’ tht) mass in tot0 with :I border of normal Cssue. For large massesit is better to remove a small section, including at the same time some of the adjacent normal tissue. Pigmented lesions such as nevi and melanomas should never be cut into : they should always be widely e?rcised if l)iopsy is attempted. ?jor sho~rld vascular lesions such as hemangiomas be cut, into ; rat her. the incision sho111tL he wide of the growth and through norntal tissue. Techniques of Biopsy.-There are foltr main methods of biopsy : the aspiration technique, the punch technique, the cautery or electrosurgical twhniquc, and the surgical technique. A.spircrtion Technique.-Aspiration biopsy as devised by Dr. Hayes Martin is used widely in the Head and Neck Section of Memorial Hospital for Cancer and Allied Diseases in New York City. It is indicated for tumors which lita below the surface of normal tissue where a specimen cannot be obtained except by surgical incision through normal tissue. This applies both to s&t tissue tlimors and tumors within bone. The objections to partial surgical biopsy in these deeply situated tumors arc that such a procedure modifies the clinical setting, and breaks down the natural local barriers to the spread of disease, favoring, in some cases,early metastasis. The special instruments needed for aspiration biopsy are an l&gauge needle 5 to 10 cm. in length, a 10 or a 20 C.C.syringe, glass slides for a smear, a small slice of Gelfoam or fibrin foam as a vehicle for some of the aspirated tissue, and a specimen bottle with 10 per cent formalin (Fig. I). The skin or mucous membrane at the site of puncture is painted with antiseptic solution and a small area is infiltrated with local anesthetic solution, raising a small wheal over the site selected for aspiration. With a sharp poi.nted scalpel a stab wound is made through t,he skin or mucous membrane. This
1036
JEROME
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ST001’.4Ct(
facilitates insertion of the needle and prevents contamination of the aspirated material by the surface epithelium. (Fig. 2.) If t,he tumor is contained within bone, a small round burr is used to penetrate the cortical plate following the initial incision through the surface epithelium. The la-gauge needle is then inserted through the stab incision, through the superficial tissues, or through the opening in the bone with the syringe attached and with the piston tightly closed (Fig. 3) until the needle is felt, to enter the tumor, With the needle held stationary, a vacuum is produced in the syringe by partly withdrawing the piston. Maintaining this vacuum, the needle is then advanced a distance of 1 to 3 cm., depending upon the size of the tumor, and then withdrawn to the starting point. This manipulation is repeated a few times changing the direction of the needle, remembering to maintain the vacuum at the same time. Here the needle is again withdrawn to the starting point and the vacuum slowly released. The syringe is then detached from the needle before complete withdrawal, and the needle, which supposedly cont,ains a specimen of tumor tissue, is withdrawn separately from the tissue. Here the needle is again attached to the syringe and the specimen expelled onto the previously prepared glass slide. A portion of the tissue is placed in the small piece of Gelfoam and then immersed in formalin for fixation and section by the usual method. The remaining portion on the glass slide is gently smeared by firm flat pressure of another glass slide, for immediate staining and study. The Punch Biopsy.-There are two types of punch biopsy to be discussed and the first was again originated by Dr. Hayes Martin, Chief of the Head and Neck Section at Memorial Hospital for Cancer and Allied Diseases. The instruments consist of a series of various-sized sharp, circular, detachable punches, resembling the ordinary leather punch (Fig. 4). The desired size punch is selected, attached to the handle, and after the selected area for biopsy has been infiltrated with a local anesthetic, the instrument is applied to the tissues and rotated back and forth several times, until it has penetrated to a depth of 5 to 7 mm. (Fig. 5). This frees the specimen from all of the surrounding tissue except at the base. The punch is put aside and the specimen is removed by grasping it carefully with the tissue forceps and severing it from the base with a scalpel or dissecting shears. It is then placed in the fixing solution. Persistent bleeding can be controlled by use of a coagulating agent such as silver nitrate, by use of the cotigulating current, or by sutures properly placed. The first method is used most often and with best results in the majority of cases. The second technique in this group is performed upon lesions or growths in areas difficult to get to by ordinary methods, such as tumors of the base or back of the tongue. The instrument used is a forceps type of tissue cutter (Fig. 6). The area is anesthetized and the cutting edges of the forceps are applied to the lesion in question and tissue snipped off in such a manner that a representative specimen is obtained. It is treated in the same manner as any other specimen by placing in the formalin bottle for future study. The Electrosurgical Technique.-This third method is mentioned only because some men still persist in its use. It presents the very obvious disadvantage
Fig
Fig.
Fig.
Fig. .-Ammamentarium for aspiration biopsy. lJnw lrft to right, ~w~lpel, kge, : all piece of Gelfoam. Fig. .-Stab incision through mucous membrane of the tongltc. .-The needle inserted through the stab incision with thv piston tightly Fig.
I
1038
Fig. 4.--S& devised by Dr. Hayes Martin, Hospital for Cancer and Allied Diseases, New 74: 107, 1947.)
Chief of the Head York, N. Y. (From Fig.
Fig. 5.-The instrument it has penetrated to a depth Fig. B.-This or similar oral cavity and surrounding
and Neck Section Martin, Hayes:
at Memorial Am. J Surg.
and
times
5.
Fig. 6. applied to the tissues, of 5 to 7 mm. instrument used for structures.
rotated
back
lesions
in
forth
difficult-to-reach
several
areas
until of
the
of cooking the tissues, even with the most judicious handling of the instrument by the operator. This results in cellular dist,ortion and destruction, making microscopic diagnosis very difficult. The only atlvant,age of electrocauter!: lit\ in it,s hemostatic qualities. tt can be twecl to cwnt tvl ht~morrliage during hiops\ mrvrtiolrcvl I)t’c\-iorlsl!-. by use of the coagulating current i1S
treatment
Fig. Fig.
7.-Typical has failed 8.-Area
Fig.
7.
Fig.
8.
tongue lesion in which carcinoma to clear it up within a two-week selected for biopsy outlinc,l.
must be ~wriwl.
ruled
out
first
if conservative
Surgical Excision.-This is the method of choice on all surface or superficially located growths of soft tissue or bone. Here again we find two types: (1) partial excision; (2) total escision or therapeutic> biopsy. Partial Excision.-Partial excision is indicated in large growths or lesions located on or just beneath the surface epithelium and in some smaller growths (Fig. 7). The reason for partial excision in these cases is obvious---some lesions
1040
JEROME
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of the mouth, found in systemic diseases, such as tuberculosis or syphilis, simulate neoplasms. Partial excision, correct diagnosis, and subsequent systemic treatment would eliminate the lesion readily. Total excision with the resulting large defect or deformity in cases of this type is certainly contraindicated. Fig.
Fig. Fig. Y.-Specimen the base. Fig. lO.-Suturing may aid the healing
carefully
grasped
with
9.
10. tissue
is unnecessary in most cases: process and control hemorrhage.
forceps, however,
lifted
and
one or two
dissected well-placed
away
from sutures
There is very little risk, in partial excision of surface lesions, of spreading disease or stimulating the growth of tumor cells because careful section does not break down the natural barriers in the surrounding normal tissue. Following selection of a representative region from which the specimen is to be obtained (Fig. 8)) the area should be infiltrated locally by injection deep to the lesion. A sharp, pointed scalpel should be used and a V-type of elliptical incision should be made. Thin, deep sections should be made rather than broad, shallow sections, and they should include normal tissue at the outer edge of the
BIOPSY
TECHSIQlVZS
Iii
ORSI,
Sil‘R(;ERS
1041
specimen. Following the initial incisions, the specimen should be very carefully grasped with tissue forceps at the normal tissue end, lifted and dissected away from the base (Fig. 9). It is then placed in the fixing solution. Suturing is usually unnecessary in partial excision; however, one or two well-placed sutures may aid in faster healing and controlling hemorrhage (Fig. 10).
Fig. Fig.
Il.-Total 1 %.-The
excision is initial incision
indicated should
Fig.
Il.
Fig.
12.
in lesions of this type. include a good border of normal
tissue
all
around.
Y’otnl Escision.-Total excision is indicat cd in small, surface lesions or sr1d1 growths of the superficial connective tissue layer (Fig. 11). The lesion is well outlined with an elliptical incision following local anesthesia, obtained in t,he same manner as described previously. The initial incisions should include a, good border of normal tissue all around (Fig. 12). The tissue is carefully lifted at one end with the tissue forceps and the growth carefully removed by means of a dissecting shears or scalpel, maintaining a good border of normal t,issue beneath the growth as well as at the sides. Persistent bleeders arc tied off with catgut or are coagulated.
1042
JEROME
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All tension on the surface sutures is relieved and the incision is closed. This can be accomplished by undermining the surface edges of the wound and then closing or by closing in layers with subcutaneous absorbable sutures. The excised growth in toto is then treated as an ordinary biopsy section and placed in the fixing solution. As a variation in the total excision technique in areas of rich vascularity such as the tongue, it is well to consider running the sutures through before excision. The sutures are placed wide and deep to the ,lesion or growth, following local anesthesia of the area. The incisions and excision are made carefully so that the sutures are not cut and the wound immediately closed subsequent to removal of the growth.
Preparing the Specimen for Study.-To avoid drying out of the specimen, the bottle of fixing solution for receiving the section should be prepared prior to completion of the operation. The bottle should be carefully labeled with the patient’s name and also with an identifying number corresponding to the number on the tissue examination request. Ten per cent formalin is used as the fixing solution; the amount of solution should be approximately twenty times the volume of the tissue. If formalin is not available, 70 per cent alcohol can be used as a poor substitute, for shrinkage of the tissue results with its use, due to dehydration. It is desirable to have a half dozen or more prepared formalin bottles on hand. The Tissue Examination Request Form.-It is important that the tissue examination request include all the information one can give that will be of use to the pathologist. The data should be checked over before the form is passed along, with the specimen. (Fig. 13.) The request should include the following : 1. 2. 3. 4. 5. 6. 7.
Doctor submitting specimen and address. Date obtained. Patient’s age. Patient’s sex. Patient’s race. Clinical description of the lesion before excision. Description of the specimen following excision.
(The last two should be a written report of the clinical appearance of the lesion and surrounding tissues. The report should be an exact description of what one actually sees, including the size, shape, color, whether raised or not, whether soft or hard, and whether ulceration and/or hemorrhage is or ever has been in evidence. ) 8. How specimen was obtained. 9. Brief clinical history. (Includes duration of the lesion, rapidity of growth, physical condition of the patient, and one’s clinical impression or diagnosis.) 10. Patient’s name and address.
Laboratory Procedures.--&zation is accomplished by placing the specimen in the preservative, 10 per cent formalin. The length of time for fixation varies
BIOPSY TECHKIQI'ES
CLINICAL RECORD
I?; ORAl, SL!R(;ERY
TISSUE EXAMINATION I DATE OBTAlNED 4 January 1951
SPEClMEN *lJBMlTTEo BY
Dr. John Doe PATIENT'SMAE sPEC'MENConsists of a yellow round shiny 20 piece of tissue about 2 cm in diameter. It is fairly firm to touch but oan be rather
HOW
OBTIINEO
Speciumn was excised surgically incision and blunt dissection.
su lb
RACE MALE 0 FEMALE
easily
w
-- .-
depressed.
in toto by means of anellf~ticslskin
WuEF C~~~l~~~l~~~~~~~~~~~~~~~~,~a
gpyyom
lmer
----border
of
mandible, 1.5 om in diameter. Is freely movable under epidermis, fibrous No pain, discharge, ulceration or discoloration. Is (hard) in nature. irritated upon occasion by shaving. Began in 1946, grew to its present size associated rith its growth. in period of a few days. No disease of infection Es8 history of cyst removal on both cheeks and behind both ears, otherwise history is irrelevant. Impression: cyst (sebaceous) FLocC"LATIONREACTION COMPLEMENT I=,XAT,ON SlONATURE ANDTkTLE
Grossr An ellipse of skin measuring 2.0x0.6 cm. and attached below it is a large grayish yellow structure resumbling a large kidney bean. It has a smooth glistening surfaoe and is of rather soft consistency. It msa8uree 2.0x1.5x0.6 cm.Cross section of the cystic-like structure reveals that it is filled with a cheesy yellow material. Micror Sections show skin on one surface and below it is a large cyst lined by oompressed etratified aqasmaua epitheliuaL The oyst is filled with desquaamted keratin. The mall of the cyst is very thin being composed of only a few strands of fibroua connective tirsue. No aeoondary skin struotures A sebaoeoua oyst and an epidermoidal cyst are are evident in the uall. usually impossible to tell apart, Because of lack of M&&gic evidence to the contrary the diagnosis of epidenuoidal cyst is favored. No neoplasm. Diagnosis:
Fig.
Epidermoidal
1X.--Tissue
examination
cyst, neck.
request form
usecl in the United
States
Navy.
JEROME
CHESTER
STOOPACK
with the size of the tissue; however, the tissue is left in the formalin solution for from six to twenty-four hours. This coagulates the protein content of the cells, thus preventing decomposition and preserving the tissue elements in as nearly the same condition that they were in at the time of fixation. Hard tissue specimens have to be decalcified before fixing. An acid such as nitric or hydrochloric is used for this process, usually 5 per cent nitric acid. Following decalcification it is treated as a soft tissue specimen and embedded in either celloidin or paraffin. Dehydration is accomplished by placing the specimen in a series of ascending alcohols, 50, 70, 95, and then 100 per cent alcohol, over a period of twentyfour hours in an especially timed machine for the purpose. Embedding.-In order that the tissue may be sectioned by the microtome, it has to have a certain rigidity to offer sufficient resistance to the cutting edge of the knife. This may be accomplished by freezing the tissues, or, as is more commonly done, by using a material which fills the interstitial spaces of the tissue, such as paraffin or celloidin. The freezing technique is used only where immediate investigation is necessary during the course of a surgical operation, for instance, questionable deep tongue biopsies ; while the patient is on the operating table, a small section is excised, sent to the laboratory, where it is quickly frozen, sectioned, examined, If the report is negative for malignancy, the rest of the tumor and diagnosed. is removed and the wound closed. However, if the report is positive, a radical tongue dissection and a possible mandibular resection on that side are performed immediately. Embedding in paraffin is a more lengthy but a far more satisfactory process, retaining the tissue in a much more adequate form for microscopic diagnosis without the distortion that is found in the freezing technique. Since the paraffin is not soluble in alcohol, the alcohol already in the tissues as a result of the dehydration process has to be replaced by xylol or chloroform, to enable the paraffin to be taken up by all of the tissue spaces. The specimens are left in two changes of solvent for a specified length of time and then paraffin is added to the solution. They remain in this solution for a few hours and then are placed in molten paraffin for another specified time. Finally they are placed in a mold filled with molten paraffin, and this is quickly cooled into a solid block. Celloidin is used in hard tissue sections containing teeth or parts of teeth. A mixture of equal parts of ether and alcohol is used as the solvent here, to fill the tissue spaces with celloidin. This type of section requires at least six weeks and as much as three months to prepare. Sectioning is accomplished by means of the microtome. types: the sliding, the rotary, and the freezing depending tissue and the embedding medium used.
There are three upon the type of
Mounting consists of placing the cut specimens on glass slides and drying on an electrically controlled heater. Staining.-Hematoxylin and eosin are uSed universally for staining in microscopic diagnosis, and consist of an initial blue stain which is followed by
partial decolorizat,ion with acid-alcohol solution ant1 then counterstaining wit11 red rosin. Aster clearing with alcohol and sylol, ant1 thorough drying:. the sliclcs is given added protection bp means of a cover glass, itnd then is ready f~)r tllc. last step. Microscopic Eznmi~ation.-Following microscopic examination sia hp the pat,hologist, his report is returned to the tloctor submitting and thus the cyc!c is completed.
and tliagno the reqncsi (
are some precautions and conin Biopsy.-Following that should be observed to insure full diagnostic and sometime:; value of the biopsy, and to avoid unnecessary di&urement of the,
Do’s and Don’t’s siderations. therapeutic patient :
1. Acoid the use of dyes: The dyes should not he used as local antiseptics for procaine infiltration, as they prevent proper preparation of the tissue for Antiseptics used should he colorless! although solutions microscopic study. with a slight amount of color such as Metaphen are permissible. 2. Include a representatiLqe av-ea of the lesion or growth: Thin, deep sections should be obtained in partial or punch biopsy rather than broad shallow ones. Superficial tissue in cases of malignancy may only show inflammatory reactions without actually getting deep enough int,o the connective tissue layer to show tumor cell invasion. Many negative reports are made in error, because of the lack of depth of the section. 3. Include an adequate border of nom& tissue: In partial biopsies, an area of normal tissue is essential for purposes of comparison in microscopic study. Material from the center of the lesion only is frequently not of valuct because of secondary changes or infection which greatly affect the microscopic picture. In total biopsies, a good border of normal t,issue should be included all around and beneath the lesion. The pathologist should be able to report adequate excision into normal tissue in cases where microscopic diagnosis proves the lesions to be premalignant or malignant. In epithelial lesions, the normal border often gives indication of whether the lesion is malignant or not. In benign epidermoid lesions, the epithelium appears pushed up above t,he line of normal epithelium, whereas in the epidermoid malignancies, the epithelium of the lesion is invasive and appears below the line of normal epithelium and well into t,he underlying connective t,issue, 1. Plan the incisions to avoid or mininzize disfiguring the patient: If the lesion from which a biopsy is to be taken is on the face or lips, the cosmetic aftereffect has to be taken into consideration: a. Follow the natural creases (Langer’s lines of tension), if operating on the face. b. Avoid reducing the vermilion border, if operating on the lips. The use of vertical incisions should be considered here. 5. Avoid the use of the electrocautery: If, of necessity, cautery must used, extreme care should be exercised to minimize cooking the tissues.
be
1046
JEROME
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6. Use care in the use of the tissue forceps: Careless handling of the tissues can crush or otherwise mutilate the specimen. This results in the appearance microscopically of large holes or of flatt,ened tissues. The specimen should be carefully held at one end with a fine forceps during excision. 7. Identify the area, if possible, in suspicious lesions: In these lesions a suture through a portion of the tissue is of great value. Placed anteriorly or posteriorly, or above or below, for that matter, a suture in a specimen that is found by the pathologist to be premalignant or malignant can often be a check on the adequacy of the excision. If the lesion has not been adequately excised, the pathologist can accurately locate for the doctor that area of the lesion by means of this previously placed suture, and the doctor can proceed accordingly without guesswork. 8. Avoid drying out of the specimen: Too frequently excised tissue is placed on the bracket table and left exposed until the operation is completed. As little as ten minutes in an area with an arid climate could cause enough cellular distortion to make microscopic diagnosis impossible. Therefore, place the specimen directly into the formalin solution following excision. 9. Use a separate bottle for each specimen: If specimens are taken from lesions in different areas on the same patient, they should be placed in separate bottles. Great confusion results if one specimen out of two or more in the same bottle proves to be premalignant, or malignant, and the pathologist cannot tell the doctor which area of the mouth is affected. 10. Accompany the specimen with a complete report: Use a comprehensive report form and fill in all pertinent, information. 11. Do not accept all negative reports: One negative report of a suspicious lesion is not necessarily diagnostic of a benign lesion. A negative report should not lull the operator into a feeling of security, when a lesion has a clinical course indicating malignant tendencies. Repeat biopsies are indicated in all There have been persistent eases in which several such suspicious lesions. negative reports on the same lesion have been followed by a positive report of malignancy. References 1. Bernier,
J. L.: An Evaluation of the Biopsy as a Diagnostic Aid, Am. J. Orthodontics and Oral Surg. (Oral Surg. Sect.) 32: 531-542, 1946. 2. Bernier, Joseph L., and Tiecke, R. W.: Biopsy, J. Oral Surg. 8: 342-348, 1950. 3. Chilton.26: N. W.: 1949.Biousv of Cancer of the Mouth, J. New Jersey I D. Sot. - ” in Diaenosis 13-15, 4. Dingman, R. 0.: Importance of Biopsy in Oral Diagnosis, J. Oral Surg. 6: 204-208, 1948. Diagnosis of Oral Tumors, J. Oral Surg. 7: 2175. Dingman, R. O., and Hayward, J. R.: 230. 1949. 6. Martin, ‘Hayes E.: Biopsy Drill Punch, Am. J. Surg. 74: 107-108, 1947. 7. Martin, Hayes E., and Ellis, E. B.: Aspiration Biopsy, Surg., Gynec. & Obst. 59: 578-589, 1934. 8. Morrey, C. W.: Responsibility of the Dentist in Mouth Cancer Detection, J. Am. Dent. A. 38: 452-453, 1949. 9. Orban, Balint: Oral Histology and Embryology, ed. 2, St. Louis, 1949, The C. V. Mosby Company. 10. Smith, J. B.: Surgical Management of Cancer of the Mouth, J. Oral Surg. 7: 95-103, 1949.
64 CLOVERFIELD
ROAD SOUTH,
VALLEY
STREAM,
N. Y.