Anatomical and pathological basis of visual cervix

Chapter 1

Anatomical and pathological basis of visualinspection with acetic acid (VIA) and with

Lugol’s iodine (VILI)

IntroductionNaked-eye visual inspection of theuterine cervix, after application of 5%acetic acid (VIA) and/or of Lugol’s iodine(VILI), provides simple tests for the earlydetection of cervical precancerous lesionsand early invasive cancer. VILI is similarto the Schiller’s iodine test, which wasused for early detection of cervicalneoplasia in the third and fourth decadesof the 20th century, but discontinued afterthe advent of cervical cytology testing.The potential difficulties in implementingcervical cytology-based screening in low-resource settings have prompted theinvestigation of the accuracy ofalternative low-technology tests such asVIA and VILI in the early detection ofcervical neoplasia.

The results of VIA and VILI areimmediately available and do not requireany laboratory support. The categorizationof the results of VIA or VILI depends uponthe colour changes observed on the cervix.A clear understanding of the anatomy,physiology and pathology of the cervix isabsolutely essential to understand thebasis and to interpret the outcome ofscreening using VIA and VILI. The objectiveof this manual is to help a range of health-care providers such as doctors, nurses,midwives and health workers to acquire

the skills and competence in administeringand reporting the results of these tests bydescribing their basis and practice.

Gross anatomy of the uterinecervixThe cervix, constituting the lower portionof the uterus, is cylindrical or conical inshape, and measures 3-4 cm in length and2.5-3.5 cm in diameter. It varies in size andshape depending on the age, parity andhormonal status of the woman. The lowerhalf of the cervix, called portio vaginalis,protrudes into the vagina through itsanterior wall, and the upper half, calledthe supravaginal portion, remains abovethe vagina (Figure 1.1). The cervix opensinto the vagina through the external os.The supravaginal portion meets the bodyof the uterus at the internal os. In parouswomen, the cervix is bulky and theexternal os appears as a wide, gaping,transverse slit. In nulliparous women, theexternal os resembles a small circular(pinhole) opening.

The portion of the cervix that isexterior to the external os is called theectocervix, which is readily visible duringspeculum examination. The portionabove the external os is called theendocervix. The endocervical canal,which traverses the endocervix, connects

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the uterine cavity with the vagina andextends from the internal to the externalos. The portion of the upper vaginalcavity that surrounds the portio vaginalisis called the fornix.

The stroma of the cervix is composed ofdense, fibro-muscular tissue traversed bythe vascular, lymphatic and nerve

supplies to the cervix. The arteries of thecervix, derived from internal iliac arteriesthrough the cervical and vaginal branchesof the uterine arteries, descend in thelateral aspects of the cervix at 3 and 9o’clock positions. The veins run parallelto the arteries and drain into thehypogastric venous plexus. The lymphatic

A Practical Manual on Visual Screening for Cervical Neoplasia

Endocervical canal

Portio vaginalis

Cervix

Bladder

Anterior fornix

Pubic bone

Urethra

FIGURE 1.1: Gross anatomy of the uterine cervix

Fundus

Fallopian tube

Body of uterus

Supravaginal cervix

Internal os

Endocervix

Lateral fornix

External os

Ectocervix

Vagina

Uterus

Posterior fornix

Rectum

Sacrum

Vagina

vessels from the cervix drain into thecommon, internal and external iliacnodes, obturator and the parametrialnodes. The nerve supply is derived fromthe hypogastric plexus. The endocervixhas extensive sensory nerve endings,while there are very few in theectocervix. Hence, procedures such asbiopsy and cryotherapy are well toleratedin most women, without localanaesthesia. Since sympathetic andparasympathetic fibres are also abundantin the endocervix, manipulation of theendocervix may stimulate these nerveendings, occasionally leading to giddiness

or fainting attacks.Microscopic anatomySquamous epitheliumThe cervix is covered by two types ofepithelium, stratified squamousepithelium and columnar epithelium,which meet at the squamocolumnarjunction. A large area of ectocervix iscovered by the stratified, non-keratinizing, glycogen-containingsquamous epithelium. It is opaque, hasmultiple (15-20) layers of cells (Figure 1.2)and appears pale pink in colour on visualexamination. It consists of a single layer ofround basal cells with a large dark-staining

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Chapter 1

FIGURE 1.2: Stratified squamous epithelium (x20)

Superficial cell layer

Parabasal layer

Basal cell layer

Intermediate cell layer

Stromalpapilla

StromaBasement membrane

FIGURE 1.3: Columnar epithelium (x40)

Columnar cells

Stroma

Basement membrane

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nucleus and little cytoplasm at thebasement membrane. The basementmembrane separates the epithelium fromthe underlying stroma. The basal cells

divide and differentiate to form theparabasal, intermediate and superficiallayers. From the basal to the superficiallayer, the cells undergo an increase in


FIGURE 1.4: Location of squamocolumnar junction (SCJ)(a) Original squamocolumnar junction (SCJ) in a young woman in the early reproductive age group. The SCJ is located

far away from the external os. Note the presence of everted columnar epithelium occupying a large portion of the

ectocervix producing ectropion.

(b) The new SCJ has moved much closer to the external os in a woman in her 30s. The SCJ is visible as a distinct

white line after the application of 5% acetic acid due to the presence of immature squamous metaplastic

epithelium adjacent to the new SCJ.

(c) The new SCJ is at the external os in a perimenopausal woman.

(d) The new SCJ is not visible and has receded into the endocervix in a postmenopausal woman. Mature metaplastic

squamous epithelium occupies most of the ectocervix.

Ectropion External os

Columnarepithelium

Original SCJ

Originalsquamousepithelium

Metaplasticsquamousepithelium

New SCJ

Columnarepithelium

External os

Mature metaplasticsquamous epithelium

External os New SCJ External os Mature metaplastic squamousepithelium

a b

c d

cytoplasm and a reduction of nuclear size.The intermediate and superficial layercells contain abundant glycogen in theircytoplasm. Since iodine readily stains withglycogen, application of Lugol’s iodine onthe squamous epithelium will result inmahogany brown or black coloration. Inpostmenopausal women, the cells in thesquamous epithelium do not maturebeyond the parabasal layer, and do notaccumulate as multiple layers ofintermediate and superficial cells.Consequently, the squamous epitheliumbecomes thin and atrophic. Thus, itappears pale and brittle, with sub-epithelial petechiae, as it is easily proneto trauma.

Columnar epitheliumThe endocervical canal is lined by thecolumnar epithelium (sometimes referredto as glandular epithelium), composed of asingle layer of tall cells with dark-stainingnuclei (Figure 1.3). On visual examination,it appears as a grainy, strikingly reddisharea because the thin single cell layerallows the coloration of the underlyingstroma to be seen more easily. It formsseveral invaginations into the substance ofthe cervical stroma, resulting in theformation of endocervical crypts(sometimes referred to as endocervicalglands). The columnar cells secrete themucus that lubricates the cervix andvagina. At its upper limit, it merges withthe endometrial epithelium in the body ofuterus, and at its lower limit, it meets withthe squamous epithelium at thesquamocolumnar junction. A localizedproliferation of the columnar epithelium inthe form of a polyp may occasionally bevisible as a reddish mass protruding fromthe external os (Figure 1.5). As columnarepithelium does not produce glycogen, it

does not change colour after theapplication of Lugol’s iodine, or remainsslightly discoloured with a thin film ofiodine solution.

Squamocolumnar junctionThe squamocolumnar junction (Figure 1.4)appears as a sharp line. The location of thesquamocolumnar junction in relation to theexternal os varies, depending upon factorssuch as age, hormonal status, birth traumaand certain physiological conditions such aspregnancy (Figure 1.4). During childhoodand perimenarche, it is located at, or veryclose to, the external os. After puberty andduring the reproductive period, the femalegenital organs grow under the influence ofestrogen. Thus, the cervix enlarges and theendocervical canal elongates. This leads tothe eversion of the columnar epitheliumonto the ectocervix, particularly on theanterior and posterior lips, resulting inectropion or ectopy. Thus, thesquamocolumnar junction is located in theectocervix, far away from the external osduring the reproductive years andpregnancy (Figure 1.4a). On visualinspection, ectropion is seen as a strikinglyreddish ectocervix (Figure 1.4a).

The buffer action of the mucus coveringthe columnar cells is interfered with whenthe everted columnar epithelium is exposedto the acidic vaginal environment. Thisleads to the destruction and eventualreplacement of the columnar epithelium bythe newly formed metaplastic squamousepithelium. Metaplasia refers to the changeor replacement of one type of epitheliumby another. As a woman passes through herreproductive life to the perimenopusal agegroup, the location of the squamocolumnarjunction progressively starts moving on theectocervix towards the external os (Figures1.4b and c). Thus, it is located at variable

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distances from the external os, as a resultof the progressive formation of the newmetaplastic squamous epithelium in theexposed areas of the columnar epitheliumin the ectocervix. From the perimenopausalperiod and after the onset of menopause,the cervix shrinks, due to the lack ofestrogen, and, consequently, the movement

of the squamocolumnar junction towardsthe external os and into the endocervicalcanal is further accelerated (Figure 1.4c).In postmenopausal women, the squamo-columnar junction is located in theendocervical canal and, hence, oftencannot be seen on visual examination(Figure 1.4d).


FIGURE 1.5: Development of squamous metaplastic epithelium(a) The arrows indicate the appearance of the subcolumnar reserve cells.

(b) The reserve cells proliferate to form two layers of reserve cell hyperplasia beneath the overlying layer of columnar

epithelium.

(c) The reserve cells further proliferate and differentiate to form immature squamous metaplastic epithelium. There is

no evidence of glycogen production.

(d) Mature squamous metaplastic epithelium is indistinguishable from the original squamous epithelium for all

practical purposes.

Immature squamous metaplasia Immature squamousmetaplastic epithelium

Original squamousepithelium

a b

c d

(x40) (x20)

(x10) (x10)

Mature squamousmetaplastic epithelium

Squamous metaplasiaThe earliest event in squamous metaplasiais the appearance of small, round, sub-columnar cells in the exposed areas of thecolumnar epithelium, called reserve cells(Figure 1.5a). These reserve cellsproliferate (Figure 1.5b) and differentiateto form a thin, non-stratified, multicellularepithelium called immature squamousepithelium (Figure 1.5c). The cells in theimmature squamous metaplasticepithelium do not produce glycogen and,hence, do not stain brown or black withLugol’s iodine solution. Numerous foci ofimmature squamous metaplasia may ariseat the same time.

Further development of the newly formedimmature metaplastic epithelium may takeeither one of two directions (Figure 1.6). Inthe vast majority of women, it developsinto a mature, stratified, glycogen-producing, squamous metaplasticepithelium, which is similar to thesquamous epithelium found on theectocervix, for all practical purposes(Figure 1.5d). Thus, it stains brown or blackafter the application of Lugol’s iodine.Several cysts, called nabothian cysts, maybe observed in the mature metaplasticsquamous epithelium (Figure 2.3). Theseare retention cysts that develop as a resultof the occlusion of the crypt openings in thetrapped columnar epithelium by theoverlying metaplastic squamousepithelium. The buried columnarepithelium in the cysts may continue tosecrete mucus, which eventually distendsthe cysts. The entrapped mucus gives anivory-white hue to the cyst on visualexamination.

In a very small minority of women, theimmature squamous metaplasia may turninto a dysplastic epithelium (an altered

epithelium showing precancerous cellularchanges), due to infection with certainhuman papillomavirus (HPV) types(Figure 1.6).

Transformation zoneThe transformation zone is the area ofthe cervix where the columnar epitheliumhas been replaced and/or is beingreplaced by the metaplastic squamousepithelium. With the naked eye, one canidentify the inner border of thetransformation zone by tracing thesquamocolumnar junction and the outerborder by locating the distal mostnabothian cysts (if present) or cryptopenings (usually visible undermagnification). In premenopausalwomen, the transformation zone isprimarily located on the ectocervix. Aftermenopause, and through old age, thecervix shrinks with the decreasing levelsof oestrogens. Consequently, thetransformation zone may move partially,

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Chapter 1

Columnar epithelium

Immature squamous metaplasia

Infection with

oncogenic HPV types

Atypical or dysplasticsquamous epithelium

Normal glycogencontaining mature

squamous metaplasticepithelium

FIGURE 1.6: A schematic diagram of further maturation of immature squamousmetaplasia.

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and later fully, into the endocervicalcanal. Almost all cervical neoplasia occursin this zone, close to the squamocolumnarjunction.

Inflammation of the uterine cervix(Figure 1.7)The most common pathological conditionaffecting a woman’s cervix is inflammation.This is caused mostly by infection (usuallypolymicrobial) and, less commonly, byforeign bodies (retained tampon, etc.),trauma and chemical irritants such as gelsand creams. The infectious agents causinginflammation in the cervix include:Trichomonas vaginalis; Candida albicans;overgrowth of anaerobic bacteria such asGardnerella vaginalis, G. mobilluncus andpeptostreptococcus; other bacterialinfections such as Haemophilus ducreyi,Neisseria gonorrhoeae, Chlamydia

trachomatis, Escherichia coli, streptococci,and staphylococci; and viral infections suchas Herpes simplex.

Columnar epithelium is more prone toinfection than squamous epithelium. Weuse the term cervicitis in this manual todenote all cervicovaginal inflammatoryconditions. Clinically, cervicitis may beassociated with symptoms such asexcessive discharge, itching of the vulvaand vagina, pain and a burning sensationduring sexual intercourse and lowerabdominal pain. Clinical signs includeexcessive, coloured (greyish, greyish-white, curdy-white (in the case of candidialinfection), yellow or greenish-yellow),malodorous or non-odorous, frothy or non-frothy secretions, tender, reddish cervixwith or without vesicles, ulcerationsand/or fibrosis; the columnar epitheliummay look flattened; and there may be


FIGURE 1.7:(a) An inflamed cervix, with ulceration, bleeding, necrosis, greenish-yellow discharge and inflammatory exudate.

(b) A reddish angry-looking, inflamed cervix with loss of the villi in the columnar epithelium and covered with

inflammatory exudate.

a b

excoriation marks on the vulva, vulvalerythema and oedema, vagina and innerthigh and perineum. Microscopically,cervicitis is characterized by cellular debrisand excessive secretions covering theepithelium, swollen and inflamed cells,desquamation of the glycogen-containingsuperficial and intermediate cells,epithelial denudation, superficial or deepulceration and congestion of the underlyingcervical stroma. Chronic inflammationresults in recurrent ulceration and maylead to healing by fibrosis.

A diagnosis of cervicitis can be madebased on the clinical features. On visualexamination, cervicitis due to non-candidial infection may be characterizedby vulval erythema and oedema,excoriation marks in the vulva and vaginaand a reddish, tender cervix withmalodorous, greenish yellow or greyish-white mucopurulent discharge, with orwithout ulceration. In the case ofgonococcal cervicitis, painful urethraldischarge is also observed. Candidialcervicitis is characterized by vulval oedemaand erythema, excoriation, and thick,curdy-white, non-odorous discharge.Herpes infection is associated with thepresence of vesicles and ulcers in theexternal genitalia, vagina and the cervix,

as well as cervical tenderness. Women withnon-candidial cervicitis may be treatedwith a combination of metronidazole 400mg plus doxycycline 100 mg orally, twotimes a day for seven days. Those withcandidial cervicitis may be treated withclotrimazole or micanazole 200 mgintravaginally, daily for three days.

Cervical neoplasiaInvasive cervical cancers are usuallypreceded by a long phase of preinvasivedisease, characterized microscopically as aspectrum of precursor lesions progressingfrom cellular atypia to various grades ofcervical intraepithelial neoplasia (CIN)before progression to invasive carcinoma.Epidemiological studies have identified anumber of risk factors that contribute tothe development of CIN and cervicalcancer. These include infection withcertain types of human papillomavirus(HPV), sexual intercourse at an early age,multiple sexual partners, multiparity, long-term oral contraceptive use, tobaccosmoking, low socioeconomic status,infection with Chlamydia trachomatis,micronutrient deficiency and a dietdeficient in vegetables and fruits. HPVtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, 59 and 68 are strongly associated with

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Chapter 1

CIN 1

Mild dysplasia

Low-grade squamousintraepithelial lesion (LSIL)

CIN 2

Moderate dysplasia

High-grade squamousintraepithelial lesion (HSIL)

CIN 3

Severe dysplasiaCarcinoma in situ

High-grade squamousintraepithelial lesion (HSIL)

Table 1: Correlation between CIN, dysplasia and the Bethesda

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CIN and invasive cancer. Persistentinfection with one or more of the aboveHPV types is considered to be a necessarycause for cervical neoplasia.

Infection with one or more of theoncogenic HPV types may result in theintegration of the viral genome into thehost cellular genome resulting in theformation of cervical neoplastic cells, theproliferation of which leads to variousgrades of CIN (synonyms: dysplasia orsquamous intraepithelial lesions (SIL)),which may progress to invasive cervicalcancer. The correlation between the CINterminology, used in this manual, and otherterminologies is given in Table 1.

Cervical intraepithelial neoplasiaThere are no specific symptoms or visiblesigns associated with CIN. However, thepresence of CIN may be suspected by thenaked-eye detection of well defined,acetowhite areas in the transformationzone, close to or abutting thesquamocolumnar junction, after theapplication of 3-5% acetic acid or of welldefined mustard or saffron yellow iodinenon-uptake areas in the transformation zone

after application of Lugol’s iodine solution. The final diagnosis of CIN is established


FIGURE 1.9: Histology of CIN 2: Atypical cells are foundmostly in the lower two-thirds of theepithelium x10.

FIGURE 1.10: Histology of CIN 3: Dysplastic cells aredistributed in the full thickness of theepithelium with loss of polarity of cells x20.

FIGURE 1.8: Histology of CIN 1: The dyplastic cells areconfined to the lower third of the epitheliumx20.

a b

by histopathological examination of tissuespecimens from the cervix. The undifferen-tiated cells in CIN are characterized byenlarged nuclei, increased intensity ofnuclear staining, nuclear polymorphismand variation in nuclear size, and adecreased amount of cytoplasm, resultingin a higher nuclear cytoplasmic ratio. Theproportion of the thickness of theepithelium showing undifferentiated cellsis used for grading CIN. In CIN 1 the undif-ferentiated cells are confined to thedeeper layers (lower third) of theepithelium (Figure 1.8). Mitotic figures arepresent, but not very numerous. CIN 2 ischaracterized by dysplastic cellularchanges mostly restricted to the lower halfor the lower two-thirds of the epithelium,with more marked nuclear abnormalitiesthan in CIN 1 (Figure 1.9). Mitotic figuresmay be seen throughout the lower half ofthe epithelium. In CIN 3, differentiationand stratification may be totally absent orpresent only in the superficial quarter ofthe epithelium with numerous mitoticfigures (Figure 1.10). Nuclearabnormalities extend throughout thethickness of the epithelium. Many mitoticfigures have abnormal forms.

It is well established that most CIN 1lesions are transient; most of them regressto normal, within relatively short periods,or do not progress to higher grades. High-grade CIN (CIN 2-3), on the other hand,carries a much higher probability ofprogressing to invasive cancer, although alarge proportion of such lesions also regressor persist. It is assumed that the meaninterval for progression of cervicalprecursors to invasive cancer may be aslong as 10 to 20 years.

Women with CIN are treated withcryotherapy, loop electrosurgical excisionprocedure (LEEP) or cold-knife conization.

Women with CIN 1 may be advised toundergo immediate treatment (e.g., insituations where follow-up of womencannot be assured) or treated later if twofollow-up visits at six or nine months apartreveal persistent or progressive disease.

The precursor lesion that arises from thecolumnar epithelium is referred to asadenocarcinoma in situ (AIS). In AIS,normal columnar epithelium is replaced byabnormal epithelium showing abnormal,irregularly arranged cells with increasedsize of cells and nuclei, nuclearhyperchromasia, mitotic activity, reductionof cytoplasmic mucin expression andcellular stratification.

Invasive cancerIn very early phases of invasion, cervicalcancer may not be associated with obvioussymptoms and signs, and, therefore, isknown as preclinical invasive cancer.Women with moderately advanced oradvanced invasive cervical cancer oftenpresent with one or more of the followingsymptoms: intermenstrual bleeding,postcoital bleeding, excessive seropurulentdischarge, recurrent cystitis, backache,lower abdominal pain, oedema of the lowerextremities, obstructive uropathy, bowelobstruction, breathlessness due to severeanaemia and cachexia.

As the stromal invasion progresses, thedisease becomes clinically obvious,showing several growth patterns, whichare visible on speculum examination.Early lesions may present as a rough,reddish, granular area that bleeds ontouch (Figure 1.11) More advancedcancers may present as a proliferating,bulging, mushroom- or cauliflower-likegrowth with bleeding and foul-smellingdischarge (Figure 1.12). Occasionallythey may present without much surface

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growth, resulting in a grossly enlarged,irregular cervix with a rough, granularsurface.

As the invasion continues further, it mayinvolve the vagina, parametrium, pelvicsidewall, bladder and rectum. Compressionof the ureter, due to advanced local disease,causes ureteral obstruction, which results inhydronephrosis and, ultimately, renalfailure. Regional lymph node metastasisoccurs along with local invasion. Metastaticcancer in para-aortic nodes may extendthrough the node capsule and directlyinvade the vertebrae and nerve rootscausing back pain. Direct invasion of thebranches of the sciatic nerve roots causeslow back pain and leg aches, andencroachment of the pelvic wall veins andlymphatics causes oedema of the lowerlimbs. Distant metastases occur late in thedisease, usually involving para-aortic nodes,lungs, liver, bone and other structures.

Histologically, approximately 90-95% ofinvasive cervical cancers in developing

countries are squamous cell cancers(Figure 1.13) and 2-8% are adenocarcinomas(Figure 1.14). It is obligatory that allinvasive cancers be clinically staged. Themost widely used staging system forcervical cancer was developed by theInternational Federation of Gynecology andObstetrics (FIGO) (see Appendix 1). This isprimarily a clinical staging system based ontumour size and extension of the disease inthe pelvis. The extent of growth of canceris assessed clinically, as well as by variousinvestigations, to categorize the diseasestages I through IV. Stage I representsgrowth localized on the cervix, while stageIV represents the growth phase in which thecancer has spread to distant organs bymetastasis.

Women with early invasive cancers(stages I and II A) may be treated withradical surgery and/or radiotherapy.Those with stage IIB and III cancers may betreated with radiotherapy with or withoutcisplatinum-based chemotherapy. Women


FIGURE 1.11: Early invasive cervical cancer: note theirregular, granular, nodular surface withbleeding on touch.

FIGURE 1.12: Advanced invasive cervical cancer: note thebulging, cauliflower-like, ulceroproliferativegrowth with bleeding and necrosis.

with stage IV cancers are usually treatedwith palliative radiotherapy and/orchemotherapy and with symptomaticmeasures.

Other conditionsLeukoplakia (hyperkeratosis) is a welldemarcated white area on the cervix(before the application of acetic acid), dueto keratosis, visible to the naked eye.Usually leukoplakia is idiopathic, but it mayalso be caused by chronic foreign bodyirritation, HPV infection, or squamousneoplasia. Condylomata or genital wartsare often multiple, exophytic lesions thatare usually found on the cervix, andoccasionally in the vagina and on the vulva,caused by infection with certain HPV typessuch as 6 and 11. They may also present asa diffuse, greyish-white lesion involvingareas of the cervix and vagina.Condylomata may be obvious to the nakedeye (before the application of acetic acid).

Pathophysiological basis of VIA Application of 5% acetic acid is believed tocause a reversible coagulation, or

precipitation of the cellular proteins. Italso causes swelling of the epithelialtissue, columnar and any abnormalsquamous epithelial areas in particular,dehydration of the cells, and it helps incoagulating and clearing the mucoussecretions on the cervix. The normalsquamous epithelium appears pink and thecolumnar epithelium red, due to thereflection of light from the underlyingstroma, which is rich in blood vessels. If theepithelium contains a lot of cellularproteins, acetic acid coagulates theseproteins, which may obliterate the colourof the stroma. The resultingacetowhitening is seen distinctly ascompared with the normal pinkish colour ofthe surrounding normal squamousepithelium of the cervix, an effect that iscommonly visible to the naked eye. Thus,the effect of acetic acid depends upon theamount of cellular proteins present in theepithelium. Areas of increased nuclearactivity and DNA content exhibit the mostdramatic white colour change.

When acetic acid is applied to normalsquamous epithelium, little coagulation

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Chapter 1

FIGURE 1.13: Histology – Keratinizing well differentiatedinvasive squamous cell carcinoma. Notethe stroma is infiltrated by sheets of malignantcells x10.

FIGURE 1.14: Histology – Well differentiated invasiveadenocarcinoma. Note the malignant cellslining the cervical crypts x20.

↓

↓

↓ ↓

↓↓

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occurs in the superficial cell layer, as this issparsely nucleated. Although the deepercells contain more nuclear protein, theacetic acid may not penetrate sufficientlyand, hence, the resulting precipitation isnot sufficient to obliterate the colour ofthe underlying stroma. Areas of CIN andinvasive cancer undergo maximalcoagulation due to their higher content ofnuclear protein (in view of the largenumber of undifferentiated cells containedin the epithelium) and prevent light frompassing through the epithelium. As a result,the sub-epithelial vessel pattern isobliterated and the epithelium appearsdensely white. In CIN, acetowhitening isrestricted to the transformation zone closeto the squamocolumnar junction, while incancer it often involves the entire cervix.

The acetowhite appearance is not uniqueto CIN and early cancer. It is also seen inother conditions when increased nuclearprotein is present, as in immaturesquamous metaplasia, in healing andregenerating epithelium (associated withinflammation), leukoplakia (hyperkeratosis)and condyloma. While the acetowhiteepithelium associated with CIN and earlyinvasive cancer is more dense, thick andopaque with well demarcated margins fromthe surrounding normal epithelium, theacetowhitening associated with immaturesquamous metaplasia, inflammation andregenerating epithelium is less pale, thin,often translucent, and patchy with ill-defined margins. Acetowhitening due toinflammation and healing is usuallydistributed widely in the cervix, notrestricted to the transformation zone andmay quickly disappear (within a minute).Leukoplakia and condylomata appearintensely greyish-white after theapplication of acetic acid.

The acetic acid effect reverses much moreslowly in CIN lesions and in early preclinicalinvasive cancer than in immature squamousmetaplasia and inflammation. It appearsrapidly and may last for 3-5 minutes in thecase of CIN 2-3 and invasive cancer.

Pathophysiological basis of VILISquamous metaplastic epithelium isglycogenated, whereas CIN and invasivecancer cells contain little or no glycogen.Columnar epithelium does not containglycogen. Immature squamous metaplasticepithelium usually lacks glycogen or,occasionally, may be partiallyglycogenated. Iodine is glycophilic andhence the application of iodine solutionresults in uptake of iodine in glycogen-containing epithelium. Therefore, thenormal glycogen-containing squamousepithelium stains mahogany brown or blackafter application of iodine. Columnarepithelium does not take up iodine andremains unstained, but may look slightlydiscoloured due to a thin film of iodinesolution; areas of immature squamousmetaplastic epithelium may remainunstained with iodine or may be onlypartially stained. If there is shedding (orerosion) of superficial and intermediate celllayers associated with inflammatoryconditions of the squamous epithelium,these areas do not stain with iodine andremain distinctly colourless in a surroundingblack or brown background. Areas of CINand invasive cancer do not take up iodine(as they lack glycogen) and appear as thickmustard-yellow or saffron coloured areas.Areas with leukoplakia (hyperkeratosis) donot stain with iodine either, andcondylomata may not, or occasionally mayonly partially, stain with iodine.


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Anatomical and pathological basis of visual cervix