Magnification and Chromoscopy with the Acetic Acid Test

Transferring knowledge and techniques that have been learned from decades of the use of acetic acid in colposcopy to the field of digestive endoscopy is a noteworthy objective. However, to be accurate, the fundamental mechanism involved in the whitening of cervical tissue after the application of acetic acid - the process known as ”acetowhitening” - has not yet been definitively clarified. In a recent paper in Endoscopy, Lambert et al. claim that ”Cytokeratins are the major cause of the acetowhite reaction, due to their filamentous architecture” [1]. This hypothesis does not explain other acetowhitening phenomena observed in nonneoplastic tissue. For example, crypt openings (resulting in so-called ”cuffed gland openings”), borders of the transformation zone, and immature metaplasia can all display whitening characteristics similar to the whitening observed with tissue classified as cervical intraepithelial neoplasia (CIN) grades I, II, and III. In fact, since nonneoplastic tissue can acetowhiten and mimic neoplastic tissue, there is a need for improved diagnostic training and/or new techniques to improve the accuracy of classifying whitened tissue when observed colposcopically.

An alternative explanation for the fundamental mechanism of the acetowhitening phenomenon is based on another concept mentioned by Lambert et al. The phenomenon of the wrapping and unwrapping of DNA around the core histones in the nucleus is often referred to as ”chromatin condensation.” Chromatin condensation and decondensation - the reversal of the process several minutes after the application of acetic acid - can be used to explain the primary mechanism of acetowhitening. Chromatin condensation occurs when the macrostructural properties of tissue allow for the permeation of acetic acid through the epithelial cell membranes to the cell nuclei. When acetic acid is introduced into highly permeable epithelial tissue, such as neoplastic tissue and certain categories of nonneoplastic tissue, the tissue whitens as a function of the nuclei/chromatin density. The intensity and rate of the acetowhitening phenomenon are proportionate to the density of the nuclei and the permeability of the tissue to acetic acid. When densely packed nuclei are accessible to acetic acid, an observable acetowhitening effect will occur. When epithelial cells are intact, highly differentiated, and acting as an effective barrier to acetic acid, little or no acetowhitening is observable. The following is a discussion supporting this macrostructurally based explanation as the key mechanism involved in the acetowhitening of both neoplastic and nonneoplastic cervical tissue.

Previously published work has discussed the acetowhitening phenomenon in cervical tissue. Drezek et al. hypothesized that the predominant effect involved in acetowhitening is ”an alteration of the refractive-index structure of the [cell’s] nucleus” [2]. Rajadhyaksha et al. hypothesized that, in addition to changes in the back-scattering properties of cells, acetic acid may also increase depolarization from intranuclear structures [3]. Both groups explain the acetowhitening phenomenon in terms of intracellular changes or relatively microstructural phenomena. The alternative, which may complement or possibly even dominate the cellular mechanism of acetowhitening, is a macrostructural intercellular mechanism.

It is well known that high-grade intraepithelial neoplasia results in a very brittle surface tissue structure. Within high-grade lesions, ”epithelial edges tend to detach from underlying stroma and curl back on themselves” [4]. This lends itself well to cell exfoliation of diseased cells, which are readily detectable in Papanicolaou smears. It also implies that intercellular cohesiveness may play an important role in the progression of neoplastic disease.

For acetic acid to penetrate cells and change the intracellular optical properties, there must be an effective surface area of cells that are exposed to the acetic acid. In normal squamous epithelial cervical tissue, the several uppermost layers of cells are highly differentiated and form a relatively impenetrable barrier against the penetration of liquids. However, other conditions exist that can increase the surface area of cells exposed to acetic acid. These include crypt openings (resulting in ”cuffed gland openings”); the borders of the transformation zone; immature metaplasia; and compromised cell junctions.

Crypt openings (resulting in ”cuffed gland openings”). Gland or crypt openings on the surface of the cervix will often whiten in a characteristic ”cuffed” pattern (Figure [1]) [5]. A Japanese group [6] went as far as to claim that the colposcopic diagnosis of cervical neoplasia could be aided by the identification of certain patterns of whitening around gland openings. Nevertheless, gland openings provide a channel or conduit for acetic acid to pass the top highly differentiated squamous-cell level and result in the increased surface area of glandular or columnar cells exposed to acetic acid.

Figure 1 An example of ”cuffed” glands in nondiseased tissue, produced by acetowhitening. From Anderson et al., Integrated colposcopy (Philadelphia, 1997) [5], reproduced with permission from Lippincott Williams & Wilkins.

Borders of the transformation zone. As with crypt or gland openings, the tissue morphology around the transformation zone often results in a cliff-like step between the high normal squamous epithelium and the lower columnar or metaplastic tissue (Figure [2]). This step provides en-face exposure - the ”side of the cliff,” as it were - to acetic acid. The cells on this cliff face therefore have a larger surface area exposed to the acetic acid and will therefore whiten in a ring-like pattern around the perimeter of the squamocolumnar junction.

Figure 2 An example of acetowhitened tissue surrounding the transformation zone. From Anderson et al., Integrated colposcopy (Philadelphia, 1997) [5], reproduced with permission from Lippincott Williams & Wilkins.

Immature metaplasia. ”When large areas of squamous metaplasia are examined at low magnification, its multifocal nature can be clearly seen. Cervical columnar epithelium often presents in a configuration of a series of ridges and clefts; if this is the case, it is the epithelium along the surface of the ridges which undergoes metaplasia first” [5]. During the metaplastic process, as the columnar villi are fusing, there is still a considerable surface area represented by the villi themselves. It is hypothesized that the nooks and crannies of the irregular growth of metaplasia contribute to the acetowhitening found in metaplastic tissue.

Compromised cell junction. Finally, with regard to actual neoplastic cervical tissue, it is hypothesized that compromised cell junctions allow for the increased permeability of acetic acid and thus a larger cell surface area effectively exposed. There are at least two sources of information that support this hypothesis - the use of toluidine blue staining to detect cervical neoplasia and the published literature on compromised cell-gap desmosomes and cadherins.

Toluidine blue was used by Richart as a vital stain for detecting cervical neoplasia as long ago as the 1960 s (Elmar A. Joura, M.D., University of Vienna; oral communication, 2000). However, this approach was not pursued, as it did not offer any information additional to that provided by acetic acid. Nevertheless, the use of toluidine blue to differentiate neoplastic tissue can be seen as another clue supporting the theory that intercellular barriers are compromised with the progression of neoplastic disease.

Further evidence of a progression of intercellular changes in neoplastic tissue has been presented by de Boer et al. [7] and Grisaru et al. [8]. De Boer et al. show that during the development of cervical lesions, substantial changes (both quantitative and qualitative) occur in cell-cell junctions. These changes make the interactions between the cells in lesions dissimilar from those of reserve cells, basal cells, or cells of immature squamous metaplasia, despite the morphological similarity that exists between all of these cell types and cells of high-grade lesions. Similarly, the experimental work by Grisaru et al. investigates the specific localization of a peptide known as connective tissue activating peptide III (CTAP-III). CTAP-III is found between adjacent epithelial cells and suggests a possible role for this chemokine in maintaining the normal architecture of epithelial tissues. Its progressive disappearance in increasingly severe CIN may explain the increased permeability of the tissue structure with the progression of cervical carcinoma.

The use of intercellular mechanisms, rather than intracellular mechanisms, to identify cervical neoplasia may provide as yet unexplored opportunities to improve the use of acetowhitening for accurate identification of neoplastic tissue. It has been suggested that other agents - monocarboxylic acids, for example, such as formic, propionic and butyric acids - may selectively whiten cervical neoplastic tissue in ways similar to acetic acid [9].

In conclusion, more work needs to be done to elucidate the fundamental mechanism underlying acetowhitening convincingly. As the acetowhitening phenomenon has been harnessed so effectively in colposcopy, it is prudent to consider its use in other areas of endoscopy and video endoscopy, as Lambert et al. propose [1]. Further research providing additional information on the fundamental mechanism involved will enhance the effective use of the technique in digestive endoscopy and help improve the efficacy of emerging new techniques using video colposcopy.

References

• 1 Lambert R, Rey J F, Sankaranarayanan R. Magnification and chromoendoscopy with the acetic acid test.  Endoscopy. 2003;  35 437-445
• 2 Drezek R, Dunn A, Richards-Kortum R. Light scattering from cells: finite-difference time-domain simulations and goniometric measurements.  Appl Opt. 1999;  38 3651-3661
• 3 Rajadhyaksha M, Meaker G, Gonzalez S. Confocal microscopy of excised human skin using acetic acid and cross-polarization: rapid detection of non-melanoma skin cancers.  SPIE Proc. 2000;  3907 84-88
• 4 Singer A, Monaghan J M, Chong S. Lower genital tract precancer: colposcopy, pathology and treatment. 2nd ed.  Oxford; Blackwell Science 2000: 115
• 5 Anderson M, Jordan J A, Morse A R, Sharp F. Integrated colposcopy: for colposcopists, histopathologists and cytologists [CD-ROM]. 2nd ed.  Philadelphia; Lippincott Williams & Wilkins 1997
• 6 Kishi Y, Yasuda M, Fujita H. et al . Cervical neoplasia and gland openings.  Gynecol Oncol. 1978;  6 333-347
• 7 De Boer C J, van Dorst E, van Krieken H. et al . Changing roles of cadherins and catenins during progression of squamous intraepithelial lesions in the uterine cervix.  Am J Pathol. 1999;  155 505-515
• 8 Grisaru D, Vlodavsky I, Prus D. et al . Connective tissue activating peptide III expression disappears progressively with increased dysplasia in human cervical epithelium.  Gynecol Oncol. 2000;  79 23-27
• 9 MacLean A B. What is acetowhite epithelium? Paper presented at the 10th World Congress of Cervical Pathology and Colposcopy. Buenos Aires, Argentina; 7-11 November 1999