A Simple Method to Evaluate the Thickness of

Collagen in Collagenous Colitis

TO THE EDITOR: In 1976 Lindstrom (1) reported the presenceof a prominent linear eosinophilic band immediately belowthe surface epithelium of the colon in a patient with waterydiarrhea. That entity was denominated collagenous colitis.

Owing to its frequent detection in later years collagenouscolitis has received much attention in the literature (2–5).Recent studies (6, 7) have estimated that the incidence ofcollagenous colitis is between 0.6/105 and 2.3/105, with a

prevalence of 15.7/105 inhabitants. This disease has a 9:1female to male predominance.

In large studies the basement subepithelial membrane ofthe normal colon measures between 4.6 and 6.9mm (6). Inpatients with collagenous colitis the extended non-continuoussubepithelial collagen band measures 10mm or more inthickness (6). Whereas the basement membrane of the normalcolon contains collagen IV, the collagen band in collagenouscolitis contains collagen I, III, IV, and VI.

The cells of the surface epithelium in collagenous colitismay be flat and vacuolated. Total detachment of the surfaceepithelium often occurs. The slight to moderate inflammation

Fig. 2. Hematoxylin and eosin (H&E)-stained section from the colonic mucosa in a patient withcollagenous colitis. The stained section was observed in a conventional fluorescence microscope (Zeissaxioscope, provided with plane neoflutar objectives, a blue 450–490 filter set, and a chromatic beamsplitter, FT 510). Note the thickness of the autofluorescent collagen band (H&E,�10).

Fig. 1. Hematoxylin and eosin (H&E)-stained section from normal colonic mucosa, observed in aconventional fluorescence microscope (Zeiss axioscope, provided with plane neoflutar objectives, a blue450–490 filter set, and a chromatic beam splitter, FT 510). Note the thin autofluorescent subepithelialbasement membrane (H&E,�10).

Scand J Gastroenterol 2000;35:223–224.

CORRESPONDENCE

Scandinavian University Press 2000

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in the lamina propria is dominatedby lymphocytesandplasmacells.Someeosinophilsandmastcellsmaybepresent.

In hematoxylin–eosin(H&E)-stainedsectionsthe inflam-matorycellsmayconcealpartof thecollagenband.Becauseof this trap,severalhistochemicalcolorantshavebeenusedtoenhancethat band, such as van Gieson, trichrome stainswithout or with safranins,andSirius red.By theaid of thosespecialstainsthebandcanreadilybeseen,althoughits lowerborder remains indistinct and shaggy,often concealedbyinflammatorycells.

Other techniqueshave beenusedto study the collagen,suchasimmunohistochemistry,in situ hybridization(8), andelectron microscopy (9). Recently, H&E-stained sectionsfrom the normal colorectal mucosa were observedin aconventional fluorescencemicroscope (Zeiss axioscope,provided with plane neoflutar objectives,a blue 450–490filter set, and a chromatic beam splitter, FT 510). Byserendipity,an apparentlycontinuous,distinct, thin, greenflorescentlinear structurewas detectedimmediatelybelowthe surfaceepitheliumof the colonic mucosa.The result ofthat observationwas confirmed in sectionsfrom 10 con-secutive,unselectednormal individuals (Fig. 1). That sharpgreenstructurewas presentabovethe fibrillar greenfluor-escentstructureof the laminapropriamucosa.

On the other hand,the studyof sevenconsecutiveH&E-stainedbiopsyspecimensfrom patientswith collagencolitisshoweda substantiallythicker, almost compact,autofluor-escentsubepithelialbandwith minornon-fluorescentroundedor elongated‘filling defects’ correspondingto capillariesentrappedin theband(Fig. 2).

The deeperborder of the thick autofluorescentcollagenband was distinct, thus contrastingwith the indistinct andshaggydeeperborderof the bandin the sameH&E sectionswhenobservedin light microscopy.

In sectionsstainedwith vanGiesonthecollagenbandwasnot fluorescent.Notwithstanding,when van Gieson-stainedsectionswere excited with a D 360/40-nmfilter (Zeiss),astrong, red–yellow fluorescentband was seen.The deeperborder of the band was distinct, thus contrastingwith theirregularandshaggydeeperborderof thebandin thesamevanGieson-stainedsectionswhenobservedin light microscopy.

H&E sectionsfrom five consecutivecasesof lymphocyticcolitis were also observedunder the sameconditions.The

distinctautofluorescentsubepithelialstructurefoundwasonlyslightly thicker thanin normalindividuals.

Sinceeosinis a fluorochromestain,which may fluoresceintensivelyin near-ultravioletandviolet light, it wasassumedthateosinwasthestainresponsiblefor thephenomenonheredescribed.However, the eosin-stainedcytoplasmaof thecolumnar cells of the surface epithelium of the colonremainednon-fluorescent.

In summary,the collagenousbandof collagenouscolitisfound in H&E sectionsmay be studied in a fluorescentmicroscope.This simplemethodpermitsinvestigationof themorphologicattributesof thatbandwithout theuseof specialstainsor of moresophisticatedtechniquesof examination.

Acknowledgement

This studywassupportedby theKarolinskaHospital,Stock-holm, Sweden.

C. A. RubioGastrointestinalandLiverPathologyResearchLaboratoryDept.of PathologyKarolinskaInstituteandHospitalSE-17176 Stockholm,Sweden(Fax:�46 8 51774524)

References

1. Lindstrom CG. ‘Collagenouscolitis’ with watery diarrhoea—anew entity?PatholEur 1976;11:87–9.

2. JessurunJ,YardleyJH,GiardelloFM, HamiltonSR,BaylessTM.Chroniccolitis with thickeningof thesubepithelialcollagenlayer(collagenouscolitis). Hum Pathol1987;18:839–48.

3. Gledhill A, Cole FM. Significance of basementmembranethickeningin the humancolon. Gut 1984;25:1085–8.

4. Pieterse AS, Hecker R, Rowland R. Collagenouscolitis: adistinctiveandpotentiallyreversibledisorder.J Clin Pathol1982;35:330–40.

5. GoldsteinNS, Gyorfi T. Focal lymphocytic colitis and collage-nouscolitis. Am J SurgPathol1999;23:1075–81.

6. JawhariA, Talbot IC. Microscopic,lymphocyticandcollagenouscolitis. Histopathology1996;29:101–10.

7. Bohr J. A review of collagenouscolitis. ScandJ Gastroenterol1998;33:2–9.

8. Aigner T, NeureiterD, Muller S,KuspertG, BelkaJ,KirchnerT.Extracellular matrix composition and gene expression incollagenouscolitis. Gastroenterology1997;113:136–43.

9. StubbeTeglbjaergPS,HessThaysenE. Collagenouscolitis: anultrastructuralstudy.Gastroenterology1982;82:561–3.

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