HISTOLOGICAL CONSEQUENCES OF GASTROESOPHAGEAL REFLUX IN MAN

Transcription

1 GASTROENTEROLOGY Copyright 1970 by The Williams & Wilkins Co Vol 58, No2 Printed in USA HISTOLOGICAL CONSEQUENCES OF GASTROESOPHAGEAL REFLUX IN MAN FARHAD ISMAIL-BEIGI, MD, PAUL F HORTON, AND CHARLES E POPE II, MD Veterans Administration Hospital, and Division of Gastroenterology, Department of Medicine, University of Washington, Seattle, Washington Esophageal suction biopsy specimens were obtained from 33 subjects with, and 21 without, subjective and objective evidence of gastroesophageal reflux From these specimens the appearance of the normal esophageal mucosa was established and histological features of the mucosa in presence of gastroesophageal reflux were determined These features are: (a) basal cell hyperplasia of the squamous epithelium, and (b) location of the papillae close to the epithelial surface A good correlation is shown between these features and symptoms, acid reflux studies, and esophagoscopy It is concluded that these features are the histological consequences of gastroesophageal reflux It has been shown that gastroesophageal reflux produces esophagitis in experimental animals 1 and generally is held to be the primary cause of esophagitis in man The gross and microscopic features of esophagitis have been described from studies of autopsy specimens by Lodge2 and Peters3 These features consist of ulceration, fibrosis, and, in the presence of an intact epithelium, inflammation in the lamina propria Palmer,4,5 by studying biopsy specimens from 61 patients with endoscopic evidence of esophagitis, concluded that the Received July 1, 1969 A cepted September 5, 1969 A preliminary report of this work was presented at the annual meeting of the American Gastroenterological Association held in Washington, D C, in May 1969 Address requests for reprints to: Dr Charles E Pope II, MD, Veterans Administration Hospital, 4435 Beacon Avenue, South, Seattie, W ashington Dr Ismail-Beigi was the recipient of Gastroenterology Training Grant 5 T01 AM from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service The authors wish to thank Dr Cyrus E Rubin and Dr Beach Barrett for their assistance, advice, and encouragement during this study 163 subepithelial accumulation of lymphocytes, plasma cells, and occasionally neutrophils is indicative of esophagitis Currently, the presence of these accumulations is considered to represent inflammation and to indicate esophagitis6-9 In many patients with clinical symptoms and signs of reflux esophagitis, no histological evidence of inflammation is present1o- 12 Conversely, inflammation has been reported in biopsy specimens from asymptomatic control subjectslo, 12 This lack of correlation between clinical findings and histology raises the following questions Are accumulations of round cells and neutrophils in the lamina propria abnormal? Are there other histological features which do correlate with symptoms and signs of reflux esophagitis? To answer these questions, esophageal biopsy specimens were obtained from subj ects with and without subjective and objective evidence of gastroesophageal regurgitation In this paper, the currently accepted histological features of esophagitis are reexamined In addition, the histology of normal esophageal mucosa is described and histological features of esophageal mucosa in presence of gastroesophageal reflux are presented

2 164 ISMAIL-BEIGI ET AL Vol 58, No2 TABLE 1 Incidence of symptoms in heartburn group Symptoms Incidence % Heartburn 100 Retrosternal chest pain 62 Dysphagia 57 Upper gastrointestinal bleeding 36 Odynophagia 12 Methods Two groups were studied The control group consisted of 21 healthy volunteers without esophageal symptoms in whom gastroesophageal reflux was not demonstrated The heartburn group consisted of 33 subjects with severe heartburn and other symptoms of esophagitis in whom reflux was demonstrated (table 1) In the process of assembling these two groups, 19 additional subjects were encountered who did not meet the criteria for the above two groups Ten of these 19 subjects had symptoms indistinguishable from the subjects in the heartburn group, yet no reflux could be demonstrated The other 9 persons were totally asymptomatic, and yet reflux was demonstrated Presence or absence of reflux was determined with the technique described below Heartburn was defined as substernal burning, radiating into the neck, coming in waves, made worse by recumbent position, and often accompanied by the regurgitation of bitter or sour fluid into the mouth In all 73 subjects, measurement of the lower esophageal sphincter pressure, a test for gastroesophageal reflux, and esophageal suction biopsies were performed An acid drip test and esophagoscopy were performed in some of the subjects An assembly consisting of a Beckman ph electrode (no 39042) and three catheters with side openings, made of polyvinyl tubing, 25 mm in outer diameter, was constructed The most distal opening was at the tip of the assembly; all openings were 5 cm apart The tip of the ph electrode was located at the same level as the distal catheter This assembly was passed via the mouth into the stomach of fasting subjects The following measurements then were made Sphincter pressure The lower esophageal sphincter pressure was determined with constant infusion and pull through techniques with the subject in the supine position Gastroesophageal reflux test A modification at the method described by Tuttle and Grossman 'S was used The ph of the esophagus was monitored with the subject in a supine position After loading the stomach with 300 cc of 01 N hydrochloric acid, the assembly was withdrawn slowly in order to position the ph electrode 2 cm above the lower esophageal sphincter The ph of the esophagus then was raised to above 60 by flushing the catheters with water Gastroesophageal reflux was considered to be present if the ph fell below 3 If reflux was not demonstrated within 3 min (free reflux), the patient was asked to perform a series of maneuvers to induce reflux These consisted of three sets of Valsalva maneuvers, leg lifts, and deep sniffs The test was graded as follows: 0, no gastroesophageal reflux 1 +, reflux demonstrated with maneuvers 2+, free reflux demonstrated Acid drip test A modified Bernstein test' was performed following the reflux test With the subject in the sitting position, the assembly was withdrawn so that the proximal tip was 25 em from the teeth Through this lumen, normal saline was run at 120 drops per min for 5 min If no symptoms were produced, the solution was changed without the subject's knowledge to 01 N Hel, dripping at the same rate The test FIG 1 Diagram of biopsy assembly, showing position of biopsy ports relative to gastroesophageal sphincter

3 February 1970 GASTROINTESTINAL REFLUX 165 was considered positive if the acid drip induced the patient's symptoms and negative if, after 30 min of acid drip, no symptoms were produced A manometric catheter was attached to a Rubin multipurpose suction biopsy tube After completion of the above studies, the biopsy assembly was introduced into the stomach and was withdrawn slowly until the upper extent of the gastroesophageal sphincter was identified manometrically Then the assembly was withdrawn further in order to position the biopsy ports 2 cm above the gastroesophageal sphincter (fig 1) From this area two suction biopsies were obtained simultaneously They were oriented, mounted, fixed in Bouin's solution, serially sectioned at 6 fl, and stained with hematoxylin and eosin By comparing biopsy specimens obtained early in the study from 7 subjects in the control group and 15 subjects in the heartburn group, certain definite histological features and differences became apparent New histological criteria were developed from this initial group and then were applied to all biopsy specimens At the end of the study all specimens were coded, mixed, and interpreted by three independent observers using the new histological criteria They were classified as either "normal" or "abnormal" When neutrophils were seen in the lamina propria, their presence also was indicated There was complete agreement between the three observers on 80% of the biopsy specimens In cases of disagreement, with the code still unbroken, the slides were reexamined and a final reading was agreed upon for each slide Then the code was broken All of the results of the biopsy specimens tabulated are from this blind review and interpretation Esophagoscopy was performed in 34 of the 43 patients who had symptoms of gastroesophageal reflux The Eder-Huft'ord esophagoscope was used in 10 of the cases and the Olympus Fiberoptic esophagoscope was used in the other 24 cases The diagnosis of esophagitis was made with the usual endoscopic criteria, ie, friability, granularity, or ulcerations of the esophageal -- --, - - I _ & " " ', _," - - r-'-";i r, : ':, - Lp - - " a- ' -'" r ' - " I-, : _,,: -,- -! l - "-, f _, -: ' : '"" / - I' : It - ' ', e,, --' --,,, " or ", - _'_:-;,;:- -- "' - " _" a " e \, - FIG 2 Normal biopsy specimen from a subject in the control group BZ, basal zone; SZ, stratified zone; P, papillae; LP, lamina propria are shown Basal zone thickness is approximately 10% of total epithelial thickness; papillae extend approximately one-half of the distance t o the epithelial surface X 170

4 166 ISMAIL-BEIGI ET AL Vol 58, No2 mucosa Esophagoscopy was not performed on the asymptomatic subjects Results The esophageal mucosa is composed of three layers: the squamous epithelium, lamina propria, and muscularis mucosae The epithelium can be divided into two portions: a basal zone consisting of several layers of basophilic cells with dark nuclei, and a stratified zone containing many layers of squamous cells with flattened nuclei A representative biopsy specimen taken from a subject in the control group is shown in figure 2 It can be seen from this specimen that the basal zone is thin and consists of only a few layers of basophilic cells Most of the epithelium is composed of stratified squamous cells The thickness of the basal zone is less than 15% of the total thickness of the epithelium Infrequently, mitotic figures are seen The papillae extend less than two-thirds of the distance to the surface of the epithelium Round cells, such as lymphocytes and plasma cells, are present in the lamina propria All biopsy specimens with these features are designated as normal A representative biopsy specimen from a subject in the heartburn group is shown in figure 3 In contrast to the normal specimen shown in figure 2, hyperplasia of the basal cells is evident The basal zone is thicker and makes up more than 15% of the total thickness of the epithelium The basal zone layers of the biopsies from the heartburn group appear to contain a few more mitotic figures than are seen in the control biopsies However, the ratio of mitotic figures to nuclei present actually would be lower in the biopsies from the heartburn group owing to the increased number of nuclei present No quantitation of mitosis was carried out in this study The papillae extend more than two-thirds of the distance to the epithelial surface As in normal spec i- - -;, I d ;i, '- f FIG 3 Abnormal biopsy specimen from a subject in the heartburn group BZ, basal zone; SZ, stratified zone Basal zone thickness is approximately 35% of total epithelial thickness; papillae extend over two-thirds of the distance to the epithelial surface X 170

5 February 1970 GASTROINTESTINAL REFLUX 167 -, - ' ' _-- " ' - "" - -,, --- ",, I,:i,,,t -,, -,- - - ",, -,, - ", - _,,,,, - -, --, - ot-'" ' -, -,, " :, " ' ;- - e ; '- " #, \ - ", - --,, \- ; '; ; " ' ': FIG 4 Comparison of a n ormal biopsy specimen (top) and an abnormal biopsy specimen (bottom) Note t he contrast between t he thick stratified zone in the normal and the t hin st ratified zone in the abnormal specimen The papillae extend completely to the surface in the abnormal specimen X 170

6 168 ISMAIL-BEIGI ET AL Vol 58, No2 "', : ' :,, ' t "" ',' - _ f _,_ " J#' : / ': -, "(II- u :, --:: ',", FIG 5 Comparison of a normal biopsy specimen (left) and an abnormal biopsy specimen (right) Round cells are present, diffusely and in collections, in the l a m propria n a of both specimens X 170 mens, round cells are present in the lamina propria All biopsy specimens with these features are designated as abnormal The thickness of the basal zone can be quantitated with the use of an eyepiece micrometer and can be expressed as a percentage of the total epithelial thickness If several readings are obtained from well oriented central core sections of each specimen, it is found that the mean basal zone thickness of the normal specimens is 10% (range, 5 to 14%) In contrast, the mean basal zone thickness of the abnormal specimens is 30%, (range, 16 to 80%) These figures are dependent upon the chosen site of measurement and are quite timeconsuming Visual estimation was used in the evaluation of most biopsy specimens in this study A comparison of a normal and an abnormal specimen as defined by the new criteria is shown in figure 4 The basal zone of the abnormal specimen makes up 80% or more of the total epithelial thickness and the stratified zone is quite thin The papillae reach the surface of the epithelium The featul:es seen in this abnormal specimen represent a severe epithelial abnormality Round cell collections are present in the lamina propria of both normal and abnormal specimens (see fig 5) Such lymphoid aggregates were found in specimens from 48% of the subjects in the control group and in 55% of the subjects in the heartburn group Thus, their presence is a normal finding and is not indicative of esophagitis The number of papillae appear equal in the normal and abnormal specimens in figures 4 and 5 It was found, however, that their number, as well as the total thickness of the epithelium, varied widely in both normal and abnormal specimens Inflammation, as indicated by the presence of many neutrophils in the lamina

7 February 1970 GASTROINTESTINAL REF LU X 169 FIG 6 Abnormal biopsy specimen (top, X 120); same specimen (bottom, X 480) Epithelium is composed largely of basal cells Inflammation is present, as indicated by presence of neutrophils in the lamina propria propria, can be seen in the biopsy specimen in figure 6 Neutrophils were not found in any of the biopsy specimens from control subjects and were present in only 18% of the biopsy specimens from the heartburn group When neutrophils were present in the lamina propria, the abnormality of the epithelium also was evident The new histological criteria of basal cell hyperplasia and extension of papillae were applied to all biopsy specimens The results are given in tables 2 to 4 In 75% of the subjects, the two biopsy specimens received the same classification If both biopsy specimens were normal, the subject was considered to have a normal biopsy result If either one or both biopsy specimens were abnormal, the subject was considered to have an abnormal biopsy result Control group (table 2) Nineteen of the 21 subjects in this group, or 90%, had normal biopsy results In the other 2 subjects, one of the two specimens was abnormal Heartburn group (table 3) Twenty-

8 ISMAIL-BEIGI ET AL Vol 58, No2 TABLE 2 Results of studies with control group (asymptomatic, without reflux) Subject Age Sex Reflux Response to LES yield testa acid dripb pressure Biopsy results' CW 42 M H K 24 M JM, 0-40 M C K 21 F AC 47 F K F 28 M M S 59 F K T 21 F LMoo 50 M C D 0 25 F WP 40 M R B ' 0 22 F T T 21 F M W 21 F MK 39 M M F 22 F S S 25 F C D 60 M J C oo 22 M A K ' 21 F C J F mmhg 0 20 N 0 Neg 16 N 0 17 N 0 Neg 30 N 0 18 N 0 Neg 30 A* 0 Neg 24 N 0 Neg 32 N 0 25 N 0 Neg 26 N 0 11 N 0 Neg 36 N 0 Neg 29 N 0 Neg 50 N 0 18 N 0 32 N 0 Neg 24 N 0 12 N 0 Neg 37 N 0 Neg 18 A* 0 Neg 26 N Total 21 32d d N, 19 (90%) A*,2 (10%) a 0, gastroesophageal reflux not demonstrated b Neg, no symptoms produced N, both biopsies normal; A*, one biopsy abnormal dmean eight of the 33 subjects in this group, or 85%, had abnormal biopsy results In 18 subjects, both biopsy specimens were abnormal and, in the other 10, one biopsy specimen was abnormal In 6 of the 33 subjects in this group, or 18%, neutrophils were present in the lamina propria, in addition to the abnormality of the squamous epithelium Miscellaneous groups (table 4) Five of the 9 subjects in the asymptomatic with reflux group, or 56%, had abnormal biopsy results In 4 of the 5, one biopsy specimen was abnormal and in the 5th both biopsy specimens were abnormal Seven of the 10 subjects in the symptomatic without reflux group, or 70%, had abnormal biopsy results Both biopsy specimens were abnormal in 6 subjects and one biopsy specimen was abnormal in the 7th subject In 2 subjects neutrophils were present in the lamina propria, in addition to the abnormality of the epithelium Discussion It currently is accepted that collections of round cells and neutrophils in the esophageal lamina propria represent inflammation and thus indicate esophagitis This study shows that round cell collections are present in many biopsy specimens from control subjects Thus, their presence is a normal finding and should not be used as evidence of esophagitis N eutrophils are seen infrequently in specimens from subjects in the heartburn group and are not seen in biopsy specimens from control subjects Therefore their presence is abnormal, but is a rather insensitive histological indicator of reflux esophagitis Use of ac-

9 February 1970 GASTROINTESTINAL REFLUX 171 TABLE 3 Results of studies with heartburn group (symptomatic, with reflux) Reflux Response LES yield I Subiect Age Sex Esophatesta to acid dripb pressure X-ray' goscopyd ---- Biopsy results' mmhg R K 56 M Normal + A-P E H 48 F Normal + A C S 50 M H H Normal A* T E 37 M H H Normal N J G 42 M HH Normal A* GL 40 F Normal + A* L J 49 M Normal A RW 57 M HH Normal A HB 54 F Normal N 1 C 56 F HH + A HD 55 M HH + A-P L B 51 M HH + A-P* A A 39 M HH + A MG 30 F HH + N E C 41 F 1+ Neg 40 Normal N E T 64 F HH + A* P C 43 M Normal + A C L 74 M Normal Normal A* J C 56 M HH + A-P HR 54 M 1+ 6 Normal Normal A M P 23 M Normal + A AM 78 F Normal + A R H 35 M Normal A F T 24 M Normal A* W S 45 M 2+ 7 HH + A J J 56 M HH + N J T 66 M HH + A* H V 59 M Normal + A BS 51 M 1+ 6 Normal + A* F F 64 M 1+ 5 Normal + A-P D T 40 M Normal + A* MD 32 M Normal + A G P 75 M HH + A-P Total ,18 +, ,22 (A), 28 (85%) 1+,15 Neg, 1 Normal,6 N,5 (15%) a 1+, reflux demonstrated with maneuvers; 2+, free reflux b +, patient's symptoms duplicated; Neg, no symptoms produced 'H H, hiatus hernia d +, esophagitis 'N, both biopsies normal; A, both biopsies abnormal; A-P, abnormal with polys; *, one biopsy abnormal; (A), total of all abnormal biopsy results I Mean cumulations of round cells and neutrophils as histological criteria for diagnosis of esophagitis might explain the poor correlation reported between clinical findings and histology The histological features of basal cell hyperplasia and closeness of papillae to the epithelial surface correlate well with the presence of symptomatic gastroesophageal reflux In 85% of the subjects with symptomatic reflux, these features were present Conversely, they were not present

10 172 ISMAIL-BEIGI ET AL Vol 58, No2 Group Subject Age Sex TABLE 4 Results of studies in miscellaneous groups Reflux Response LESyield X-ray Esophatest to acid drip pressure goscopy Biopsy results -- mmhg Asympto- MW 23 M N matic, with P H 52 M N reflux K W 24 M A J J 42 M N T R 23 M 1+ Neg 32 A* P T 24 M 1+ Neg 16 N F K 51 M A* WB 51 M Normal A* c H 45 M A* Total 9 37 b 1+,9 Neg, 2 22b Normal, 1 (A), 5 (56%) N,4 (44%) Sympto- A B 59 M 0 9 Normal + N matic, D M 49 F Normal Normal A without A F 45 M 0 14 Normal Normal N reflux WH 49 M Normal A-P K S 20 F 0 22 Normal A HH 52 M Normal Normal A-P R R 45 M 0 17 Normal + A c F 63 M 0 13 Normal + A* E L 43 F 0 12 H H N WB 44 F HH A Total b 0,10 +,4 15b H H, 2 Normal,3 (A), 7 (70%) Normal,8 +,3 N,3 (30%) a For explanation of symbols, see footnotes to table 3 b Mean in 90% of the control subjects These features are distinct and allow a high level of agreement between different observers It therefore is concluded that these features are histological characteristics of the clinical syndrome of reflux esophagitis What is the correlation between these histological features with esophagoscopy, another means of examining the esophageal mucosa? Esophagoscopy was performed in 34 of the subjects who were symptomatic In 25 subjects the diagnosis of esophagitis was made endoscopically; in 22 of them, or 88%, biopsy also was abnormal In 9 subjects the esophageal mucosa appeared normal endoscopically; the biopsy, however, was abnormal in 7 Therefore, it would appear from this study that biopsy is a more sensitive method for evaluation of the esophageal mucosa than endoscopy The relationship between a positive history of heartburn, a positive reflux test, and an abnormal biopsy result is shown in figure 7 All of the subjects in this study are shown in the diagram There is a total of 54 subjects inside the three circles who at least had one of the above three characteristics The 19 subjects outside these three circles are the asymptomatic controls with normal biopsy results The lack of complete overlap of these three circles might be explained by the following points 1 Gastroesophageal reflux does not produce symptoms in all subjects There are 9 asymptomatic subjects in this study with evidence of gastroesophageal reflux; in 5 of these subjects, esophageal biopsies were abnormal It is possible that some of these subjects may become symptomatic or

11 February 1970 GASTROINTESTINAL REFLUX 173 might present with hemorrhage or an esophageal stricture at a later date never having experienced any symptoms of esophagitis 2 There are 10 subjects with heartburn in whom the test for reflux was negative Esophageal biopsies were abnormal in 7 of the 10 persons in this group It is possible that these subjects have intermittent reflux which was not present on the day that they were tested 3 In this study an area 2 cm proximal to the lower esophageal sphincter was chosen for obtaining all biopsy specimens This area was selected because endoscopically the mucosal abnormalities are most severe at the lower end of the esophagus The histological lesions in this selected area are present in a patchy distribution This is shown by the fact that 25% of the subjects studied had one normal and one abnormal biopsy specimen taken from this area Such patchiness may explain the failure to obtain abnormal histology in all subjects with significant gastroesophageal reflux It is possible that, if more biopsy specimens were obtained, the correlation between reflux and histology might be improved The histological features presented suggest the possibility of an increased rate of loss and replacement of squamous epithelium in the presence of gastroesophageal reflux One can hypothesize that more superficial layers of the epithelium are desquamated as the result of injury from gastric juice and thus the papillae appear close to the epithelial surface The hyperplasia of basal cells could be the histological manifestation of increased rate of replacement An identical type of histological response to injury has been reported in the squamous epithelium of skin It was found that, when the superficial horny layers of the epithelium are stripped experimentally with tape, hyperplasia of the basal cells is produced17, 18 The proximity of the papillae to the surface of the epithelium suggests a possible mechanism for production of heartburn This proximity might allow the refluxed gastric juice to come in closer contact with FIG 7 Venn diagram showing relationship of history of heartburn, objective evidence of gastroesophageal reflux, and abnormal histology The numbers indicate number of,subjects in each area The 19 subjects in the lower right corner are the asymptomatic controls with normal biopsy results the lamina propria of the esophageal mucosa This contact may stimulate chemoreceptors or unmyelinated nerve fibers located in this region Such stimulation of receptors rather than inflammation might produce heartburn To date, over 300 biopsies have been performed in this laboratory They are relatively easy to perform, safe, and painless There have been no complications With the use of these new histological criteria for interpretation, esophageal biopsy may serve as a useful tool in the diagnosis and investigation of reflux esophagitis REFERENCES 1 Ingram, P R, R K Keswani, and W H Muller, Jr 1960 A correlative histopathologic study of experimental surgical reflux esophagitis Surg Gynec Obstet 111: Lodge, K V 1955 The pathology of nonspecificbesophagitis J Path Bact 69: Peters, P M 1955 The pathology of severe digestion oesophagitis Thorax 10: Palmer, E D 1954 Subacute erosive (peptic) esophagitis, clinical study Arch Intern Med (Chicago) 94:

12 174 ISMAIL-BEIGI ET AL Vol 58, No 2 5 Palmer, E D 1955 Subacute erosive (peptic) esophatitis, histopathologic study Arch Path (Chicago) 59: Winkelstein, A, B S Wolf, M L Som, and R H Marshak 1954 Peptic esophagitis with duodenal or gastric ulcer J A M A 154: Ballem, C M, H W Fletcher, and R D McKenna 1960 The diagnosis of esophagitis Amer J Dig Dis 5: Svoboda, A C, C M Knauer, C N Gamble, S C Sommers, and L S Monroe 1967 Problem in the early diagnosis of peptic esophagitis Gastroint Endosc 13: Bettarello, A, T Brito, and S Zaterka 1967 Progressive systemic sclerosis II Esophageal involvement Amer J Dig Dis 12: Siegel, C I, and T R Hendrix 1963 Esophageal motor abnormalities induced by acid perfusion in patients with heartburn J Clin Invest 42: Schuman, BM, and J A Rinaldo 1966 Relative frequency of esophagitis and gastritis in patients with symptomatic hiatus hernia Gastroint Endosc 12: Goldman, M S, J R Rasch, D S Wiltsie, and M Finkel 1967 The incidence of esophagitis in peptic ulcer disease Amer J Dig Dis 12: Winans, C S, and L D Harris 1967 Quantitation of lower esophageal sphincter competence Gastroenterology 52: Pope, C E, II 1967 A dynamic test of sphincter strength: its application to the lower esophageal sphincter Gastroenterology 52: Tuttle, S G, and M I Grossman 1958 Detection of gastroesophageal reflux by simultaneous measurements of intraluminal pressure and ph Proc Soc Exp Bioi Med 98: Bernstein, L M, and L A Baker 1958 A clinical test for esophagitis Gastroenterology 34: Pinkus, H 1951 Examination of the epidermis by the strip method of removing horny layers J Invest Derm 16: Pinkus, H 1952 Examination of the epidermis by the strip method J Invest Derm 19: