Tnt JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright 1568 by The Williams & Wilkins Co.VOl. 50, No. 2

Printed in U.S.A.

SUCTION BLISTER DEVICE FOR SEPARATION OF VIABLEEPIDERMIS FROM DERMIS*

IJRPO KIISTALA, M.D.t

There is need for technic by which epi-dermis can be separated from the dermis bypurely mechanical forces avoiding chemical orthermal damage. Of the in vitro separationtechnics, Van Scott's stretch method (1) andits modifications (2, 3) have reached widestapplication. They require, however, compara-tively large surgical or necropsy specimensand have the disadvantage that the epidermis,scraped off with a scalpel, may lack the basalcell layer (4).

As with unidirectional stretching, dermal-epidermal separation has been achieved bythe application of pressure gradients causingmultidireetional distension. As long ago as1900 Weidenfeld (5) reported that subepi-dermal blisters could be produced when hydro-static water pressure was mediated on thedermal side of neeropsy specimens of humanskin. In 1950 Blank and Miller (6) publisheda reversal of this method. They applied aforceful suction on the epidermal side of im-mersed necropsy skin pieces tightly wiredacross a vacuum thistle. In the hands ofBurbach (7) this method yielded inconsistentresults.

In vivo separation of the complete humanepidermis by suction was accomplished in1964 (8). Comparatively low suction pressuresfor prolonged periods succeeded in consistentlyraising subepidermal blisters.

A literature search revealed that Unna hadexpressed the use of pressure forces for

in 1878 (9).In a study of eantharidin blistering he men-tioned that blisters can be produced by"troekenes Sehröpfen", a certain kind of drycupping of the intact skin. Apparently Unnadid not develop this, not even mentioning itin his review of blister formation (10).

Presented at the Twenty-eighth Annual Meet-ing of the Society for Investigative Dermatology,June 18—20, 1967, Atlantic City, New Jersey.

* From the Department of Dermatology, Fm-versity Central Hospital, Helsinki, Snellmaninkatu14, Finland.

t This study forms a part of the author's thesisand was made under supervision of T. Putkonen,M.D., M.P.H. and K. K. Mustakallio, M.D.

For suction blistering Kiistala and Musta-kallio (8) initially used a conventional capil-lary resistance meter, by which blisters couldbe raised within two hours by a pressure of200 mm Hg below the atmospheric pressure.By employing a slightly different suction cupBielick'r (11) bad attempted to quantitate theNikolsky phenomenon, but never was able toproduce suction blisters on healthy humanskin. He did, however, work with too shortsuction times (up to 30 mm.) and applied tooforceful a suction (500 mm Hg), the latterresulting in bruising.

After comparing several types and severalsizes of suction cups designed for measuringcapillary resistance (12, 13, 14, 15) or intra-ocular pressure (16, 17), the present authorcame to the conclusion improvements wereneeded. Hence, a special suction blister devicewas developed. This enables production of sub-epidermal blisters of preselected number andsize with a simultaneous general decrease ofunnecessary suction trauma. This suctionblistering has been extended to the furry ani-mal skin and stratified epithelium.

MaTHOD AND AppLIcATIONs

In vivo blistering can be produced by pressuredifferences ranging from about one hundred toseveral hundred millimeters of mercury and bycups of a few millimeters to several centimeters indiameter. Strong suction forces of long durationdamage the skin. In order to provide suctionblisters of good quality, i.e. free of unnecessarytrauma, only low pressures can be used. In spite ofthese low pressures, the blistering produced by theolder cups could still be followed by visible traumaas shown by petechiae, persistent flush or edema ofthe area as well as by histological tearing damage.

Ta the construction of a device for atraumaticblistering the main problem was to avoid toodeep an impression of the cup edge, excessivesliding in and overstretching of the skin. First, thecup edge was remodeled to a comparatively broadflange and was equipped with several shallowgrooves. Using this type of contact with the skin,undue impressions could be prevented and anincreased air-proofness acquired. During mostsuctions, a properly shaped bulge of the suckedskin also resulted. However, in regions where theskin is slack and free to slip over the underlying

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FIG. 1. The suction blister device, Dermovac®. The three separate parts of the suctioncup are presented at bottom left, lying upside down. For use the flange-piece is screwedto the chamber-piece. The adapter plate can be placed inside if required. The suction cupready for use is shown at bottom right. It is coupled via a stop-cock and a cone-connectorto a polyethylene tubing leading to the vacuum pump.

tissue, its resiliency could still cause an excessivefilling of the cup despite the grooved cup flange.This adverse phenomenon is exaggerated with anincrease of the inner diameter of the cup.

As a rule, larger cups adhere better to the skinthan do smaller cups, which is particularly evidentwith low suction forces. They are also more prac-tical for large samples. However, the displacementof the sucked tissues and the danger of overdis-tension are greater. In addition, several bodyregions with rounded outer contours or with bonytissues closely underneath restrict their use. Thussmaller suction cups are also needed.

Most of the properties of human skin could beplaced under reasonable control when the broadand grooved flange was combined with the useof different sizes of interchangeable flange pieces.A final improvement was obtained when a perfo-rated, concave diaphragm was placed inside thesuction cup as a mould to check the bulging skin.A properly shaped bulge was empirically deter-mined from selected suctions which had resultedin optimally atraumatic blisters. If the perforateddiaphragm plate was made plane, the cup didnot adhere well to the skin. The use of concavediaphragms across the cup opening inhibited thestill existing possibility of an excessive slipping-inof the outside skin into the cup. In addition, byvarying the number and size of the round holesin the diaphragm, standard blisters could be raisedat will. These improvements are the basis of the

construction of the suction blister device Der-movac.*

The Suction Device DermovacThe suction blister device Dermovac is shown

in Fig. 1. It consists of a suction cup and of aseparate hand pump with an aneroid gauge.

The suction cup, made of transparent plexiglass,is composed of three separate parts: a flange-piece,a chamber-piece and an adapter plate, the latterbeing adjustable inside the cup as a diaphragmto prevent excessive bulging.

On the skin side of the flange-piece, whichserves as the cup border, are concentric roundedgrooves each approximately 1 mm in depth. Theflange-pieces, varying in size from 15 mm to 50mm in inner diameter, are interchangeable and canall be screwed to the standard chamber-piece. Amounted gasket ring of polyethylene ensures anair-proof linkage between the flange-pieces and thechamber-piece.

The chamber-piece, on the other hand, leads viaa stop-cock and a cone connector to the tubingof the pump device. When the pressure maintainsthe desired level, the pump tubing can be re-leased after closure of the stop-cock (Fig. 2) andthe pump used for separate suctions. For mostpurposes, however, it is more useful to have the

* Dermovac® is available from Instrumentarium,Helsinki, Finland.

SEPARATION OF VIABLE EPIDERMIS 131

Fcc. 2. Three suction cups attached by suction to the forearm. For additional supportshort strips of adhesive tape running over the flat cup margins may be also used. Thecup on the left is disconnected from the pump device. The remaining cups demonstratesimultaneous suction mediated by T-tubes leading to a 1000 cc vacuum stabilizer and tothe pump device. Note the filter fitted in front of the pump device to prevent moistureand small particles from reaching its interior.

pump connected for the entire suction period. Theblistering process can be visually followed throughthe transparent roof of the chamber-piece.

The adapter plate is a concave plexiglass mouldand is placed as a diaphragm inside a correspondingflange-piece (Figs. 1, 2, 5). The adapter plateshave been perferated by either one central or bymultiple round holes from one to several milli-meters in diameter (Figs. 1, 2, 5), determining thenumber and size of the suction blisters (Figs. 4, 5).

The pump device consists of the manuallyworked pump, an aneroid gauge, an air lockoperated by a spring, a valve by which thepressure may be regulated at will and the outletto the tubing. All these are mounted in plexi-glass. The aneroid meter is scaled for suctionsfrom 0 to 400 mm Hg below the atmosphericpressure. For in vitro experiments a meter scaledup to 650 mm Hg may be utilized. By coupling alarge vacuum container (Fig. 2) the effect ofincidental air leaking may be overcome. Forperiodic calibration of the gauge or for special

studies a conventional mercury or water manome-ter can be easily coupled.

Instructions for the Use of the Suction DeviceThe size of the suction cup to be used depends

on the quantity of epidermis or blister fluidneeded, but also on the outer contour, thicknessand resiliency of the skin region selected. For largesamples the 25 mm suction cup is useful for thecubital fossa, mid-volar forearm and the trunkskin. This cup size is also the largest applicableto the skin of extremities. For the trunk skinsuction cups up to 50 mm in diameter can be stillused, especially together with adapter plates pre-venting otherwise painful tensions. 15 to 20 mmsuction cups are practical for taut skin (shins) andfor baby skin. Miniature cups 5 to 15 mm indiameter have to be used for the skin of smallanimals and for the mucous membranes.

For cadaver skin suction cups 15 to 70 mm indiameter have all been successfully tested. Com-paratively large cups are advisable because a

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Fio. 3. Without employment of an adapter plate, the suction blisters of varying sizegradually coalesce. Of the blisters produced within 90 minutes, three unite 20 minutesand the fourth 30 minutes later to form a single bulla about 20 mm in diameter. The25 mm suction cup was removed momentarily for photography.

flu. 4. When an adapter plate with 5 mm-bores was used inside the cup, standard suctionblisters, each 4 mm in diameter, could be pro-duced by a suction of 150 mm Hg within 100 min-utes.

sufficient suction force to attach the flange-pieceairtight upon very dehydrated skin requires not onlyhigher pressures, but larger areas. Generally,gluing of the skin with the flange should beavoided, but may be necessary in dehydrated orhardened skin, as well as in hairy areas.

The suction pressure at the onset should be onlyslowly increased to the maintenance level, i.e. inabout one minute. In placing the cup upon theskin, only gentle manual pressure should be ap-plied. Within a few minutes the cup will adhereto the glabrous skin; in hairy areas an airtightsituation is obtained somewhat more slowly. There-after, attachment of short strips of adhesive tape

over the flat borders of the flange secures thestability of the cup, especially if the test subjectis allowed to move freely.

Suction blister times* are not dependent onthe suction pressures alone. In old age accelerationof blister formation is observed. Great individualvariations and certain regional differences are alsoevident.

Independent of the size of the cup, suctions of150 to 200 mm Hg cause initial blistering withinthree hours on the mid-volar forearm, chest orabdomen. By the use of these comparatively lowsuction pressures, the dermo-epidermal cleavagebegins smoothly in the plane between the base-ment membrane and the basal plasma membraneof the basal cells, cellular ruptures being mainlyconfined to the epidermal appendages (18).

The development of the suction blisters can besomewhat hastened by suction pressures of 250 to350 mm Hg, at the expense, however, of the in-creased tendency of trauma formation. By stillstronger suctions painful stretching and bruisingoccur regularly.

The adopter plate accelerates the formation ofblisters and hinders trauma to some extent. It isnecessary when large suction cups arc applied onloose skin areas. Moreover, without an adapterplate the blisters appear, grow and coalesce in ahaphazard manner (Fig. 3), and a suction bulln ofstandard size, corresponding to the inner diameterof the cup, is obtained only when the epidermalseparation has reached the inner circumference ofthe flange. With proper choice of an adapter plate,the numbcr and size of suction blisters can be de-termined at will (Figs. 4 and 5). The smalleststandard blisters, developing between the epider-mal appendages (Fig. 5), arc particularly suited for

* Suction blister time denotes the period of suc-tion from the onset until the first vesicles becomevisible.

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SEPARATION OF VIABLE EPIDERMIS

FIG. 5. Suction vesicles produced within 60 minutes, when an adapter plate with 1.5mm-bores and a suction of 150 mm Hg was employed. The picture taken immediatelyafter the cup was released shows the gentle impressions of the grooved flange. Two ofthe vesieles formed show an indentation due to an anchoring follicle.

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studies where both epidermal and dermal traumashould be at minimum.

The fate of a suction blister depends on itssize. Less than one millimeter vesicles disappearwithin some minutes. Larger blisters do notvanish spontaneously after the release of suctionbut become somewhat flaccid with time. Whensheltered, for instance by the suction cup fixed withadhesive tape, the suction blisters remain unrup-tured at least for one week. The initially water-clearand acellular (19) blister fluid turns within a fewdays to an amber-color and contains cells.

Investigative Applications of Suction Blistering

In vivo separation of full-thickness viable epi-dermis by suction has versatile applications. Thesheets of epidermis, uncontaminated by fibroblastsand other dermal cells, provide material for cultureof epidermal cells (20). The suction separated epi-dermis might contribute to the studies in organculture of mutual interactions of epidermal anddermal components in the formation of the base-ment membrane material. Future studies will alsoshow, whether or not the epidermal cells of suctionblister roofs are adequate for immunological test-ing and for transplantation.

The suction blister roof suits several kinds ofbiochemical studies of epidermis (21, 22, 23, 24),for quantitation of epidermal bacteria (25) and forin vitro studies of cpidermal permeability (26).

Not only the epidermis but also certainstratified mucous epithelia can be separated bysuction in living human subjects (Fig. 6). Puresamples of oral epithelium may thus provide

material for biochemical comparison with theepidermis.

The diseased human skin does not always re-spond with blistering to suction. In acute eczemawith the loss of the healthy epidermal barrier, onlyoozing of fluid through the epidermis occurs. How-ever, from certain skin lesions suction bipsies havebeen successful, for instance, in cases of lichenruber planus, basal cell epithelioma and discoidlupus erythematosus (Fig. 7).

The determination of suction blister time incertain bullous diseases might be a quantitativesubstitute for the Nikolsky phenomenon. In ourpreliminary studies suction times down to as lowas two to five minutes were obtained on theerythematous skin of some patients with pemphi-gus foliaceus or pemphigus erythematosus by sue-tions of 200 mm Hg. In skin areas near spontaneouslesions of patients with pcmphigus vulgaris, Hailey-Hailey, bullous pemphigoid, erythema multiformeand acute urticaria, the blistering times variedgreatly from one patient to another, i.e. from thatof the normal skin to as low as 20 minutes (27).On the other hand, in several young adults withdermatitis herpetiformis (27) and in two patientswith hereditary epidermolysis bullosa the blisteringtime was always comparable to that of the healthyskin. The possible value of this test can be furtherstudied, when the individual variations and theeffects of sex and age on the suction blister timewill be established in large enough series.

Suction blisters can be raised on human skin invivo and also in vitro, i.e. on the cadaver skin atroom temperature and even at 00 C. The suctiontime here is generally greatly retarded, to as long

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damaged state (Fig. 10). Also the blood corpusclescan he visualized and their circulation studied. Theblister base is well suited for testing of pain pro-ducing substances, including plasma kinins, hista-mine and allergens.

Regeneration of the epidermis can be studiedunder controlled conditions, because the suctiontrauma of the dermis is small and of standardnature and the denuded area can be measured.

SUMMARY

Dermo-epidermal separation by applicationof suction on normal human skin has beenaccomplished. Subepidermal blisters are reg-ularly raised when low suction pressures arcmaintained for sufficiently extended periods.Previously used suction cups were all de-signed for other purposes and caused diffi-culties in consistent production of suctionblisters in a minimally traumatic and standardway.

By construction of a special device, standardsuction blisters of preselected size and num-ber can be raised on human skin withoutdiscomfort in about two hours by a suction

Fro. 6. Two suction blisters, 3 mm in diameter,produced on the buccal epithelium of the lowerlip within 80 minutes by a suction of 80 mm Hg.The miniature suction cup witb a 7 mm inner-diameter and the adapter plate used are shownbelow.

as six to 24 hours if suction pressures of 200 mmHg are used.

Suction detachment of small specimens of epi-dermis can be achieved also on furry animal skin.After clipping the hair and gluing a small cupto the skin, blistering could be accomplished invivo on adult guinea pigs (Figs. 8 and 9) and onthe downy skin of newborn rats.

The blister fluid of minimally traumatized blis-ters is a water-clear transudate, presumably amolecular filtrate of blood serum and lymph,containing about 2.0 per cent protein and neitherred cells nor inflammatory cells. It may serve asa substitute for tissue fluid or lymph (28). Theacellularity of the fluid in small fresh blisters isthe prerequisite for studies of cellular dynamics intoxic or allergic inflammation (19).

The base of the suction blister functions as a Fio. 7. Suction biopsy of a lesion of discoidskin window, permits visualization of the capillaries erythematosus showing characteristic keratinousand even of deeper vessels in a comparatively un- plugs.

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Fio. S. Histologic appearance of a suction blister produced on clipped abdominal skinof an adult living guinea pig. This section from the blister border shows dermo-cpidermalseparation of the interfollicular areas. Fibrinous exudate within the cavities and cellularinfiltration of the corium are indications of moderate suction trauma in the furry skin.This suction blister of 3 mm in diameter was produced by the miniature suction cup seenin Fig. 6, after gluing of the cup to the skin.

Fm. 9. At higher magnification the separation of the complete epidermis is shown moreclearly.

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Fm. 10. The base of the suction blister forms an actual skin window. Thus even deepervenous plexus can easily he brought into focus.

of 150 mm Hg. The blister roof consists ofviable, full-thickness epidermis; tbe clearfluid is a non-inflammatory transudate, andthere is no resultant scarring.

By this suction device it is also possible toseparate the epidermis from corpse skin andcertain furry animals, as well as the stratifiedepithelium of buccal mucosa.

The suction separated epidermis should beuseful for tissue culture and biochemicalanalyses. Some other uses of the suctionblister method are suggested.

REFERENCES1. Van Scott, E. J.: Mechanical separation of the

epidermis from the corium. J. Invest. Derm.,18: 377, 1952.

2. Gilbert, P., Mier, P. D. and Jones, T. E.: Animproved technic for the isolation of epi-dermis from human skin. J. Invest. Derm.,40: 165, 1963.

3. Spruit, D.: An improved technic for, the iso-lation of epidermis from larger specimens ofhuman skin. J. Invest. Derm., 42: 285, 1964.

4. Ciovanella, B. C. and Heideiberger, C.: Mouseepidermal cells and eareinogenesis. I. Iso-lation of skin constituents. Cancer Research,25: 161, 1965.

5. Weidenfeld, St.: Zur Physiologie der Blasen-bildung. I Mittheilung. Arch. Derm., 53: 1,1900.

6. Blank, I. H. and Miller, 0. C.: A method forthe separation of the epidermis from dermis.J. Invest. Derm., 15: 9, 1950.

7. Burbach, J. P. E.: Bijdrnge tot de Kennis vanBlaarvorming in de Huid. Akademisch Pro-esehrift, Amsterdam, 1957.

8. Kiistala, U. and Mustakallio, K. K.: In-vivoseparation of epidermis by production ofsuction bhsters, Lancet, 1: 1444, 1964.

9. Unna, P.: Zur Anatomie der Blasenbildung ander menschlichen Haut. Vjschr. Derm. Syph.,5: 1, 1878.

10. Unna, P. 0.: Die Histopathologie der Haut-Krankheiten, Berlin, 1894.

11. Bielickfr, T.: Messung der Zusammenhalt-barkeit der Hautschichten mittels Saug-druek. Dermatologiea, 112: 107, 1956.

12. Hecht, A. F.: Experimentell-klinische Unter-suchuagen ueber Hautbiutungen im Kindes-alter. Jb.f.Kinderh. (Erg.-Heft), 65: 113, 1907.

13. Borbély von, F.: Ueber die Blutungsbereit-schaft der Haut. Mtinch. Med. Wehsehr., 21:886, 1930.

14. Dalldorf, C.: A sensitive test for subclinicalscurvy in man. Am. J. Dis. Child., 46: 794,1933.

15. Brown, E. E.: Evaluation of a new capillaryresistometer: the Petechiometer. J. Lab.Clin. Med., 34: 2, 1949.

16. Rosengren, B.: A method for producing intra-

SEPARATION OF VIABLE EPIDERMIS 137

ocular rise of tension. Acta Ophthalm., 12:403, 1934.

17. Langham, M. E. and Maumenee, A. E.: Thediagnosis and treatment of glaucoma basedon a new procedure for the measurement ofintraocular dynamics. Trans. Amer. Acad.Ophthalm. Otolaryng., 68: 277, 1964.

18. Kiistala, U. and Mustakallio, K. K.: Dermo-epidermal separation with suction. Electronmicroscopic and histochemical study of ini-tial events of blistering on human skin. J.Invest. Derm, 48: 466, 1967.

19. Kiistala, U., Mustakallio, K. K. and Rorsman,H.: Suction blisters in the study of cellulardynamics of inflammation. Acta derma-tovener. (Stockholm), 47: 150, 1967.

20. Ingemansson-Nordqvist, B., Kiistala, U., andRorsman, H.: Culture of adult human epi-dermal cells obtained from roofs of suctionblisters. Acta dermatovener. (Stockholm),47: 237, 1967.

21. Mustakallio, K. K., Kiistala, U., Nieminen, E.and Leikola, E.: Les lipides de l'Spidermehumain. Separation de l'Spiderme in vivopar la technique de succion pour l'Stude deslipides par chromatographie en couche mince.Arch. biochim. cosmét., 7: 11, 1964.

22. Nieminen, E., Leikola, B., Koljonen, M.,Kiistala, U. and Mustakallin, K. K.: Quan-titative analysis of epidermal hpids by thin-layer chromatography with special reference

to seasonal and age variation. Acta derma-tovener. (Stockholm), 47: 327, 1967.

23. Mustakallio, K. K., Kiistala, U., Piha, H. 3.and Nieminen, E.: Epidermal lipids in Bes-nier's prurigo (Atopic eczema). Ann. Med.exp. Biol. Fenn., 45: 323, 1967.

24. Zenisek, A., Kral, 3. A., Strych, A., Hais, I. M.,Petranova, 0. and Hovorka, 3.: Le taux del'acide urocanique dans l'Spiderme des sujetsnormaux de differente origine. Arch. biochim.cosmét., 9: 49, 1966.

25. Mustakallio, K. K., Salo, 0. P., Kiistala, R.and Kiistala, U.: Counting tbe number ofaerobic bacteria in full-thickness humanepidermis separated by suction. Acta path.microbiol. acand., 69: 477, 1967.

26. Mustakallio, K. K., Kiistala, U., Piha, H. 3.and Nieminen, B.: Lipids extracted by di-methylsulfoxide (DMSO) from full-thicknesssheets of epidermis. Scand. 3. din. Lab. In-vest., 19, Suppl. 95: 50, 1967.

27. Putkonen, T., Mustakallio, K. K. and Kiistala,U.: Per Gebrauch einer Saugglocke bei derUntersuchung des Nikolsky-Phhnomens. Pre-sented at the Hungarian Dermatol. SocietyCongress, April 22, 1965, Budapest.

28. Hansson, H., Kiistala, U., Rorsman, H. andTryding, N.: Diamine oxidase in blisterfluid. Acta dermatovener. (Stockholm), 47:94, 1967.