BRONCHIAL BIOPSY CHRONIC AND ASTHMA - ThoraxBRONCHIAL BIOPSY IN CHRONIC BRONCHITIS AND ASTHMA and...

Thorax (1960), 15, 142.

BRONCHIAL BIOPSY IN CHRONIC BRONCHITISAND ASTHMA

BY

A. A. GLYNN AND L. MICHAELSFrom the Wright-Fleming Institute of Microbiology, St. Mary's Hospital Medical School and St. Mary's

Hospital, and the Department of Pathology, St. Mary's Hospital Medical School, London

(RECEIVED FOR PUBLICATION OCTOBER 24, 1959)

Chronic bronchitis is responsible for a greaterannual mortality in Great Britain than any othersingle respiratory disease, and its incidence is farhigher in this country than anywhere else in theworld (Oswald, 1958), yet relatively few compre-hensive studies of the pathogenesis of the conditionhave been published.

Considerable attention has been paid recentlyto the factor of infection in chronic bronchitis,especially with regard to the role of Haemophilusinfluenzae, since Mulder's work in 1938 (Mulder,1956; May, 1958). Brumfitt, Willoughby, andBromley (1957) have emphasized the preponder-ance of H. influenzae over other organisms whenswabs are taken directly from the bronchi in casesof chronic bronchitis. One of us (A. A. G.) hasfound antibodies to H. influenzae in high titre intwo-thirds of patients with the disease. The titreswere not significantly affected by intercurrentattacks of acute bionchitis.

It has long been recognized clinically thatsymptoms of chronic bronchitis may persist in theabsence of infection, and clinicians have also formany years been aware that a fundamentalcharacteristic of chronic bronchitis is the excessiveproduction of mucus in the respiratory tract.That infection may thereby be facilitated, andthat the association of infection and excess mucusproduction may ultimately cause emphysema, hasbeen suggested by Reid (1954). Evidence of ahistological basis for excess mucus production isas yet lacking, and there are few studies of thedetailed pathology of chronic bronchitis asdistinct from that of emphysema.

Rokitansky in 1838 described six cases ofchronic bronchitis in which grossly dilated mucousglands were present in the posterior wall of thetrachea and in the bronchi. This condition isnow recognized as an occasional radiological(Simon and Galbraith, 1953) and pathological(Duprez and Mampuys, 1953) inanifestation ofchronic bronchitis. Rokitansky also described

less severe degrees of this mucous glandulardilatation in some of his cases. Huber andKoessler (1922) gave a very detailed account ofthe pathology of " bronchial asthma " based onsix patients of their own and 15 culled from theliterature. It is clear from their description thatan infective element was present in many.Bronchitis was definite or probable in 12 of the13 in whom they describe mucous gland hyper-trophy, while only in one patient with bronchitiswas there mucous gland atrophy. Florey,Carleton, and Wells (1932) mentioned that histo-logical examination of the bronchi in a group of14 patients with chronic bronchitis showed dilateddeep glands, the majority of which were mucousin type. Reid, on the basis of post-mortem studies,stated that mucous gland hypertrophy and gobletcell hyperplasia are early changes in chronicbronchitis, and she has stressed the importance ofthis view in subsequent papers (Reid, 1958). Ingeneral, however, the pathological basis of chronicbronchitis is referred to merely in terms of thename as a chronic inflammation of the bronchialwall.We report here the results of a study of

bronchial biopsies in 45 patients. They fell clini-cally into two main groups: those with chronicbronchitis (27 cases) and those with asthma (18cases). A more detailed classification is givenbelow.From these biopsies from living patients we

hoped to establish and describe specific histo-logical changes in chronic bronchitis and inasthma, and to determine if possible their relation-ship to the severity and to the duration of thesedisorders. Secondly, in patients with clinicalevidence of both asthma and bronchitis we hopedthat histological evidence might throw light uponthe interrelationship of these two disorders.

MATERIAL AND METHODSBronchoscopy was carried out on 45 patients who

were admitted to St. Mary's Hospital for investigation

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BRONCHIAL BIOPSY IN CHRONIC BRONCHITIS AND ASTHMA

and treatment between August, 1957, and January,1959.CLASSIFICATION OF PATIENTS.-The patients were

divided on clinical grounds into 27 with chronicbronchitis and 18 with asthma.The diagnosis of chronic bronchitis was based on

a history of productive cough for at least two years,associated with periodic febrile exacerbations, particu-larly in winter. Eight patients had pure chronicbronchitis, 10 had started with pure bronchitis butafter a number of years had developed wheezingdyspnoea, and nine had wheezed from the onsetof bronchitis. The asthmatics all suffered fromparoxysmal attacks of wheezing dyspnoea, often withprolonged wheezing between attacks.

Evidence of an allergic basis for the asthma (clearclinical history of sensitivity and at least one of thefollowing: family history of allergy, skin tests, oreosinophilia) was present in eight patients. Theaetiology of the asthma in the remaining 10 isunknown.

In six, including four of the allergic patients,infection had become superimposed on the asthma.The diagnosis is discussed in more detail in relation

to the pathological findings.The patients were graded as mild, moderate, or severe

(1-3) by clinical assessment of their functional state.Those in grade 1 (13 cases) suffered little incapacity,those in grade 3 (11 cases) were frequently in hospitalor in bed at home and unable to do their normalwork. The remainder (21 cases) were put in grade 2.

BRONCHOSCOPY.-Patients were bronchoscoped in theward after premedication with omnopon and scopol-amine and under local anaesthesia with " lignocaine."Usually no difficulties were encountered and mostpatients were not distressed though some neededfurther sedation. There was one death (Case 22) in apatient with severe bronchopneumonia whose bronchiwere obstructed by secretions. Secretions weresucked out and the appearance of the mucosaobserved. A snippet of mucosa was taken, alwaysfrom the right middle lobe carina, using Brock'sforceps. Only a small amount of capillary bleedingof short duration was produced.

HISTOLOGICAL METHODS.-The fragment of bron-chial mucosa was fixed in formol saline, embedded inparaffin, and cut through its entire thickness at 5microns, all sections being mounted. In all casessections were stained by the following methods:haematoxylin and eosin, Van Gieson's stain, Verhoeff'sstain for elastic, mucicarmine, alcian blue, periodic-acid-Schiff, Gram's and methylene blue stains forbacteria, and toluidine blue for gamma meta-chromasia. In five cases of asthma and of chronicbronchitis sections were stained for reticulin byFoote's method and by the phloxine tartrazine methodfor inclusion bodies.

In one case of chronic bronchitis the material wasfixed in cold acetone and sections were stained foralkaline phosphatase by Gomori's method and fornon-specific esterase by the method of Nachlas and

Seligman (1949). Sections from all cases wereexamined with the oil immersion (1/12 in.) objectiveas well as by the usual magnifications.A record of the main histological features in each

case was made without knowledge of the clinicaldiagnosis.

Sections were also cut of the right middle lobecarina from necropsy specimens of 15 normal lungs,stained by the same methods and used as controlsin assessing variations seen in the biopsy material.

RESULTSBRONCHOSCOPIC APPEARANCES. -A variable

amount of mucus or mucopus was seen in thebronchi during bronchoscopy, but its presence,nature, and quantity bore inconstant relationshipsto the clinical diagnosis. In general, however, inpatients presenting primarily with asthma themucosa of the trachea and bronchi was seen onbronchoscopy to be smooth and moist. In somechronic bronchitics the mucosa was extremelysoft and thick so that it bulged over the edge ofthe bronchoscope. We do not feel, however, thatany definite relationship can be establishedbetween the naked-eye bronchoscopic appearancesand the clinical diagnosis.NoRMAL HISTOLOGY OF THE BRONCHUS.-The

normal bronchial epithelium consists of a surfacelayer mainly of ciliated cells with occasional gobletcells, an intermediate, and a basal cell layer(Fig. 1). The basement membrane on which theepithelium rests is composed of reticulin andcollagen fibres and varies from about 4 to 7

-~\ ;9 - - -V.._

_za } P. _~~~~~~~~P

FIG. 1.-Normal bronchial epithelium. Note surface, intermediateand basal layers and cilia arising from surface cells. Haema-toxylin and eosin x 830.

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A. A. GLYNN and L. MICHAELS

microns in thickness. The normal lamina propriausually shows some degree of lymphocytic infil-tration ; its deeper part consists of elastic fibreswhich rest on the smooth muscle layer. Beneaththe smooth muscle lie the deep bronchial glands.Two types of glandular acini may be

distinguished, mucous and serous. The latter arenormally more frequent, the mucous acini oftenbeing few and scattered. The serous acini arecharacterized by the presence of minute granuleswhich fill the cytoplasm. In most serous acinithese granules are basophilic and P.A.S. positive,but in occasional acini the granules are eosino-philic and do not stain with P.A.S. In the mucousacini there are usually some serous cells arrangedin demilunes. The cytoplasm of the mucous cellsconsists of large, irregular, weakly-basophilicglobules; it stains strongly with P.A.S. and muci-carmine. Flat, elongated myo-epithelial cellsadhere to the periphery of both mucous andserous glandular acini (Figs. 2 and 3). Ductslined by goblet cells pass through the smoothmuscle layer, lamina propria, and epithelium toopen on the surface. Peripheral to the deepbronchial glands is the bronchial cartilage.

HISTOLOGICAL CHANGES IN BIOPSIES OF CILIA.-Cilia were carefully examined, but we found noconstant qualitative alteration nor anything charac-teristic of bronchitis. In some, cilia were absentas a result of loss of the superficial and inter-

f.b.., ,.~. t ,. s \Age

t. jE - .. .

FIG. 2.-Normal bronchial glands in bronchial biopsy of a case ofasthma. There are numerous serous acini whicth stain moredeeply than the mucous acini. Haematoxylin and eosin x 104.

FIG. 3.-High-power view of part of Fig. 2. The serous cells containnumerous dark granules in their cytoplasm. These are eosino-phils and plasma cells between the glands. Haematoxylin andeosin x 830.

mediary layers of the epithelium, a loss which wasprobably an artefact and due to the trauma of theoperation. Sometimes, however, although thewhole thickness of the epithelium was intact, ciliawere deficient or absent. This finding was usuallyassociated with severe goblet cell hyperplasia, yetcilia were sometimes seen arising from surfacegoblet cells.

SQUAMOID METAPLASIA.-Squamoid metaplasiawas found in seven patients, six of whom hadasthma, the other chronic bronchitis. Thechange was not uniform throughout the epitheliumin any, and the remainder of the epithelium wasof normal columnar secretory type.

Typical " prickle cells " were not seen, so thatthe term squamous metaplasia cannot be applied;but in some cases intercellular connexions werepresent and perhaps represented early "prickle-cell" formation. Keratinization was not seen. InP.A.S.- or mucicarmine-stained sections mucoidsecretion was sometimes seen in the cytoplasm ofthe surface cells in squamoid areas; it was notvisible in haematoxylin and eosin sections (Figs. 4and 5). Mitoses were occasionally present in cellnuclei in the squamoid areas of epithelium.

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| ;&B.:B ^ ............... ; . Z ....~~~~~~~~~~~~~~~~~..........FIG. 4.-Bronchial biopsy from a case of asthma. The epithelium

shows squamoid change on the left and is of the normal ciliatedvariety on the right. Haematoxylin and easin x 403.

GOBLET CELL HYPERPLASIA.-A considerableincrease in mucus-containing goblet cells in thesurface epithelium was observed frequently in thisseries. This feature is shown in Tables 1-111 as

+, denoting that a majority of the surface cells

FtG. 5.-High-power view of part of squamoid area of Fig. 4. Theepithelial c:lls are flattened towards the surface. Haematoxylinand eosin x 830.

are goblet cells, and as + + when all surface cellsare goblet cells. The sign + indicates that an

occasional surface cell is a goblet cell, compatiblewith the appearance in the normal bronchus(Fig. 6).

TABLE IPATHOLOGICAL AND CLINICAL FEATURES IN CASES WITH MUCOUS CHANGE IN GLANDS (GROUP 1)

Cas Dep Eosinophils Golt DanssLength of Clncl H. influenzaeCaseN

Deep. Lmina Goblet DiagnosisG n

A SexaHistory Girandel in TobaccoNo. Gands Propria Cells I(Years) (Years) Gae sputum

14 M+ ++ + B 54 F 30 2 + 017 ,, 4 ±+3 58 F 20 2 + 012 +i B-IA 66 F 40 3 + 044 - ++ B- B+A 53 M 45 3 + 0

26 M+±+ 0 B 57 M 16 2 + +19 ,, 0 - B+A 54 M 12 3 0' +27 ± + B-B+A 60 M 18 3 + +31 4- +± B -B+A 53 M 17 2 + 034 ,, + + A-A+B, 26 M 9 2 + 022 ,, ± _ A2 57 F 10 2 0 029 , + A3 57 M 2 0 +

13 M+ + + B 65 M 3 2 + +11 0 ± B+A 58 M 6 2 016 + + + B+A 66 F 4 1 + _20 + ± B+A 64 F| 12 2 0 039 ,, 0 ± B+A 46 F 16 2 0 036 + ++ B--*B+A 58 M 3 2 + +25 4- + A 30 M 8 2 0 +

1 Acute infection treated with antibiotics before admission. 2 Plus pneumococcal bronchopneumonia. 3 Plus staphylococcal pneumonia.Mucous change in deep glands: M+ + +=All deep acini mucous. No serous acini. No demilunes. M+ + =Deep glands predomin-

antly mucous. Occasional serous acini. Occasional demilunes. M+=Increase of mucous glands. Numerous serous acini and demilunes.M± Small numbers of mucous glands only in section. S=Serous glands predominant. No mucous gland increase. S±=Small numberof serous glands only in section. - =No glands in section.

Diagnosis: A=Asthma. B=Chronic bronchitis. B+A=Chronic bronchitis with asthma. B- B+A=Chronic bronchitis developingasthma later. A- A+B=Asthma developing bronchitis later.

Goblet cells: + + =All surface cells are goblet cells. + =Most surface cells are goblet cells. ± =Occasional surface cells are goblet cells.- Surface epithelium absent .

Eosinophils in lamina propria: +=Numerous. ±=Occasional. 0=Absent.

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TABLE II

PATHOLOGICAL AND CLINICAL FEATURES IN CASES WITH PREDOMINANTLY SEROUS GLANDS (GROUP 2)

Dep EosinophilsDeep in Lamina

Glands Propria

3 Serous4 ,,7 1

33353743

2 ,.10 ,,18 ,,24 ,.9 ,.

21 ,,6 ,,

38 ,41 ,,42 ,,

GobletCells

I +

I + +

Diagnosis

AAAAAAA

A-A+BA-A+BA- A+BA-,A+BA-A+BB+A

B- B+AB,B+AB- B+A

BB

Age(Years)

654355521652344960475547605367496661

Length of Clinical H. influenzaeSex History Grade in Tobacco

(Years) Sputurn

F 6 3 0 0F 20 1 + _M 4 3 0 0F I11 2 0 0F 8 1 0 0F 25 2 0 0F 4 1 0 a-F 7 1 + _F 20 2 0 0FFFFF

FFF

M

318

133492

22

2

2

3

600

0

0

0

0

0

0

0

0

Mucous change in deep glands: M+++=All deep acini mucous. No serous acini. No demilunes. M+a+=Deep glands pre-dominantly mucous. Occasional serous acini. Occasional demilunes. M+=Increase of mucous glands. Numerous serous acini anddemilunes. Ma-=Small numbers of mucous glands only in section. S=Serous glands predominant. No mucous gland increase. SISmall number of serous glands only in section. -=No glands in section.

Diagnosis: A=Asthma. B=Chronic bronchitis. B+A=Chronic bronchitis with asthma. B-B+A- Chronic bronchitis developingasthma later. A-A+BA Asthma developing bronchitis later.

Goblet cells: +a + =All surface cells are goblet cells. + =Most surface cells are goblet cells. = Occasional surface cells are goblet cells.-=Surface epithelium absent.

Eosinophils in lamina propria: +=aNumerous. 4--Occasional. 0=Absent.

JTABLE IIIPATHOLOGICAL AND CLINICAL FEATURES IN CASES WITH FEW OR NO GLANDS IN SECTION (GROUP 3)

Case Deep Goblet DaAge Clinical H.influenaCase.\ Glandls in Lamina Cells Diagnosis (Yeae) Sex History Grade in Tobacco~Propria Clsr)(Years) Sputum

15 M 0 + B+A 46 M 4 3 +a 030 M± + B-B+A 43 F 17 2 + +40 Mt + + B 65 M 20 3 + +5 sE L ++ B-B+A

M40 3

0

8 A 38 M 7 2 0

23 S + + + A 57 F 6 1 0 032 S+ + _ B+A 34 F 4 1 0 028 0 + + B-.Ba+A 45 M 20 3 + +45 0 B 44 M 3 1 + 0

Mucous change in deep glands: M + + +± All deep acini mucous. No serous acini. No demilunes. Ma+ + =Deep glands predomin-antly mucous. Occasional serous acini. Occasional demilunes. M+-Increase of mucous glands. Numerous serous acini and demilunes.Ma--Small numbers of mucous glands only in section. S=Serous glands predominant. No mucous gland increase. S =Small numberof serous glands only in section. --No glands in section.

Diagnosis: A=Asthma. B=Chronic bronchitis. B+A-Chronic bronchitis with asthma. B-B+A=Chronic bronchitis developingasthma later. A-A+B=Asthma developing bronchitis later.

Goblet cells: + +-All surface cells are goblet cells. + =Most surface cells are goblet cells. Occasional surface cells are goblet cells.-=Surface epithelium absent.

Eosinophils in lamina propria: +aNumerous. I=Occasional. 0=Absent.

BACTERIA. Bacteria, including bacilli resemblingH. influenzae, were not seen in the epithelium on

examination of Gram- or methylene-blue-stainedsections, despite repeated search.BASEMENT MEMBRANE.-Measurements were

made of the thickness of basement membrane inVan-Gieson-stained sections using an eyepiecemicrometer. Care was taken to make measure-

ments in portions of basement membranesubjacent to vertically cut epithelium.

Abnormalities noted were: (1) thinning,(2) reduplication, and (3) thickening.

(1) Thinning of basement membrane was seen

in 10 cases. It was always associated with severe

inflammatory congestion of the lamina propria,with numerous congested capillaries in contactwith the outer surface of the basement membranegiving an appearance suggesting absorption of thelatter by encroachment of capillaries on it (Fig. 7).This change was found in cases of both asthmaand chronic bronchitis.

(2) Reduplication occurred in two patients withasthma in whom the basement membrane consistedof numerous collagenous layers each of which

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FIG. 8.-Bronchial biopsy from a case of asthma showing reduplica-tion of the basement membrane. Instead of a single homogen-eous layer the basement membrane now consists of numerouslayers of collagen separated by fibroblasts. Haematoxylin andvan Gieson x 830.

FIG. 6.-Bronchial epithelium from biopsy of a case of asthma-There is a layer of mucus on the surface, appearing black in thephotograph but red in the original. Goblet cells are increasedin number and constitute most of the surface cells. Note thebasement membrane beneath the epithelium. Periodic-acid-Schiff x400. B- - * 4f _ ibR _S : ,ie. i q g --- NL

t*W F-! sS z # N ^_. '.s'...s. .. t :b ^' .^s - ..__o j WE . &. gt - § | < N 9'i-- ' ; . _ts .pa 3 - Lit' > ffl>,<ie 9 ffi if; .....e

-lWj#i < Y 9 jijSlW <jR = . - _t IIts , -t * sCo w t - -s @';Il-" . SLi . ;wk. :vt1 . j _ r: ,,, we.. .. - . Y- w . ef _ -<Fz IXW _; =sV.:B . g e FP |f; _ * -11 iFip $X SX ^ ; s _w Xlkt- w- je;;e j '? .o in; itMj f W -* | -] b w w

_|ip ;, i w b Z 't t-ta . :4 __ t * wH ... S: Wt, ............... ..... to . - o' -- P:'_ ..... *FIG. 7.-Bronchial biopsy from a case of asthma showing thinning

of the basement membrane. Note the inflammatory congestionand exudate encroaching on basement membrane. Haematoxy-lin and van Gieson x 830.

was thinner than normal basement membrane.Fibroblasts were present between the layers ofcollagen (Fig. 8).

(3) A definitely thickened basement membraneof 9 to 10 ,u in width was seen in only threepatients, two with asthma and one with bronchitis(Fig. 9).LAMINA PROPRIA.-A degree of leucocytic infil-

tration was present in the lamina propria in every

FIG. 9.-Bronchial biopsy from a case of asthma showing thickeningof the basement membrane (about 11 p) which is here representedby an unstained zone between epithelium and lamina propria.Haematoxylin and eosin x 830.

case, but the extent of the observed infiltrationwas unrelated to infection or to the severity ofthe clinical condition. In asthma, the exudateusually consisted predominantly of eosinophil andplasma cells. The former did not need specialstaining, being readily recognized in haematoxylinand cosin-stained sections, using an oil immersion

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objective, by their large pink granules. Theeosinophil cells were sometimes disintegrated, and _.were then represented only by numerous free .Agranules in some areas. Eosinophil cells werecommonly found within the lumina of capillariesas well as in the connective tissue. Occasionally ,they were seen straddled across the basement imembrane; they were also often present inmoderate numbers within the epithelium andaround the deep glands. They were alwaysaccompanied by a variable, often heavy, infiltrate A

of plasma cells. Seven of the cases showing heavy NY

eosinophilia of the lamina propria were under i*treatment with prednisone when the bronchial -biopsy was taken. In chronic bronchitis theexudate was variable in extent and type, and largenumbers of polymorphs, plasma cells, lymphocytes,and macrophages with an occasional eosinophilwere usually found. 4v

Variation was also found in the thickness of + .the elastic layer of the lamina propria. It was inno way characteristic either of asthma or of *ibronchitis.

vs4^-* 114-{...............v_ X ~....... o o

LZ;. :8 .'. @ I. High-powr.m.41-,_S f E _ t FIG. 1I1.-High-power view of part of Fig. 4. There is a leucocyticinfiltration between the glandular acini. Haematoxylin and

4. .e e effl Jostin x400.

t .TXr ''tXsfW t >8 S MUSCLE.-The smooth muscle layer, comparedwith normal controls, was not modified in

t -^j4 bronchitis or in asthma.GLANDS.-In most biopsies a considerable

amount of deep glandular tissue was present.V4~ k id ^;>vMarkedchanges were found in the deep glandsild,i in most chronic bronchitics in this series. These--s ½|*j89)*=vjw<6 idpi changes consisted of a variable degree of replace-= g E fSt-* = eF7ment of the normally predominant serous glands

§ by hypertrophied mucous glands. The following~sw~ g./Ai4+s ~ description applies to all cases in this study found

to have severe mucous gland hypertrophy and4 g g g ';f. ff- , designated in the tables as + + + or + +. Serousv<85;,|^X* ;i + iZ: glands were greatly reduced in number or absent;

if present their morphology was normal, being'I .b ' usually of the basophilic variety, though in some

cases a few eosinophilic granular acini wereg tspresent. Demilunes were absent. Only an

¶Vzoccasional glandular acinus in any field showedFiG. I0.-Deep bronchial glands from biopsy of a case of severe evidence of active mucus discharge; the appear-

chronic bronchitis (30-year history) showing severe mucous ances are then of small flat cells enclosing achange (+ + +). The acini are all mucous in type and are lumen filled with mucus. Most of the mucouslarger than normal. Magnification and staining are the sameas in Fig. 2. acini showed overdistension of the cytoplasm with

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mucus rather than its excessive discharge intothe lumen. The mucous acini were greater indiameter than normal, their component cells tallerand somewhat wider, and had cytoplasm stainedin a less basophilic fashion than in normal bronchi.Mitoses were not seen (Figs. 10 and 11).

Differences were not found between the stainingproperties of normal and hypertrophied mucousglands. A strong reaction with P.A.S. and muci-carmine and gamma metachromasia with toluidineblue was shown by the cytoplasm of the mucousglands; this latter reaction is often seen in themucous glands found in the normal bronchus andit cannot be taken to denote any chemical changein the mucous secretion in cases of chronicbronchitis. Inclusion bodies were not seen inphloxine-tartrazine- stained sections. No esterasewas found in the biopsy from the single casetested in this way. Alkaline phosphatase waspresent only in its customary situation aroundblood vessels.The ducts were often lined by goblet cells

showing a more distended, less basophilic mucuscontent than normal, and in several specimensthere was an apparent increase in ducts centralto the muscle layer, with glandular acini, includingserous glands, arising from the ducts in thisabnormal situation (Fig. 12).

Lesser degrees of mucous change in the deepglands are designated as + in the tables.

"4~~~~~~~~~~~~A

s-'4VRi R.tk"4. 4. e

FIG. 12.-Bronchial biopsy from cases of long-standing chronicbronchitis. Small acini, some of them serous in type, are

arising from ducts in the lamina propria. Haematoxylin andmucicarmine x 140.

CORRELATION OF CLINICAL WITH PATHOLOGICALFINDINGS. -The clinical diagnoses, made in-dependently of the histological appearances, arecomplicated, ranging from patients with purebronchitis through those with varying degrees ofbronchitis and asthma to those with pure asthma.We have therefore tabulated our results on thebasis of the major histological finding-mucouischange in the deep glands-into three groups.Group 1 consists of 18 patients with mucouschange in the bronchial glands, group 2 of 18patients with predominantly serous glands, andgroup 3 of nine patients in whom too few glandswere present in the sections for them to beproperly classified. The clinical features and theother histological findings in the three groups arepresented in Tables I to III and discussed morefully below.

Group 1.-Four teen of the 18 patients (Table I)in this group had chronic bronchitis. In 10 ofthese some asthmatic symptoms were also present,but all had well-established cough with purulentor mucopurulent sputum. H. influenzae wasisolated from the sputum in 10 of the 14 cases.The remaining four patients were all asthmatics.

Case 34, though primarily suffering from pollenasthma, had had, over the previous 18 months,two severe and prolonged attacks of bronchitiswith H. influenzae in his sputum. At the timeof biopsy Case 22 had a severe pneumococcalbronchopneumonia, and Case 29 a staphylococcalpneumonia with abscess formation. Only in Case25, who had asthma with a minor allergic and amajor psychogenic element, was there no clinicaland bacteriological evidence of infection.With a few exceptions the degree of mucous

change was related to the length of history. Thusall patients with severe mucous change (+ + +)had a history of 20 years or more of chronicbronchitis. Lesser degrees of the change wereassociated with a shorter history.

Although it is a little surprising that three ofthe four with severe mucous change were women,the probability of this occurring by chance is 1 in23, given the relatively high proportion of women(seven out of 18) in the whole bronchitis group.This excess of women compared with the numbersfound in special bronchitis clinics is almostcertainly due to local factors in selection. Manypatients were referred via the allergy clinic, andthere were more female than male beds available.The reports of Fry (1954), Goodman, Lane, andRampling (1953), and others show that chronicbronchitis is more common in women than issometimes supposed.

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The early age of onset (8 years) in Case 44 raisesthe possibility of some inherent abnormality ofmucous secretion, but, in the absence of anysupporting evidence for this hypothesis and takingthe series as a whole, it seems better to regard themarked changes as the result of prolonged disease.

Increased numbers of goblet cells were foundin 10 patients, three with bronchitis, five withbronchitis and asthma, and two with asthma alone.All but the two last had H. influenzae in theirsputum.

Excessive infiltration with eosinophils was foundin only four patients with bronchitis, all of whomalso had asthmatic symptoms. Three of these(Cases 12, 20, and 34) had an allergic history.Only one (Case 20) had ever had a blood eosino-philia (1,000/c.mm.=8%) though not at the timewhen the biopsy was taken.

Group 2.-All but two of the 18 cases in thisgroup were predominantly asthmatic. Of the twoexceptions one, Case 42, had had mild bronchitisfor two years, the other, Case 41, had had severebronchitis for many years.Of the asthmatics three (Cases 6, 21, and 38)

had started with mild bronchitis, but when seenhad had little or no cough and sputum for someyears. In none of the three was there evidenceof infection. Five more asthmatics had developed,after several years of asthma, mild attacks of acutebronchitis in the 12 to 18 months before beingbronchoscoped.H. influenzae was isolated from the sputum in

five patients, including one asthmatic with noclinical evidence of infection. Ten patients hadlarge numbers of goblet cells; in seven of thesethere were symptoms of mild bronchitis.

Eosinophils were numerous in the laminapropria in 17 cases including both the purebronchitics. Nine of the 17 (Cases 2, 3, 4, 9, 18,21, 24, 38, and 42) at some time had a bloodeosinophil count of over 9%.

Group 3.-In two of these no deep glands at allwere seen in the sections; in the remainder veryfew glands were present.

In three patients, all with bronchitis, the fewglands present were mucous. In four, two withbronchitis and asthma and two with pure asthma,the glands were serous.

Five patients, four of them with H. influenzaein the sputum, had increased numbers of gobletcells.

Three in this group showed an excess ofeosinophil infiltration in the lamina propria.

TOBACCOSmoking was more common (seven out of 16)

among the patients having mucous gland excessthan in those with serous glands (two out of 14)(Tables I and II).However, as information about smoking habits

was not complete it would be unwise to stress thedifference between the two groups. Moreover,Table I shows that there is no relation betweensmoking and the degree of mucous conversion,which suggests that tobacco is not the immediatecause of mucous change, though it has been shownby Oswald and Medvei (1955) and Palmer (1954)that bronchitis is more frequent among smokersthan among non-smokers. The present distribu-tion of smokers may simply be a reflection of thefact that 10 of the 13 were men, among whomboth bronchitis and smoking are more common.

Goblet cell hyperplasia was not foundsignificantly more frequently among smokers.

DISCUSSIONIn general the histological pattern in chronic

bronchitis would seem to be quite distinct fromthat in asthma. In chronic bronchitis few or noeosinophils were found in the lamina propria, andthe deep glands showed an increase in numberand size of mucous acini as compared with normal.In asthma, on the other hand, eosinophils wereplentiful in the lamina propria and the deep glandswere morphologically normal, with a preponder-ance of serous glands.The mucous gland change was the most striking

feature in chronic bronchitis. We usually obtainedsufficient glandular material to see this changeclearly, because the biopsy was taken at thebifurcation of a bronchus, where we had a firmgrasp of the whole thickness of the mucosa.Sometimes when the mucosa was seen to bedistinctly thickened at bronchoscopy, microscopicexamination showed severe "mucous " change,namely, a particularly large number of deepmucous glands.The pathological features comprising mucous

change consisted of an increase both in thenumber of mucous relative to serous glands, andan increase in size of the mucous acini due to theswelling of their component cells and their lumina.We think it is unlikely that new formation ofglandular acini takes place; the change is prob-ably the result of transformation of serous intomucous acini followed by increase in size of thelatter. The granules of the majority of serouscells gave similar staining reactions to the cyto-plasm of the mucous cells, and interchange from

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one type of cell to the other might be a recurrentprocess in their normal activity. The terms" mucous " and " serous" as applied to bronchialglands are derived from the morphologicalsimilarity that these glands show to salivary glandacini, where each type of acinus has been shownto produce its own distinct form of secretion. Nosuch relationship has ever been shown with regardto the glands in the respiratory tract, and it seemsinherently more likely that the two histologicalvarieties represent different grades of activity inthe production of mucous secretion. We considerthe mucous change, therefore, as being in thenature of a work hypertrophy of the secretorytissue.

Probably the mucous glandular transformationtakes place not only at the site examined in ourbronchial biopsies but also at all levels of thebronchial tree. We have studied histologicallypieces of trachea removed from the anteriortracheal wall at tracheostomy in two chronicbronchitics. Both showed appreciable increasein mucous glands as compared with the normal.In one, a man of 40 with one year's history ofbronchitis and a recent bronchopneumonia, themucous change was of moderate degree (corre-sponding to between + and + +). In the other,a woman of 55 with a history of chronicbronchitis for four years and recent broncho-pneumonia, the mucous change in the trachealglands was more severe (corresponding to between+ + and + + +). Studies of the bronchial glandsin cases of chronic bronchitis coming to necropsyare being carried out by one of us (L. M.) andhave already shown in three cases a striking degreeof mucous glandular change (+ + +) in all partsof the trachea and bronchial tree, the deep glandspresenting a far greater volume of tissue thannormal.We have obtained histological material from

each patient on a single occasion only, so thatwe cannot state whether the mucous change ispermanent or reversible. We think that some withmild mucous change do revert to normal, but insevere chronic bronchitis, in view of the persistenceof symptoms, the mucous change is permanent.The diagnosis of chronic bronchitis has hitherto

only been possible on clinical grounds by a processof exclusion of other respiratory or cardiovasculardiseases in a patient with a chronic cough(Scadding, 1959). The evidence we present heresuggests that the mucous change in the deep glandsmay represent the pathological basis of chronicbronchitis, which correlates well with the clinicalview that an increased production of mucoidsputum is a basic feature of the disease (Oswald,

1958). The pathological changes vary in degreeaccording to the severity of the disease, which mayoften be measured by the length of history, sincemost patients become progressively worse withpassage of time. In four of our patients with verylong histories of chronic cough, a maximum degreeof mucous change was present. Lesser degrees ofthis change are not specific and may be producedby relatively acute respiratory infections, since theywere seen not only in chronic bronchitis of shorterduration but also in two patients with asthma inwhom bronchopneumonia had developed at thetime when the biopsy was taken (Table I, Cases22 and 29). Because of this clear relationship ofthe glandular changes to the severity of the disease,we feel that bronchial biopsy in which glandulartissue is included may prove to be a valuable aidin the diagnosis and prognosis of chronic bron-chitis as well as in the scientific study of thedisease. The functional grading as defined aboveof the cases of chronic bronchitis shows less rela-tionship to the degree of mucous change than doesthe length of history, but it should be rememberedthat such a grading is a very rough assessment ofthe severity of the disease and is probably anindex more of the degree of coexistent emphy-sema than of chronic bronchitis.

Increase in surface goblet cells was seen in manycases in this series. This feature was describedby Reid (1954) as an early change in chronicbronchitis. The tables show that many cases ofasthma as well as chronic bronchitis havemoderate (+) or marked (+ +) increase of gobletcells. Engel (1958) suggests that increase in gobletcells may simply be a manifestation of mild irrita-tion to the bronchus. Since infection may be animportant type of irritant producing this change,we have tabulated (Table IV) the incidence ofinfection in patients with and without goblet cellhyperplasia. The positive association betweengoblet cell hyperplasia and H. influenzae infection

TABLE IVRELATIONSHIP OF GOBLET CELL HYPERPLASIA TO

INFECTION

No. of No. ofTotal Cases with Cases withToa H. influenzae PneumococciCases Isolated Isolated

from Sputum from Sputum

Normal numbers of gob-let cells (N) I1 2 4

Goblet cells increased(+ or ++) 27 17 9

No surface epithelium insection .. 7 3 4

Cf. goblet cell hyperplasia in patients with and without H. influenzaeinfection y2=6-2. n 1, P-=0<002.

Cf. goblet cell hyperplasia in patients with and without pneumo-coccal infection x2= 0-02, n = I. P=0> 0 9.

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is significant at the 2% level, but the distributionof pneumococci could easily have occurred bychance. Brumfitt et al. (1957) have stressed thatnot all organisms found in the sputum arise fromthe bronchi. Pneumococci are frequently contami-nants from the throat. In the present series thepneumococci were found in the throat or nose ofeight of the 17 patients with pneumococci in thesputum. In only four cases did the course ofillness and the response to antibiotics suggest thatpneumococci were playing a significant part. Thegoblet cells were increased in two of these.However, although in this series infection withH. influenzae appears to have been important inthe production of goblet cell hyperplasia, it is clearfrom Table IV that other factors must also some-times operate.We founid eosinophils were numerous in the

lamina propria in most asthmatics, and thisfeature was more nearly constant than the presenceof a blood eosinophilia or of numerous eosinophilsin the sputum. Numerous eosinophils wereoccasionally seen in the lamina propria of ourchronic bronchitics, but these usually hadasthmatic symptoms as well, and it is possiblethat there was an added allergic factor in thesecases. Two with symptoms of bronchitis only(Cases 41 and 42, Table II) had a heavy eosinophilinfiltrate and no mucous change in the bronchialglands. A condition of " allergic eosinophilicbronchitis" has been described by Mulder (1956)in which patients with symptoms of bronchitis andnot asthma have numerous eosinophils in theirsputum. Cases 41 and 42 would seem to fit intothis category.The presence of a local eosinophilia in the seven

cases in which therapy with prednisone was inprogress when the biopsy was taken is of interest.The blood esosinophils are as a rule con-siderably reduced in such patients, suggestingthat the eosinophils seen may have migrated fromthe blood or have been produced locally inthe bronchial wall. Numerous eosinophils arecommonly seen in the lumina of capillaries in thelamina propria of asthmatics, and it is possiblethat the new formation of eosinophils takes placeclose to the capillary walls. This may explain thepersistence of eosinophils in the sputum after theyhave disappeared from the blood under theinfluence of steroid therapy.Thickening of the basement membrane beneath

the bronchial epithelium is described as a constantfeature in bronchial asthma (Cardell, 1956), butwas seen in only two of the 18 patients withasthma in the present series. In most reports

available to us on the state of the basementmembrane in asthma it has been described asthickened on the basis of a study of randomsections usually of small bronchi included withlung material. With few exceptions no attempthas been made in the published reports tostandardize the site at which the membrane wasobserved or to attempt measurements in verticallycut areas. Our measurements on biopsy materialat a standard site in the bronchial tree suggest thatthickening of the basement membrane is not ascommon in cases of asthma as would be supposedfrom the literature, which is based mainly onreports of cases of status asthmaticus coming tonecropsy. Huber and Koessler (1922) foundthickening of all elements of the bronchial wallin their patients with asthma (and bronchitis)compared with bronchi of similar outside diameterfrom normal controls. However, Kourilsky,Decroix, Blondeau, and Ganter (1952) observed" fibrosis " of the lamina propria in 10 of their19 cases in which biopsy material from the rightmiddle lobe bronchus was obtained. This appearsto be similar to or identical with the reduplicationof the basement membrane which we havedescribed in two of our asthmatics. Thickeningof the basement membrane was also seen in onechronic bronchitic in our series. This is in keepingwith the observations of Crepea and Harman(1955), who have shown that basement membranethickening is not confined to asthma but may befound in a wide variety of lung diseases.

SUMMARYBiopsy of the bronchial mucosa at the right

middle lobe carina was performed in 45 patientswith chronic bronchitis or asthma or a combina-tion of the two.The bronchitics frequently showed changes in

the deep glands, characterized by an increase insize and number of mucous acini and a disappear-ance of serous cells. The degree of mucous changeappeared to be related to the duration of thedisease. We suggest that mucous change of thedeep glands is the cardinal pathological featureof severe chronic bronchitis. Patients with asthmaconsistently showed a heavy infiltration of eosino-phils into the lamina propria.

Hyperplasia of surface goblet cells was seen inboth asthma and chronic bronchitis and wasprobably related to infection.Squamoid metaplasia of the epithelium was seen

in seven patients, but true squamous change wasnot. Thinning, reduplication, and thickening ofthe basement membrane were found.

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We wish to thank Dr. W. D. W. Brooks, ProfessorD. M. Pryce, and Dr. A. W. Frankland for muchencouragement and advice, Mr. L. L. Bromley forinstruction in the use of the bronchoscope, Mr. P.Abbey for the provision of the tracheostomy speci-mens, and Miss G. Latham for invaluable technicalassistance.

REFERENCESBrumfitt, W., Willoughby, M. L. N., and Bromley, L. L. (1957).

Lancet, 2, 1306.Cardell, B. S. (1956). Int. Arch. Allergy, 9, 189.Crepea, S. B., and Harman, J. W. (1955). J. Allergy, 26, 453.Duprez, A., and Mampuys, R. (1953). Thorax, 8, 141.Engel, S. (1958). J. clin. Path., 11, 302.Florey, H., Carleton, H. M., and Wells, A. Q. (1932). J. exp. Path.,

13, 269.

Fry, J. (1954). Brt. med. J., 1, 190.Goodman, N., Lane, R. E., and Rampling, S. B. (1953). Ibid.,

2, 237.

Huber, H. L., and Koessler, K. K. (1922). Arch. intern. Med., 30,689.Kourilsky, R., Decroix, G., Blondeau, M., ana Ganter, Y. (1952).

J. franc. Med. Chir. thor., 6, 201.May, J. R. (1958). In Recent Trends in Chronic Bronchitis, ed.

Oswald, N. C. Lloyd-Luke, London.Mulder, J. (1938). Acta med. scand., 94, 98.

(1956). Proc. rov. Soc. Med.. 49. 773.Nachlas, M. M., and Seligman, A. M. (1949). J. nat. Canicer Inst., 9,

415.Oswald, N. C. (1958). Ed. of Recent Trends in Chronic Bronchitis.

Lloyd-Luke, London.and Medvei, V. C. (1955). Lancet, 2, 843.

Palmer, K. N. V. (1954). Brit. med. J., 1, 1473.Reid, L. McA. (1954). Lancet, 1, 275.- (1958). Postgrad. med. J., 34, 24.Rokitansky, C. (1838). Ost. Jb., 16, 382.Scadding, J. G. (1959). Lancet, 1, 323.Simon, G., and Galbraith, H.-J. B. (1953). Ibid., 2, 850.

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BRONCHIAL BIOPSY CHRONIC AND ASTHMA - ThoraxBRONCHIAL BIOPSY IN CHRONIC BRONCHITIS AND ASTHMA and...

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