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LIBRAR Y
Michigan State
Univ «any '

     
   

  

This is to certify that the

thesis entitled

Selective Bronchial Catheterization and
Bronchial Brush Biopsy in the Dog

presented by

Gary Wayne Thayer

has been accepted towards fulfillment
of the requirements for

Master Science

degree in

 

 

[2 , , .

Major professor ,A

:/

I

Date /{/i/7f

0-7 639

SELECTIVE BRONCHIAL CATHETERIZATION AND
BRONCHIAL BRUSH BIOPSY IN THE DOG

BY

Gary Wayne Thayer

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Small Animal Surgery and Medicine

1978

ABSTRACT

SELECTIVE BRONCHIAL CATHETERIZATION AND
BRONCHIAL BRUSH BIOPSY IN THE DOG

BY
Gary Wayne Thayer

Principle, lobar and segmental bronchi were selectively Catheterized
via the cricothyroid membrane or endotracheal tube using a polyethylene
catheter . Cytologic specimens were collected using nylon and stainless
steel bronchial brushes attached to flexible guide wires . Cytologic prepara-
tions made by touching or smearing the brush on the slides were compared.

Inflammatory disease was created in 1 lung of a group of dogs .
Bronchial brush biopsies were compared by evaluating the relative number
of inflammatory cells seen in the cytologic specimens from the control lung ,
the affected lung , and bronchial washings . The procedure was performed
on 19 dogs with spontaneous respiratory tract disease .

Selective bronchial catheterization via the endotracheal tube was found
to be the method of choice. Nylon bronchial brushes with cytologic
specimens made by the smearing technique were superior in the dog . The
bronchial brush biopsy was shown to be a selective procedure in identifying
an area of pathology within the lungs .

Fifteen of 19 (79%) dogs with spontaneous respiratory tract disease

had bronchial brush biopsies consistent with the final diagnosis.

This work is dedicated to my wife and parents for their
inspiration, encouragement, and sacrifices that I

might obtain my career goals as a veterinarian.

ii

ACKNOWLEDGEMENTS

I would like to recognize the following for their contribution
to this work:

Dr. Colin B. Carrig as major professor and for his encouragement
and guidance.

Dr. George Eyster for encouraging his clients to allow this biopsy
procedure to be performed on their dogs.

Mrs. Cleo Taggart for her assistance in the administrative tasks
necessary to complete this work.

The Department of Small Animal Medicine at the University of

Georgia for allowing time to complete this thesis.

TABLE OF CONTENTS

I. INTRODUCTION --------------------------------------------- 1
II. LITERATURE REVIEW ---------------------------------------- 4
History of Bronchial Brushing+-------------------7-7 --------- 4
Indications for Bronchial Brushing --------------------------- 4
Contraindications for Bronchial Brushing --------------------- 5
Applied Gross Anatomy -------------------------------------- 5
Applied Microscopic Anatomy -------------------------------- 7
Technique of Selective Bronchial Catheterization and
Bronchial Brushing ---------------------------------------- 10
Complications of Bronchial Brushing ------------------------- 13

Cytology Obtained from the Lower Respiratory Tract

of Animals ------------------------------------------------ 14

Results Obtained by Bronchial Brushing in Human Patients---- 16

III . MATERIALS AND METHODS ----------------------------------- 21
Equipment for Selective Bronchial Catheterization and

Bronchial Brushing ---------------------------------------- 21

Technique of Selective Bronchial Catheterization and

Bronchial Brushing ---------------------------------------- 25

A . Catheterization via the Cricothyroid Membrane ---------- 25

B . Catheterization via the Endotracheal Tube -------------- 32

Preparation of Slides for Cytologic Evaluation ---------------- 35

iv

Evaluation of Cytologic Preparations ------------------------- 35

Evaluation of Nylon Bronchial Brushes vs . Steel Bronchial
Brushes -------------------------------------------------- 35

Evaluation of Nylon Bronchial Brushes vs . Bronchial
Washing in Experimental Inflammatory Disease --------------- 41

Evaluation of Bronchial Brushing in Dogs with Clinical
Disease --------------------------------------------------- 43

IV . RESULTS ---------------------------------------------------- 44

Technique of Selective Catheterization and Bronchial
Brushing -------------------------------------------------- 44

Preparation of Slides for Cytologic Evaluation ----------------- 45

Evaluation of Nylon Bronchial Brushes vs . Steel Bronchial
Brushes --------------------------------------------------- 45

Evaluation of Nylon Bronchial Brushes vs . Bronchial
Washings in Inflammatory Disease --------------------------- 51

Evaluation of Bronchial Brushings in Dogs with Clinical

Disease --------------------------------------------------- 60
V0 DISCUSSION ------------------------------------------------- 76
VI. BIBLIOGRAPHY ---------------------------------------------- 83

10.

11.

LIST OF TABLES

BRONCHIAL BRUSH BIOPSY IN HUMAN PATIENTS WITH PRIMARY
PULMONARY NEOPLASIA ------------------------------------- 17

BRONCHIAL BRUSH BIOPSY IN HUMAN PATIENTS WITH .
SECONDARY PULMONARY NEOPLASIA--------------=--, --------- 18

BRONCHIAL BRUSH BIOPSY IN HUMAN PATIENTS WITH
CONFIRMED INF LAMMATORY DISEASE ------------------------- l8

CRITERIA FOR EVALUATION OF CYTOLOGIC PREPARATIONS---- 40

CRITERIA FOR EVALUATION OF INFLAMMATORY RESPONSE IN
CYTOLOGIC PREPARATIONS ----------------------------------- 42

EVALUATION OF CYTOLOGIC PREPARATIONS OBTAINED BY
THE TOUCH AND SMEAR TECHNIQUES ------------------------- 47

EVALUATION OF CYTOLOGIC PREPARATIONS OBTAINED BY
NYLON AND STEEL BRONCHIAL BRUSHES ---------------------- 50

INF LAMMATORY RESPONSE OBSERVED WITH BRONCHIAL
WASHING AND BRONCHIAL BRUSHING IN DOGS WITH
INF LAMMATORY DISEASE ------------------------------------- 56

EVALUATION OF CYTOLOGIC PREPARATION: BRONCHIAL
BRUSHING VS . BRONCHIAL WASHING -------------------------- 61

PERTINENT DATA OF CLINICAL PATIENTS WITH NEOPLASTIC
DISEASE EVALUATED BY BRONCHIAL BRUSH BIOPSY ----------- 62

PERTINENT DATA OF CLINICAL PATIENTS WITH

NON-NEOPLASTIC DISEASE EVALUATED BY BRONCHIAL .
BRUSH BIOPSY ............................................... 64

vi

LIST OF FIGURES

POLYETHYLENE CATHETERS USED IN SELECTIVE BRONCHIAL
CATHETERIZATION FOR BRONCHIAL BRUSHING --------------- 22

1 . 72mm NYLON BRONCHIAL BRUSH MOUNTED ON A 100cm
DEFLECTABLE TIP GUIDE WIRE ------------------------------- 23

1.72mm NYLON (A) AND STEEL (B) BRONCHIAL BRUSHES ----- 24

NORMAN MASK ELBOW (arrow) INSERTED IN THE ANESTHETIC
SYSTEM WITH BRONCHIAL CATHETER INSERTED . THE DRAPE
HAS BEEN REMOVED FOR PHOTOGRAPHIC ILLUSTRATION ------- 26

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF

THE THORAX OF A 1 YEAR OLD BEAGLE DOG DEMONSTRATING
SELECTIVE CATHETERIZATION OF THE CRANIAL SEGMENTAL
BRONCHUS OF THE LEFT CRANIAL LOBE ---------------------- 28

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF THE
THORAX OF A 1 YEAR OLD BEAGLE DOG DEMONSTRATING
SELECTIVE CATHETERIZATION OF THE CAUDAL SEGMENTAL
BRONCHUS OF THE LEFT CRANIAL LOBE ---------------------- 29

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF THE
THORAX OF A 1 YEAR OLD BEAGLE DOG DEMONSTRATING
SELECTIVE CATHETERIZATION OF THE CRANIAL SEGMENTAL
BRONCHUS OF THE RIGHT CRANIAL LOBE --------------------- 30

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF THE
THORAX OF A 1 YEAR OLD BEAGLE DOG DEMONSTRATING
SELECTIVE CATHETERIZATION OF THE CAUDAL SEGMENTAL
BRONCHUS OF THE RIGHT CRANIAL LOBE --------------------- 31

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF THE
THORAX OF A 1 YEAR OLD BEAGLE DOG DEMONSTRATING
SELECTIVE CATHETERIZATION OF THE VENTRAL SEGMENTAL
BRONCHUS OF THE RIGHT MIDDLE LOBE ---------------------- 33

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF THE
THORAX OF A 1 YEAR OLD BEAGLE DOG DEMONSTRATING
SELECTIVE CATHETERIZATION OF THE VENTRAL SEGMENTAL
BRONCHUS OF THE INTERMEDIATE LOBE ---------------------- 34

BRONCHIAL BRUSH (arrow) BEING INSERTED INTO THE
CATHETER PREVIOUSLY THROUGH THE ENDOTRACHEAL TUBE
VIA THE NORMAN MASK ELBOW ------------------------------- 36

CATHETER WITH BRONCHIAL BRUSH INSIDE BEING REMOVED
FROM THE DOG FOLLOWING BRUSHING OF A BRONCHUS -------- 37

PREPARATION OF SLIDE WITH BRUSH PUSHED BEYOND THE

TIP OF THE CATHETER ---------------------------- . g- --------- 38
TOUCH (A) AND SMEAR (B ) TECHNIQUES FOR PREPARATION

OF BRONCHIAL BRUSH CYTOLOGIC PREPARATIONS ------------ 39

CYTOLOGIC PREPARATIONS FROM A BRONCHIAL BRUSH

BIOPSY OF A NORMAL DOG. CILIATED EPITHELIAL CELLS (A),
NEUTROPHIL (B), GOBLET CELL (C), MAST CELL (D) CAN

BE IDENTIFIED IN THESE PREPARATIONS MADE USING THE

SMEAR TECHNIQUE . MODIFIED POLYCHROME METHYLENE

BLUE STAIN; 450x (1), 720x (2) ------------------------------ 46

BRONCHUS OF A 1 YEAR OLD BEAGLE DOG 24 HOURS FOLLOWING
BRONCHIAL BRUSH BIOPSY USING A STEEL BRONCHIAL BRUSH .
THERE IS A DEEP ABRASION IN THE MUCOSAL LAYER OF THE
BRONCHUS (arrow) ------------------------------------------ 49

SELECTIVE BRONCHOGRAM OF A 1 YEAR OLD BEAGLE DOG .

SIX m1 OF 60% PROPYLIODONE SUSPENDED IN PEANUT OIL WAS
DEPOSITED IN THE BRONCHI OF THE RIGHT CRANIAL, MIDDLE,
INTERMEDIATE, AND CAUDAL LOBES . NOTICE THAT NO
CONTRAST MEDIA IS PRESENT IN THE LEFT LUNG ------------- 52

POST-MORTEM APPEARANCE WITH PARTIAL CONSOLIDATION OF .
THE RIGHT LOBES OF A DOG'S LUNGS 72 HOURS AFTER
BRONCHOGRAPHY OF THE RIGHT LUNG LOBES WITH 6m] OF
PROPYLIODONE SUSPENDED IN PEANUT OIL ------------------- 53

CYTOLOGIC PREPARATION OF A BRONCHIAL BRUSH BIOPSY
FROM LUNG 72 HOURS AFTER 6ml OF PROPYLIODONE SUSPENDED
IN PEANUT OIL WAS INJECTED INTO ITS PRINCIPLE BRONCHUS .
RED BLOOD CELLS (A), RUPTURED MACROPHAGES (B),
NEUTROPHIL (C), EOSINOPHIL (D) WERE IDENTIFIED IN THIS

PREPARATION. MODIFIED POLYCHROME METHYLENE BLUE
STAIN; 720x ------------------------------------------------ 55

viii

20.

21.

22.

23.

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF THE
THORAX OF A 4 YEAR OLD GERMAN SHORTHAIR POINTER DOG
WITH CHRONIC BRONCHITIS ---------------------------------- 70

CYTOLOGIC PREPARATION FROM A BRONCHIAL BRUSH BIOPSY

OF A DOG WITH BRONCHITIS; DEGENERATIV E NEUTROPHILS (A)
AND MACROPHAGES (B) ARE PRIMARY CELL TYPES . MODIFIED
POLYCHROME METHYLENE BLUE STAIN; 720x ----------------- 71

LATERAL (A) AND DORSO-VENTRAL (B) RADIOGRAPHS OF THE
THORAX OF A 3 YEAR OLD GERMAN SHEPHERD DOG WITH
PULMONIC INF ILTRATION AND EOSINOPHILIA ------------------ 72

CYTOLOGIC PREPARATION FROM A BRONCHIAL BRUSH BIOPSY

OF A 3 YEAR OLD GERMAN SHEPHERD DOG WITH PULMONIC
INFILTRATION AND EOSINOPHILIA. THE CELLS ARE
PREDOMINANTLY EOSINOPHILS. MODIFIED POLYCHROME
METHYLENE BLUE STAIN; 720x ------------------------------- 73

ix

INTRODUCTION

Bronchopulmonary disease accounts for a significant number of the
medical problems seen in small animal practice. At Michigan State
University's Small Animal Clinic, 465 canine patients admitted during the
years 1970 through 1975 had clinical lower respiratory tract disease . Until
the late 1960's , the veterinary clinician had few aids with which to examine
the lower respiratory tract. To diagnose, prescribe and monitor therapy,
he had to rely on history and the physical examination. In addition, diag-
nostic aids including radiography and basic laboratory examinations such
as complete blood counts, fecal examination, microfilaria check, and evalua-
tion of fluid obtained by thoracocentesis , (in the event pleural fluid was
present) were also utilized. Thoracotomy was the only means of visualizing
the tissues of the lower respiratory tract and obtaining biopsy specimens.

To allow more specific evaluation of the lower respiratory tract
without risk of thoracotomy, bronchoscopy and bronchial washing were
developed. These techniques visualized the airways for collection of
cytologic and culture specimens from the dog and cat (O'Brien, 1970) .
Various percutaneous needle biopsy techniques for obtaining lung and
pleural tissues for histopathologic evaluation were also described (Reif,
1974) . In addition, transtracheal aspiration for collection of cytologic

and cultural specimens from the lower respiratory tracts of dogs and cats

1

2
was introduced (Creighton and Wilkens, 1974a) . The development of the

flexible fiberoptic bronchoscope made possible the visualization and
sampling of more peripheral lung tissue from known locations .

The development of more sophisticated diagnostic equipment and
techniques made possible advances in diagnostic capabilities and the
understanding of lower respiratory tract diseases . None of these
techniques, however, allowed collection of biopsy material from small
diameter bronchi. In 1964, a technique was described for use in human
patients that allowed selective catheterization of specific bronchi with the
aid of an image intensified fluoroscope, and the collection of cytologic
samples from the peripheral bronchial tree by the use of a nylon brush
attached to a flexible wire that could be passed through the catheter
(Hattori, 1964) . This procedure has been subsequently modified and
improved, and is now used in hospitals throughout the United States .

The objectives of this work are to describe and evaluate the technique
of selective catheterization of bronchi in the dog and the collection of cyto-
logical specimens from the peripheral bronchial tree using a bronchial
brush .

More specific aims of the project are to:

1 . Evaluate procedures for the selective catheterization of primary,
secondary, and tertiary bronchi using an image intensified fluoroscope.

2 . Collect cytologic specimens by means of the bronchial brush

through the preplaced catheter .

3

3 . Document the amount of trauma at the biopsy site and post-biopsy
complications for both nylon and stainless steel brushes .

4 . Evaluate methods of preparation of the biopsy specimens for
cytologic evaluation .

S . Evaluate the cytologic specimens obtained with nylon and steel
brushes with regards to the number of cells obtained and cellular
morphology.

6. Evaluate the cytologic specimens obtained by bronChial washings
and nylon bronchial brushings with regards to the number of cells
obtained and cellular morphology .

7. Evaluate the selectivity and sensitivity of the nylon bronchial
brush in the detection of experimental inflammatory disease .

8. Apply and evaluate the techniques of selective bronchial catheteri-
zation and bronchial brush biopsy to clinical patients with respiratory

disease .

LITERATURE REVIEW

History of Bronchial Brushinj_

 

Selective bronchial catheterization was first described in France
(Metras, 1947) . The first description of this procedure in the American
literature was in 1964; in it the first description of the technique of
bronchial brush biopsy appeared (Hattori, 1964) . In 1966, the use of
arterial catheters for the purpose of selective bronchography was described
(Fennessy, 1966) . One year later, this technique was described in conjunc-
tion with bronchial brush biopsy (Fennessy, 1967) . In recent years, the
procedures have been used extensively for the diagnosis of lower
respiratory disease in medical centers throughout the United States
(Backus, 1971; Bean, 513.1." 1968; Bibbo, $3.11." 1973; Fennessy, 33.1.,
1973; Murphy and Komorowski, 1974; Penido, 9321., 1972; Rockoff, 1974;
Skitarelic and von Haam, 1974; Smith and Warrock, 1972; Solomon, gt a' ,
1974; Willson and Eskridge, 1970; Zavala, gal. , 1972) .

Indications for Bronchial Brushing

 

Many authors have reported the usefulness of selective bronchial
catheterization and bronchial brush biopsy in the diagnosis of solitary
peripheral pulmonary lesions that cannot be visualized by bronchoscopy
(Fennessy, e_t__al. , 1970; Fennessy, ital. , 1973; Fennessy and Kittle, 1973;

Firestone, 1973; Hattori, Eta” 1964; Janower and Richard, 1971;
4

5
Rockoff, 1974) . Recently, this technique has been successful in the

diagnosis of diffuse lung disease (Fennessy and Kittle, 1973; J anower and
Richard, 1971) and has been used to drain lung abscesses (Rockoff, 1974) .
In addition, bronchial brushes can be used to obtain cultures from the

lower respiratory tract for bacteria, (Backus, 1971; Bibbo, gt a_1_. , 1973;
Fennessy, e; a_1_. , 1973; Fennessy and Kittle, 1973; Murphy and Komorowski,
1974; Rockoff, 1974), viruses (Fennessy, 9131., 1973; Fennessy and

Kittle, 1973) , and fungi (Fennessy and Kittle, 1973) .‘ Two authors have
found selective bronchial Catheterization and bronchial brush biopsy useful
in the diagnosis of infectious lung disease, secondary to irnmunosuppressive
therapy in transplant patients (Fennessy, _e_t a. , 1973; F ennessy and

Kittle, 1973) .

Contraindications for Bronchial Brushing

 

In the human patient, contraindications to this technique include
recent severe hemoptysis, suspected vascular lesions, thrombocytopenia,
or other bleeding disorders, and dyspnea at rest (Fennessy, gt a_1_. , 1973;

F ennessy and Kittle, 1973) . These authors emphasize that the contraindica-
tions should be ignored if the need for a definitive diagnosis is imperative
to the well-being of the patient.

Applied Gross Anatomy

 

The right lung of the dog has four lobes, cranial, middle, caudal,

and intermediate lobes . The left lung , being smaller than the right, has
only two lobes, the cranial and caudal lobes. The bronchial tree begins as

the trachea bifurcates at the level of the fifth intercostal space, just to the

right of the midline and dorsal to the base of the heart.

6

The right principle bronchus begins at the tracheal bifurcation and
enters the dorsal aspect of the hilus of the lung . The right cranial lobar
bronchus leaves the principle bronchus just distal to the hilus from its
lateral aspect and bends cranially . The right cranial lobar bronchus then
gives off a large bronchus just distal to the hilus from its dorsal aspect, the
caudal segmental bronchus, which ventilates the caudal bronchopulmonary
segment of the cranial lobe . The right cranial bronchus continues cranio-
ventrally as the cranial segmental bronchus and ventilates the cranial

bronchopulmonary segment .

After the exit of the cranial lobar bronchus, the right principle
bronchus gives off the right middle lobar bronchus from its ventrolateral
aspect. Shortly after its beginning , the middle lobar bronchus gives off a
large bronchus from its lateral aspect which ventilates the dorsal broncho-
pulmonary segment of the middle lobe; it is named the dorsal segmental
bronchus. The middle lobar bronchus continues as the ventral segmental
bronchus, which ventilates the ventral bronchopulmonary segment of the

right middle lobe .

After giving off the right middle lobar bronchus, the principle
bronchus gives off the intermediate lobar bronchus from its ventral medial
wall. The intermediate lobar bronchus passes caudad and divides into the
dorsal and ventral segmental bronchi. These bronchi ventilate the dorsal
and ventral bronchopulmonary segments of the intermediate lobe respec-
tively .

Following the exit of the intermediate lobar bronchus, the right

principle bronchus continues as the caudal lobar bronchus and gives off two

7

bronchi from its ventrolateral aspect. The first of these is the ventral basal
bronchopulmonary segment of the caudal lobe . The second ventrolateral
bronchus is named the lateral segmental basal; it ventilates the lateral basal
bronchopulmonary segment .

From its dorsal surface, the caudal lobar bronchus gives off two
bronchi, the cranial dorsal segmental bronchus and the caudal dorsal
bronchopulmonary segment and caudal dorsal bronchopulmonary segment of
the caudal lobe respectively . The caudal lobar bronchus continues as the
dorsal basal segmental bronchus ventilating the dorsal basal bronchopul-

monary segment of the caudal lobes.

The left principle bronchus enters the hilus of the left lung and gives
off the left cranial lobar bronchus from its lateral aspect, which divides into
the cranial segmental bronchus and the caudal segmental bronchus . These
bronchi ventilate the cranial bronchopulmonary and caudal bronchopulmo-
nary segments of the left cranial lobe respectively . The left principle
bronchus continues as the left caudal lobar bronchus and branches as in the

right caudal lobe .

The segmental bronchi in all lobes branch into subsegmental bronchi

and terminate in bronchioles described under microscopic anatomy (Hare,
1975) .

Applied Microscopic Anatomy

 

The mucous membrane of bronchi are arranged in longitudinal folds
and are lined by pseudostratified columnar ciliated epithelium with goblet
cells on a prominent basement membrane . The epithelium changes to a

Simple columnar ciliated epithelium with goblet cells as the bronchi

decrease in size.

The lamina pr0pria consists of a thin subepithelial layer composed of
loose connective tissue with few elastic fibers, many leukocytes, and a
prominent capillary network. Just beneath the connective tissue is a thick
layer of longitudinally oriented elastic fibers arranged in bundles in the

larger bronchi and having a spiral course in smaller bronchi.

The muscularis mucosa is composed of smooth muscle circularly
arranged in large bronchi and tends to be spirally arranged in smaller
bronchi. Elastic tendons connect the smooth muscle cells of the muscularis

mucosa to the elastic fibers of the lamina propria and submucosa.

The submucosa is primarily loose connective tissue containing many
elastic fibers, blood vessels, and the bulk of the simple cuboidal tubulo-
acinar mucous glands . These glands decrease in number as the Size of the

bronchi decrease and are absent in the smallest bronchi of the dog .

The bronchi are surrounded by a fibroelastic cartilage layer consis-

‘ ting of hyaline and elastic cartilage plates interconnected by dense irregular
elastic tissue. Hyaline cartilage predominates in the larger bronchi, while
in the smaller bronchi elastic cartilage is more prevalent. The adventitia
surrounds the fibroelastic layer and consists of elastic fibers and loose

connective tissue with a few tubular acinar glands in the larger bronchi.

The terminal bronchi branch dichotomously giving rise to primary
bronchioles which continue to branch to secondary, tertiary and respira-
tory bronchioles. The mucous membranes of the primary and secondary
bronchioles appear histologically in longitudinal folds which decrease in

height toward the end of the secondary bronchioles. The lumen is lined by

9

simple columnar epithelium in the primary and secondary bronchioles and
simple cubiodal epithelium in the tertiary and respiratory bronchioles . The
number of goblet cells and ciliated cells decrease as the bronchioles get
smaller and are rare beyond the secondary bronchioles . The lamina propria
is composed of loose connective tissue with many lymphocytes and elastic
fibers which decrease in number toward the respiratory bronchiole. The
muscularis mucosa consists of a few layers of spirally wound, smooth
muscle, which are reduced to one layer and to occasional cells in the
respiratory bronchioles . The adventitia] layer is composed of loose
connective tissue with many elastic fibers that are continuous with those of
the surrounding lung tissues. Respiratory bronchioles are distinguished
from tertiary bronchioles by having direct communication with alveoli. In
the dog , the respiratory bronchiole represents the typical distal airway

because the tertiary bronchiole is very short.

The alveolar ducts extend distad to the respiratory bronchiole and
open directly into alveoli. They are lined by simple squamous or simple
cuboidal epithelium identical to that of the alveoli. Sparse amounts of
elastic connective tissue fibers and occasional muscle cells are found

beneath the epithelium .

Large sac-like structures terminate the alveolar ducts made up of
alveolar septa. Many alveoli open into these structures; they are called
alveolar sacs. The alveoli are lined by squamous alveolar epithelial cells
(type I cells) . Interspaced between these cells are cuboidal epithelial cells

(type II cells) that are thought to produce surfactant. A third cell type is

the alveolar marcophage (dust cell) (Dellman, et 11: , 1976) .

10

Technique of Selective Bronchial Catheterization and Bronchial Brushing
Selective bronchial catheterization and bronchial brush biopsy are
accomplished in the human patient by one of two techniques after the affected
area of the lung has been identified by radiography. The radiopaque poly-
ethylene catheter is introduced into the trachea by passage through natural
airways, either the nose (Backus, 1971; Bean, e_t_a_l_. , 1968; Bibbo, 3 al. ,
1973; Fennessy, 1966; Fennessy, 1967; Fennessy, 1968; Fennessy, _e_t_ 31.,
1970; Genoe, 1974; Janower and Richard, 1971; Penido, gal” 1972;
Pierce and Jacobs, 1969; Willson and Eskridge, 1970; Zavala, 9311.. , 1972)
or mouth (Fennessy, g Q. , 1970; Willson and Eskridge, 1970) . In the
alternative technique, catheterization is accomplished by the passage of
the catheter through the cricothyroid membrane (Rockoff, 1974) . Anesthesia
for both techniques is accomplished by premedication with atropine, a
narcotic or non-narcotic tranquilizer, and local anesthesia of the respira-
tory tract (Backus, 1971; Fennessey, 1966; Fennessy, 1967; Fennessy,

1968; Fennessy, 9131.” 1970; Janower and Richard, 1971; Penido, eta_1_.,

1972; Rockoff, 1974; Willson and Eskridge, 1970) .

The catheter is directed to the desired bronchus under fluoroscopic
control. Some clinicians prefer to preshape the catheter to facilitate

entry of the diseased bronchus (Bibbo, _e_t 11.. , 1973; Fennessy, 1966;

Fennessy, 1967; Janower and Richard, 1971; Rockoff, 1974; Smith and
Warrack, 1972) . Introduction of this preshaped catheter can be facilitated

if a guide wire is used (Backus, 1971; Bean, 2131' , 1968; Fennessy, 1967;
Fennessy, 1968; Fennessy, e_t_£., 1970; Genoe, 1974; Zavala, 32.1., 1972).

Other authors use a two catheter system , the larger being preshaped to the

11

lobar bronchus and the smaller or inside catheter being straight. A
controllable guide wire is used to guide the catheter to the lesion (Willson

and Eskridge, 1970) . Some clinicians have developed guided catheter

systems in which the shape of the catheter can be changed mechanically
while inside the bronchial tree (Pierce and Jacobs , 1969; Zavala, e_t_ §° ,

1972) . After placement of the catheter, radiographs are taken in multiple

views to insure the desired area of lung has been catheterized. A small
nylon brush is passed through the catheter and into the lesion (Backus,
1971; Bean, e_t_a_l_., 1968; Bibbo, e_t_a_l., 1973; Fennessy, 1967; Fennessy,.
1968; Fennessy, e_t_fl. , 1970; Fennessy, gt a_1_. , 1973; Fennessy and Kittle,
1973; Genoe, 1974; Hattori, eta}, 1964; Janower and Richard, 1971;

Rockoff, 1974; Willson and Eskridge, 1970) . Steel brushes are recom-

mended when the catheter is against the lesion (Backus, 1971; Bibbo, et £° ,
1973; Fennessy, e_t_a_l_. , 1970) or in diffuse lung disease (Fennessy, 1968;
Fennessy, 1974; Fennessy and Kittle, 1973) . The brushes may be deflect-

able, meaning that the brush tip can be deflected independently of the

catheter (Bibbo, etal. , 1973; Fennessy and Kittle, 1973; Fennessy, e_t_ e_l. ,
1973; Genoe, 1974; Hattori, Stain 1964; Willson and Eskridge, 1970) or
of the non-deflectable variety (Bean, gt fl' , 1968; Fennessy, 1968;
J anower and Richard, 1971) . One article described the use of an acrylic
sponge in place of the brush (Smith and Warrack, 1972) . After brushing
the lesion vigorously, the brush is removed from the catheter and slides
are prepared for microscopic evaluation .

Many authors have combined selective bronchial catheterization and

bronchial brush biopsy with bronchial washing for cytology and culture,

12
Fennessy, e_t_a_l_., 1970; Genoe, 1974), forceps biopsy (Fennessy, 1968),

selective bronchography (Bean, e_t_a_l_. , 1968; Fennessy, 1968; Genoe, 1974;
Hattori, e_t_;al. , 1964; Janower and Richard, 1971; Penido, Eta” 1972), or
a combination of all four techniques using the same catheter (F ennessy,
1967; Fennessy, eta}, 1973; Fennessy and Kittle, 1973; Rockoff, 1974).
Some authors have reported submitting the actual brushes for bacterial
culture (Backus, 1971; Bibbo, 2131” 1973; Fennessy, 1967; Fennessy,
1968; Fennessy, eta” 1970; Fennessy, gal” 1973; Fennessy and

Kittle, 1973; Murphy and Komorowski, 1974; Rockoff, 1974) or viral

culture (Fennessy, e_t_ 3.1. , 1973; Fennessy and Kittle, 1973) .

Slides and histologic specimens are prepared by various methods,
depending on the laboratory . Slides are made directly from the nylon
brush and fixed in 95% ethyl alcohol (Bean, _e_t_ a_1_. , 1968; Bibbo, 93 a_1_. ,
1973; Fennessy, 1967; Fennessy, 1968; Fennessy, e_ta;l_., 1970; Fennessy,
.3111: , 1973; Fennessy and Kittle, 1973; Hattori, e_ta_1_l_. , 1964; Janower and
Richard, 1971; Murphy and Komorowski, 1974; Skitarelic and Worthamm,
1974; Solomon, 9131., 1974; Zavala, e_tgl. , 1972) . Some workers submit

air dried specimens, along with the alcohol-fixed slides, for bacterial

staining (Bean, $9.1.“ , 1968; Fennessy, g 31. , 1973; Fennessy and Kittle,
1973) . Wet preparations are most often stained by the papanicolaou method
(Bibbo, 9111., 1973; Hattori, e_t_g._l_., 1965; Murphy and Komorowski, 1974;
Skitarelic and von Hamm, 1974; Zavala, e_t_ 3.1: , 1972) . Steel brushes are
placed in 10% formalin, and the tissue adherent to the brush is teased off
with a forceps after the tissue is fixed. The tissue is then collected,

embedded in a paraffin-block, sectioned and stained by routine hemotoxylin

13
and eosin techniques (Bibbo, gt al. , 1973; Fennessy, gt gl. , 1970;

Fennessy, e_t_ a_1_. , 1973; Fennessy and Kittle, 1973) .

One author suggests soaking the brushes in saline and spinning down
the cellular material to be passed through a millipore filter to collect the
cells for staining (Zavala, gt gt. , 1972) . The clinician using the sponge
submits the sponge to be embedded, sectioned, and stained with hemo-

toxylin and eosin (Smith and Warrick, 1972) .

Complications of Bronchial Brushing

 

Several minor complicafions have been reported in the human includ-
ing slight febrile reactions (Fennessy, 1967; Fennessy, 1968; Fennessy,
g _a_l_. , 1970; Janower and Richard, 1971) , blood tinged sputum (Fennessy,
gt gl_. , 1970) , and slight epistaxis due to catheter trauma (Fennessy, 1967;
Fennessy, 1968; J anower and Richard, 1971) . More serious complications
include pneumothorax, which has not resulted in deaths (F ennessy and
Kittle, 1973; Fennessy, gtg_1_., 1973; Janower and Richard, 1971; Rockoff,
1974) , gross hemoptysis (Bean, gtgt. , 1968) , and hemorrhage, which has
resulted in the death of two patients (F ennessy and Kittle, 1973; Fennessy,
gt _a_1_ . , 1973).

When the trachea is approached through the cricothyroid membrane,
about one-third of the patients will develop subcutaneous emphysema that
resolves spontaneously in 24 to 48 hours (Rockoff, 1974) . Massive
hemorrhage occurred when an anomalous right cricothyroid artery, which
crossed the cricothyroid membrane, was lacerated with the needle and

pushed into the tracheal lumen drowning the patient (Shillaci, gt gl. . 1976) -

14

Some clinicians report loosing the tip of a brush in the bronchial
tree. These were retrieved by forceps inserted through the catheter
(Fennessy, 1968; Fennessy, gt_g_l_., 1970; Fennessy and Kittle, 1973) .
Septicemia, pneumonia, and lidocaine reactions have been reported
(Fennessy and Kittle, 1973) .

Qymlogy Obtained From the Lower Respiratory Tract of Animals

 

Roszel described the use of cytology in the diagnosis of disease of
several organ systems of animals. Included in this series were five cases of
respiratory neoplasia of which 2 were tumors of the lower respiratory

tract with neoplastic cells in bronchial washings (Roszel, 1967) .

Creighton and Wilkins (1974a) described ciliated columnar to cuboidal
epithelial cells in aspirates from normal and abnormal dogs . Reactive
hyperplastic epithelial cells (histiocytes) were seen in chronic inflamma-
tory processes . Neoplastic cells were observed in some cases where cancer
was suspected. Many types of inflammatory cells including neutrophils,
eosinOphils, lymphocytes , plasma cells, and mast cells were observed.
Also, macrophages were seen. Foreign material such as bacteria, parasi-
tic microfilaria, fungal elements, and anthracitic pigments were observed.
Viral inclusion bodies were identified in 2 dogs (Creighton and Wilkins,

1974a).

In another paper evaluating the transtracheal aspiration biOpsy on 75
canine patients, Creighton and Wilkins (1974b) defined eight cellular
patterns they felt to be indicative of specific types of neoplastic or inflamma-
tory disease. The cytology was compared with the other data available

on these patients . They concluded that cytology is useful in further

15

defining diseases of the lower respiratory tract, but should be used only in
conjunction with other diagnostic techniques (Creighton and Wilkins, 1974b) .
One author reported that the greatest percentage of cells, in bronchial
wash solutions from normal dogs, were either macrophages or epithelial
cells . Neutrophils averaged 1 .395 of the cells seen in bronchial washings
from normal dogs; with lymphocytes, basophils and mast cells also being

observed (Patterson, gt 3.1: , 1974) .

Washings obtained through a rigid bronchoscope, from patients which
were eventually confirmed to have primary pulmonary neoplasia, contained
neoplastic cells. These tumors included squamous cell carcinoma, adeno-
carcinoma, mixed squamous and adenocarcinoma, large and small cell
undifferentiated carcinoma, and alveolar cell carcinoma. Aspirates from
dogs with metastatic disease were not so rewarding because most of these

tumors did not involve the tracheobronchial tree (O'Brien and Roszel, 1974) .

A cellular pattern suggestive of canine distemper has been proposed
and is characterized by intracytoplasmic inclusion bodies in the bronchial
epithelial cells (O'Brien and Roszel, 1974) . Chodosh, gt é' , suggests that
in washings from human patients in which the predominant cells are eosino-
phils, a diagnosis of allergic bronchitis is appropriate and those in which
neutroPhils predominate (60-90%) are suggestive of an inflammatory disease.
He also stated that the alveolar macrophage is a sensitive indicator of
cellular response, and a paucity of these cells is commonly the first sign of
acute inflammatory disease processes. A marked increase in these cells
denotes that the cellular response is good and is usually followed by

recovery (Chodosh, EE£° , 1970) .

16
No reports of cytology obtained by bronchial brush biopsy were

found from animal patients .

Results Obtained by Bronchial Brushing in Human Patients

The results of bronchial brush biopsies performed on human patients

have been reported by several clinicians and are summarized in Tables 1,

2, and 3.

The accuracy of this technique in the diagnosis of primary pulmonary
disease in the human patient has been reported as low as 62% and as high as
92%, with most clinicians reporting about 70% of the confirmed cases to have
malignant cells in the bronchial brush biopsy. Tabulation of the data in
Table 1 demonstrates that 73% of the patients with confirmed primary pul-

monary neoplasia had malignant cells in the biopsy specimens .

Data on the effectiveness of this technique for diagnosis of metastatic
lung disease is less abundant. Tabulation of the reports listed in Table 2
indicates that 51% of those patients biopsied that were later confirmed to
have metastatic pulmonary disease had neoplastic cells in the bronchial
brush biopsy .

Reports of bronchial brush biopsy as a diagnostic tool in non-neo-
plastic disease are few and difficult to interpret because in many cases, no
definitive diagnosis is obtained . Tabulation of the data in Table 3 shows the
bronchial brush biopsy to be helpful in the diagnosis of 16% of the confirmed
cases of inflammatory disease. One clinician states that the most common
radiographic diagnosis of pulmonary infiltrates is that of a non-specific
inflammation that usually does not extend into the bronchial tree, making the

results of bronchial brush biopsy unrewarding (F ennessy, 1968) .

17

TABLE 1 . BRONCHIAL BRUSH BIOPSY IN HUMAN PATIENTS WITH PRIMARY
PULMONARY NEOPLASIA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Confirmed
Primary Neoplastic
. Pulmonary Cells in False Positives

Reference N eqplasia Biopsy For N ecplasia
Hattori, gta. , 1964 14 13 (92%) 0
Fennessy, gtgl. , 1973 224 157 (70%) ‘ — 4‘ 9
Willson and Eskridge, 1970 44 35 (80%) 0
Solomon, (231;, 1974 47 41 (87%) 0
Fennessy, 1967 37 23 (62%) 0
Murphy and Komorowski, 1974 20 13 (65%) 0
Fennessy, 1968 50 32 (64%) 0
Fennessy and Kittle, 1973 238 182 (76%) 0
Fennessy, e_t_flu 1970 117 83 (71%) 0
Penido, gtg_l_., 1972 56 40 (71%) 1
Bean, e_tgl” 1968 57 39 (68%) 0
Zavala, gtgl. , 1972 73 57 (78%) 0
Genoe, 1974 23 16 (70%) 0
Smith and Warrack, 1972 27 18 (67%) 4
Skitarelic and von Haam, 1974 47 40 (85%) 0

 

 

1074 789 (73%) 14

 

18

TABLE 2 . BRONCHIAL BRUSH BIOPSY IN HUMAN PATIENTS WITH

SECONDARY PULMONARY NEOPLASIA

 

Confirmed Metastatic

Neoplastic Cells

 

 

 

 

 

 

 

 

Reference Pulmonary NeoRlasia in Biopsy
Fennessy, e_t_a_1_. , 1973 30 16 (53%)
Willson and Eskridge, 1970 l 0 (0%)
Fennessy, 1967 6 2 (33%)
Murphy and Komorowski, 1974 2 2 (100%)
Fennessy, 1968 9 4 (44%)
Fennessy, ggt. , 1970 15 8 (53%)
63 32 (51%)

 

TABLE 3 . BRONCHIAL BRUSH BIOPSY IN HUMAN PATIENTS WITH

CONFIRMED INF LAMMATORY DISEASE

 

Confirmed Inflammatory Brush Biopsy

 

 

 

 

 

Reference Disease Aided Diagnosisa
Fennessy, 1967 50 4 (8%)
Murphy and Komorowski, 1974 15 5 (33%)
Fennessy, 1968 27 6 (22%)

92 15 (16%)

 

 

aCytology or culture of brush for virus, bacteria, or fungi was helpful

in diagnosis .

19

Several clinicians comment that the most common cause for failure of
this technique is improper catheter placement and failure to verify place-
ment of the brush by radiographs in at least 2 views (Bibbo, gt gt. , 1973;

F ennessy, 1967; F ennessy, 1968) . Another cause for failure is a sharp
curve (Bibbo, 33 gl. , 1973) or stenosis (Skitarelic and von Haam, 1974) in
the diseased bronchus making it impossible to catheterize. Also, placement
of the brush in the necrotic center of a tumor produces a lack of neoplastic
cells in the biopsy and a false negative result (Bibbo, gt gli, 1973) . Bean
emphasizes that the lesion must extend into the bronchus for the brush

biopsy to be successful (Bean, gt gt. , 1968) .

One clinician suggests that the bronchial brush biopsy is most useful
when the lesion is at the level of the second or third generation bronchi
(Firestone, 1973) , while another clinician emphasizes the usefulness of the

technique in the diagnosis of more peripheral pulmonary lesions (Fennessy,

3591., 1970).

It has been shown that bronchial brush biopsy increases the number
of pulmonary cancer patients with neoplastic cells in their sputum . In one
group of patients, 106 had neoplastic cells in their Sputum after bronchial
brush biOpsy of 160 that were negative before the procedure (F ennessy and

Kittle, 1973) .

The cells obtained by bronchial brushing were well preserved and in
sheets, while those obtained by washing showed some degree of degenera-
tion and were isolated or in well defined clumps . This study suggested
there is no advantage to performing a bronchial wash combined with a well

performed bronchial brush (Bibbo , gt a_1_. , 1973) .

20
One clinician showed that in contrast to brushing , the diagnosis

yield obtained by bronchial washing was small. In 46 bronchial washings,
only 7 contained malignant cells , while 46 of these confirmed cases of
pulmonary neoplasia had diagnostic bronchial brush biopsies (Solomon,

e_t g_1_. , 1974) .

Some authors report that controllable brush (deflectable tip) systems
decrease the time necessary to perform the procedure and increase the
range of lung field accessible by this technique (Willson and Eskridge,
1970) .

Zavala states that a mobile catheter system increases the diagnostic
accuracy of this technique by 10% (Zavala, gt _a_l.. , 1972) .

F ennessy and his co-workers found that transtracheal brush or
forceps biopsy were most successful in the diagnosis of primary pulmonary
neoplasia and of less value in metastatic pulmonary disease . Also, it was of
value in the diagnosis of opportunist pulmonary infections such as
aspergillosis or Pneumocystis carinii when other diagnostic methods failed.
They suggest that the lack of diagnostic biopsy in many cases of inflamma-

tory disease may be due to previous antibiotic therapy (Fennessy, gal fl° ,

1973) .

MATERIALS AND METHODS

Equipment for Selective Bronchial Catheterization and Bronchial Brushing

Selective bronchial catheterization and bronchial brush biopsy
required the use of an image intensifiédfluoroscope. It was necessary to
perform this technique under general anesthesia . The capability of inhala-
tion anesthesia was not essential but was preferred, as usually dogs with
respiratory disease are less than ideal anesthetic risks .

Bronchial catheters used with nylon brushes were made from 75cm
lengths of .071 inch inside diameter and .093 inch outside diameter poly-
ethylene tubinga. Stainless steel bronchial brushes required a larger
catheter (.085 inch inside diameter and .110 inch outside diameter) . The
tip of the catheter to be inserted into the bronchial tree was curved approxi-
mately 65° at a distance of 1 .5cm from the tip . This allowed easier entry
into some of the second and third generation bronchi (Figure 1) .

Two types of bronchial brushes were employed; a disposable 1 .72mm
diameter nylon brushb mounted on a 100cm deflectable tip guide wire
(Figures 2 and 3), and a disposable 1.72mm diameter stainless steel

bronchial brushb attached to a 100cm non-deflectable guide wire.

 

aFormocath, Becton-Dickson, Rutherford, NJ.

bMill-Rose Company, Mentor, OH .

21

22

.oszmsmm SHHmozomm
. H amps:

mom 20HH<NHmMHmmB<O Q<Hmozomm m>HHomqmm zH 9mm: mmmemme<o mzmqwmhquom

 

22

.oszmDmm Q<Hmozomm
mom ZOHH<NHmMBmmH¢o H<Hmozomm m>HHomqmm 2H QmMD mmmpmme<o mzmqwmequom

 

.H mmpon

’.

23

mmHS mDHDo mHB Mdm<eomqmma EOOOH < 20 QmHZDOE mmbmm A<Hmozomm zoqwz EENb.H

.m mmbon

 

.24

 

FIGURE 3. 1.72m NYLON (A) AND STEEL (B) BRONCHIAL BRUSHES.

25

Access to the endotracheal tube while maintaining ventilatory support
was accomplished by the use of a Norman Mask Elbowc (Figure 4) or a dis-
posable swivel adapterd inserted in the anesthetic system between the
endotracheal tube and the anesthesia machine delivery hoses.

For selective bronchial catheterization through the cricothyroid mem-
brane, a No. 10 scalpel blade, 14 ga. 11: inch needle, and a 100cm guide
wire are required in addition to the above material.

A fenestrated surgical drape was used in both techniques to prevent
contamination of the catheter and brushes . Several standard microscopic
slides were available at the time of biopsy and slides were prepared directly
from the brush immediately upon removal from the bronchus .

Technique of Bronchial Brushing

 

No attempt was made to perform this procedure in a sterile manner,
although all equipment was sterilized between animals and an effort was
made to keep contamination to a minimum .

Two routes of bronchial catheterization were performed and evaluated.
A. Catheterization via the cricothyroid membrane

Four dogs were anesthetized with thiamylal sodiume, intubated and

maintained by inhalation anesthesia using halothanef. The dogs were placed

 

cNorth American Drag er, Telford, PA .
dOlympus Corporation of America, New Hyde Park, NY .

eSurital, Park Davis, Co. , Detroit, MI.

tFluothane, Ayrest Laboratories Incorporated, New York, NY .

.QMHmmmzH mmemme<o

.ZOHH<mHmDAAH onm<mooeomm mom Qm>ozmm zmmm m<m mm<mo mme
q<Hmozomm :EHB EHHmwm oHBmmHmmz< mmb 2H mmemwmzH ABOHLmv gomqm xm<§ z<§moz .v mMDon

 

27

in dorsal recumbency and their laryngeal areas clipped and surgically
prepared. A fenestrated surgical drape was placed over the dog and
table to prevent contamination of the catheter and brushes . The crico-
thyroid membrane was palpated betWeen the cricoid and thyroid cartilages
of the larynx with a gloved finger and a small stab incision made over the
membrane. The endotracheal tube was removed and a 14 gauge needle
passed through the cricothyroid membrane and into the larynx . A guide
wire was passed through the needle into the larynx and directed into the
trachea . The needle was removed and the catheter placed over the guide
wire and into the trachea, after which the guide wire was removed. The
endotracheal tube was replaced and the dog positioned under the fluoro-
scope.

With the aid of the image intensified fluoroscope, the catheter was
manipulated to the desired bronchus . The majority of the catheter manipu-
lation was done with the dog in ventral recumbency. The right and left
principle bronchi along with the caudal lobar bronchi of the caudal lobes
were catheterized using a straight catheter . All other bronchi were
catheterized with a curved catheter .

The right or left cranial lobar bronchi were catheterized by directing
the catheter into the principle bronchus of the side desired and its tip

placed beyond the orifices of the cranial lobar bronchi. The tip of the

catheter was then directed towards the lateral wall of the bronchus and the

catheter withdrawn until the tip entered the cranial lobar bronchus. The

catheter was then directed either dorsal or ventral and advanced depending

on the segmental bronchus desired (Figures 5, 6, 7, 8) . To catheterize the

28

 

.mmOA A<HZ<MQ EMMA mmH mo mbmozomm Q¢Hzmzwmm A<Hz<mo mmh mo ZOHB<NHmMHmmH<O m>HBomqmm ozHH<mHmzoEmm
com mqo<mm Odo m<mw H < mo x<mOmB Mme mo mmm<mUOHQ<m Amv Q<mezm>lommoa Qz< A<V u<mme<q .n mmwam

<

29

.mmog A¢Hz<mo Hmmq mme mo mbmozomm A<Bzmfiomm Q<Qb<o Mme mo onH<NHmmemmB¢o m>HHomqmm 02H9<mhmzozmo
UOQ mqu<mm GAO m<m~ H < mo x<momh axe mo mmm<mcoHQ¢m Amv H<MB2M>IOWEOQ Qz< A<v A<mme<q .0 mmbon

 

3O

.mmoq A<Hz<mo Hmon mmB mo mamozomm A<Bzm26mm q<Hz¢MQ mme mo ZOHB<NHmMHmmH<o m>HHomAmm UZHH<EHWZC§MQ
Goo mgu<mm DAG m<mw H < mo x<fiomh mmh mo wmm<moOHQ<m Amv A¢mezm>lommom Qz< A<V Q¢MMH<A .b mmbcmm

 

31

.mmoq H<Hz<mo Bmch mme mo mbmozomm H<Hzm§omm H<QD<Q MIR mo ZOHB<NHmMHmmH<o m>HHom4mm UZHH<mHWZOEMQ
GOO MHO<Mm QHO m<mw H < mo x<mome Mme mo mmm<mooHQ<m Amy H<mbzm>lommom 02¢ A<V H<mme<q .w mmbon

 

32
right middle lobar bronchus, the tip of the catheter was directed down the

right principle bronchus past the orifice of the right middle lobar bronchus .
The tip was directed ventro-lateral and the catheter withdrawn until the tip
entered the middle lobar bronchi. The catheter was then advanced into the
ventral segmental bronchus (Figure 9) . The intermediate lobar bronchus
was catheterized by directing the catheter down the right principle bronchus
with the tip directed ventro-medial until the intermediate lobar bronchus
was entered . The catheter tip was then directed either dorsal or ventral
(Figure 10) depending on the segmental bronchus desired. The animal was
then positioned in lateral recumbency to insure proper placement of the
catheter . The bronchial brush was passed through the catheter and into

the bronchus and the biopsy collected by moving the brush vigOrously over
the bronchial wall. The brush was pulled back into the tip of the catheter

to prevent contamination of the biopsy material during withdrawal. The
catheter, along with the brush, was then withdrawn from the animal. Slides
were prepared as described later . The entry into the trachea through the

cricothyroid membrane was not sutured . The animals were observed for
complications .

B. Catheterization via the endotracheal tube

The second method was to catheterize the bronchus through the endo-

tracheal tube . Four dogs were anesthetized as previously described. An
adapter was placed in the anesthetic system at the junction of the endo-
tracheal tube and the hoses (Figure 4). A fenestrated surgical drape was
then placed over the dog and table to protect the catheter and brushes from

contamination. The catheter was introduced through the adapter, into the

33

.mmog MHQQHE HmUHm MHZ. no 950295 q<ezm20mm 392m; Mme mo onB<NHmmemmH<o m>HHomHmm 02HB§HWZOSHHQ
COD mqo<mm QHO g H < mo acme EC. ho mmgong Am: Héezglommoa Qz< :3 Ewe: .0 mmDon

 

34

.mmoq MH<HQmEmMH2H MIR mo mbmozomm H<ezm20mm H<mezm> mmB mo ZOHH<NHmmHmEB<o m>HHomqmm OZHH<mHm20HEQ
OOQ MHU¢Mm GHQ m<mw H < mo xdmome mmb mo mma<mooHQ<m Amv H<mezm>lommom Qz< A<V A<mme<q .OH mmbon

 

35
endotracheal tube and positioned with the aid of the image intensified

fluoroscope as was previously described. The animals were observed in
two views to confirm catheter position. The brush was passed through the
catheter and into the bronchus (Figure 11) . The biopsy was taken by
vigorously brushing the bronchial wall. The brush was then pulled back
into the tip of the catheter to protect the biopsy while the brush and
catheter were removed from the dog (Figure 12) . Slides were prepared
from the brush after it was pushed from the tip of the catheter (Figure 13) .
The dogs were allowed to recover from anesthesia and observed for compli-

cations .

Preparation of Slides for Cytologic Evaluation

 

Slides were prepared from nylon bronchial brushes using 2 different
techniques . The touch technique was performed by touching the brush to
the slide in several areas . The smear technique was performed by rolling
the brush as it was pushed along the slide resulting in smearing of the
biopsy material (Figure 14) . The slides were air dried, stained with

8

modified polychrome methylene blue , and cover slips applied.

Evaluation of Cytologic Preparations

 

Comparison of the cytology obtained by each technique was
accomplished utilizing the criteria listed in Table 4 . All slides were
evaluated without knowledge of their sources . These criteria allowed
numerical scores to be alloted to each slide for each area of interest. The

numerical scores were then added for all slides from the same technique and

 

Hema-Tek Stain Pak, Ames Co. , Division of Miles Labs, Inc . ,
Elkhardt, IN .

36

.Somqm x0

H<mmo<meoazm mme mODommB wHwboH>mmm mmamme<o mme OBzH QMEmmmzH oszm Aachpw

V mmbmm H<Hmozomm

 

<2 Z<Emoz mmB <H> mmDH

.HH mmbon

37

< no oszwbmm OZHSOHHOm COD mme 20mm Qm>02mm oszm monzH mmbmm A<Hmozomm mHHB mmemmh<o

 

.mbmozomm
.NH mmDUHm

38

mmemme<o mmh mo AHH Mme Ozowmm Qmmmbm mmbmm EHHE MDHHm mo onH<m<mmmm

 

.MH mmwam

39

 

A 8

FIGURE 14. TOUCH (A) AND SMEAR (B) TECHNIQUES FOR PREPARATION OF
BRONCHIAL BRUSH CYTOLOGIC PREPARATIONS. '

40

TABLE 4 . CRITERIA FOR EVALUATION OF CYTOLOGIC PREPARATIONS

Numerical Score

 

Readable Fields With 4 X Objective:

 

Excellent - Approximately 10 Readable Fields 3
Adequate - Approximately 5 Readable Fields 2
Poor - 2 or Less Readable Fields 1

Hemorrhage:

Excessive - Number of RBC's Exceeds Nucleated Cells 1
Moderate - Equal Number of RBC's and Nucleated Cells 2
Minimal - Nucleated Cells Exceed RBC'S 3

Nuclear Detail:

Excellent - Nuclei Stain Darkly Basophilic 3
- Good Differential Staining of Nucleus
- Even Distribution of Nuclear Chromatin
- Nuclear Membranes Clearly Defined

Adequate - Nuclear Membranes Generally Intact 2
- Nuclear Staining Less Basophilic
- Nucleoli Somewhat Prominent

Poor - Poorly Defined Nuclear Membranes 1
- Bare Nuclei Present
- Cells Appear Nonviable With Predominant

Nucleoli

 

Cytoplasmic Detail
Excellent - Cytoplasmic Membranes Intact 3
- Cilia Present on Many Cells
- Pink to Blue Background Stain
- Granulocytes Exhibit Well Defined
Granules
Adequate - CytOplasmic Membranes Generally Intact 2
- Differential Staining is Fair (Lighter
Background)
- Some Epithelial Cells Exhibit Cilia
- Granulocytic Granules Identifiable
Poor - Bare Nuclei l
- Cytoplasmic Membranes Not Intact
- Excessive Dispersion of Granulocytic
Granules

 

41

their means compared by application of the student 'tl test. If the variance
of the means were not equal, a modification of the test was used where no
pooled variance term is used (Steel and Torrie, 1960) . The level of signifi-
cance was established at .05 (p 0.05) for all statistical analysis of data.

Evaluation of Nylon Bronchial Brushes vs. Steel Bronchial Brushes

 

Four dogs had biopsies performed with nylon brushes at 4 different
anatomical locations within the bronchial tree. These dogs were killed 24,
48, 72, and 96 hours after biopsy . Gross and histologic examinations of the
biopsy sites were performed. Four dogs were biopsied with steel brushes
and the same observations made.

Ten slides were randomly chosen for comparison of the cytology from
each of the 2 groups and were evaluated as previously described.

Evaluation of Nylon Bronchial Brushingvs. Bronchial Washing in
Experimental Inflammatory Disease

 

 

Four clinically normal Beagles with normal thoracic radiographic
patterns were used in this portion of the study. The dogs were anesthetized

with pentobarbital sodium and endotracheal tubes inserted.

A bronchial wash was performed by instilling 10-20cc of normal
saline into the bronchial tree through a catheter that had been passed
through the endotracheal tube. Suction was applied to the catheter recov-
ering as much of the saline from the bronchial tree as possible. The
bronchial washings were prepared for cytological examination by centrifug-
ing at 3,000 rpm for 7-10 minutes and removing the supernate by pippette.
The sediment was smeared on glass slides and air dried for staining and

application of cover slips as previously described.

42

Two bronchial brush biopsies were obtained from each of the right
and left lungs using the endotracheal tube technique previously described .
Slides were prepared using the smear technique and stained with modified
polychrome methylene blue.

The right main bronchus was selectively catheterized and 6ml of 60%
propyliodone suspended in peanut Oilh was instilled in the right lung with
the intent to create inflammatory disease (Myer, gt _a_l_. , 1974) . Its distribu-
tion was recorded radiographically . The left lung Served as a control.

The dogs were evaluated at daily intervals for 4 days. At each
evaluation, the clinical status of the dogs was noted and thoracic radio-
graphs taken . Two bronchial washings were performed and two bronchial
* brush biopsies from each lung were obtained . On the fourth day the dogs
were killed. The lungs and bronchial tree were examined for gross lesions
and tissues were collected for microscopic examination .

The bronchial brushings and bronchial washings were evaluated
using the criteria in Table 4. The inflammatory response was evaluated in
the bronchial washings and brushings according to the criteria in Table 5 .
TABLE 5 . CRITERIA FOR EVALUATION OF INFLAMMATORY RESPONSE IN
BRONCHIAL CYTOLOGIC PREPARATIONS

Numerical Score

 

Excessive - Number of Inflammatory Cells Exceeds

Epithelial Cells 3
Moderate - Number of Inflammatory Cells Equal

to Epithelial Cells 2
Minimal - Number of Epithelial Cells Exceeds

Inflammatory Cells 1

 

h‘Dionosil Oily, Glaxo Laboratories, Ltd. , Greenford, England.

43

The inflammatory response in the bronchial washings and brushings were
compared to find differences in their ability to detect inflammatory disease.
The cytologic preparations obtained by bronchial brushing were compared
to the radiographic appearance and post-mortem findings in the lung of
origin . Brush biopsies from affected and non-affected lungs in the same
animal were compared to evaluate the selectivity of the bronchial brush
biopsy. The cytologic preparations from both the bronchial brush biopsies
and bronchial washings were compared to the clinical status. and radio-
graphic appearance of the thorax in each dog to measure the sensitivity of
each method to detect sub-clinical inflammatory disease .
Evaluation of Bronchial Brushing in Dogs with Clinical Disease
Selective catheterization and nylon bronchial brush biopsy was per-
formed in 19 clinical patients . Results of the bronchial brush biopsy were
compared to clinical; endoscopic and radiogrpahic findings; hematology;

gross pathological and histo-pathological findings when available.

RESULTS

Technique of Selective Bronchial Catheterization and Bronchial Brushiri

Transtracheal catheterization via the cricothyroid membrane resulted
in the following observations . While performing the technique on one dog,
difficulty was encountered in passing the needle through the Cricothyroid
membrane and into the trachea. In 3 of 4 dogs, it was difficult to pass the
catheter over the guide wire and into the trachea because of resistance by
the tissues around the guide wire. In one dog, this became so time consum-
ing, this approach was abandoned and the bronchi were catheterized
through the endotracheal tube. One dog presented no difficulty during any
part of the procedure. N o difficulty was encountered in manipulating the
catheter after it was in the trachea and the endotracheal tube replaced .
Forty to 60 minutes were needed to perform catheterization by this
technique.

One larynx was examined 24 hours after the catheterization revealing
no significant gross lesions.

While performing the bronchial catheterization through the endo-
tracheal tube, resistance was met when attempting to pass the catheter
through the adapter and into the endotracheal tube in 2 dogs. This was
overcome by disconnecting the adapter and passing the catheter through

the adapter and into the endotracheal tube. Subsequently, the adapter was
44

45

reconnected to the endotracheal tube. Bronchial catheterization by this
technique requires 20 to 30 minutes to complete.

The right and left main bronchi and the right middle lobar bronchus
were the easiest to catheterize. The right and left cranial lobar bronchi
were the most difficult to catheterize and required the curved tipped
catheters . The bronchial brush could be passed to the periphery of all
lobes.

Cytology obtained by selective bronchial catheterizatiOnand
bronchial brush biopsy from normal dogs has as its primary cell type
ciliated bronchial epithelial cells . Goblet cells were seen in fewer numbers .
A few neutrOphils were present in most fields , with lesser numbers of
eosinophils and mast cells observed (Figure 15) .

Preparation of Slides for Cytologic Evaluation

Technically, there was no difference in difficulty in preparing the
slides by the touch or smear technique .

The results of evaluation of the cytologic preparations resulting from

these techniques are presented in Table 6. No difference was seen in the
amount of hemorrhage, nuclear detail, or cytoplasmic detail. A signifi-
cantly greater number of readable fields was seen in the slides prepared by

the smear technique .

Evaluation of Nylon Bronchial Brushes vs. Steel Bronchial Brushes

When using steel brushes, the need for a larger catheter resulted in
more difficulty catheterizing the desired bronchus than with nylon
bronchial brushes . Since steel brushes are more difficult to pass through

the catheter and do not have a deflectable tip , collection of the biopsy is

46

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on!
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47

TABLE 6. EVALUATION OF CYTOLOGIC PREPARATIONS OBTAINED BY THE

TOUCH AND SMEAR TECHNIQUES

 

 

 

 

 

 

 

 

Slide Readable Fields IIemorrhagg Nuclear Detail Cytoplasmic Detail
Touch Smear Touch mear Touch Smear Touch Smear
l 3 3 3 2 3 3 2 3
2 2 3 2 3 3 2 3 2
3 2 3 3 3 2 3 2 2
4 2 3 1 2 2 2 2 2
5 2 3 2 3 3 2 2 3
Mean 2.2a 3a 2.2 2.6 2.8 2.6 2.2 2.4
Standard
Deviation .44 0 .84 .55 .45 .54 .44 .55

 

 

 

 

 

 

 

 

 

 

aSignificant Difference p < 0 . 05 .

48

more difficult. If only a small specimen was collected by the steel brush, it
was difficult to transfer this material to a slide.

The results of the comparisons of cytologic preparations obtained with
nylon and steel bronchial brushes are presented in Table 7 . There was
significantly less hemorrhage with the nylon brushes, and therefore nucle-
ated cells were more easily evaluated. The cytoplasmic detail was
significantly better with the nylon brushes . No difference was seen in the
number of readable fields or nuclear detail.

In the 4 dogs biopsied with nylon brushes and evaluated 24, 48, 72,
and 96 hours after biopsy, no gross or histologic lesions were noted.

In the 4 dogs in which the biopsies were performed with steel
brushes, 3 lesions were observed on the bronchial walls in the area the
biopsy was obtained. Two lesions were seen in the dog evaluated 24 hours
after the biopsy was performed. These lesions were abrasions of the
bronchial wall .5cm in width and 1 .5cm in length with blood clots filling
the defects (Figure 16) . Histologically these areas were denuded of
epithelium and the basement membranes were ruptured. Blood clots were
adhered to the mucosa and hemorrhage was present in the adjacent tissues .
The third lesion was seen in the dog evaluated 96 hours after the biopsy
was performed. The gross and histologic appearance of this lesion was
essentially the same as the lesions in the other dog . There were no compli-

cations observed clinically in these 8 dogs at any time after the biopsy.

49

 

FIGURE 16. BRONCHUS OF A 1 YEAR OLD BFAGLE DOG 24 HOURS FOLLOWING
BRONCHIAL BRUSH BIOPSY USING A STEEL BRONCHIAL BRUSH. THERE IS A
DEEP ABRASION IN THE MUCOSAL LAYER OF THE BRONCHUS (arrow).

50

TABLE 7. EVALUATION OF CYTOLOGIC PREPARATIONS OBTAINED BY
STEEL AND NYLON BRONCHIAL BRUSHES

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Slide Readable Fields Hemorrhagg uclear Detail Cymlasmic Detail
Steel Nylon Steel Nylon llylon Steel Steel Nylon

1 3 3 2 3 3 3 2 3

2 3 3 1 2 2 2 1 3

3 2 3 1 3 2 - 2 1 2

4 3 3 2‘ 2 2 3 1 3

5 2 3 l 2 1 2 2 2

6 2 3 3 3 2 3 2 2

7 3 3 1 3 3 2 1 2

8 3 3- 2 3 2 2 1 3

9 2 3 2 3 2 2 1 2

10 3 3 1 '3 2 Z 1 2
Mean 2.4 3 1.63 2.7“l 2.1 2.4 1.3ID 2.4b
Standard . 84 0 . 69 . 48 . 56 . 51 . 40 . 51
Deviation

 

 

 

 

 

 

 

 

 

a'bSignificant Difference p < 0 .05 .

51

Evaluation of Nylon Bronchial Brushirivs. Bronchi'al'Wa‘shifl in
Eagerimental Inflammatory Disease

 

 

122g;

On day 0, the bronchogram revealed contrast media present in the
right lung with only a small amount of residual contrast media present in
the trachea (Figure 17) .

On day 1, there were no abnormalities on the physical examination.
Radiographically, an increased interstitial density was present throughout
the right lung . An alveolar pattern was present in the right middle lobe
characterized by airbronchograms . No abnormalities were observed in the
left lung .

On day 2, the dog's temperature was 103 .2°F and a productive cough
was observed. Radiographically, the left lung appeared normal. The
alveolar pattern was still present in the right middle lobe and the right
cranial lobe . The interstitial density in the right caudal lobe had decreased.

By day 3, the temperature returned to normal and the cough was
improved. The radiographic appearance of the thorax was similar to that
noted on day 2 .

Post-mortem examination revealed consolidation of the caudal portion
of the right cranial lobe, the right middle lobe, and the cranial portion of
the right caudal lobe (Figure 18) . No gross lesions were seen in the left
lung . On histologic examination, the affected areas were characterized by
bronchial and alveolar exudate, which consisted primarily of neutrophils,
with a significant increase in the number of macrophages . Vacuoles of

various sizes were present in the macrophages and were attributed to the

52

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.OOQ MAU<MQ GAO m<mw H < mo E<moomozomm M>Hhomqmm .bH mmbon

 

53

 

FIGURE 18 . POST-MORTEIVI APPEARANCE WITH PARTIAL CONSOLIDATION OF THE
RIGHT LOBES OF A DOG'S LUNGS '72 HOURS AFTER BRONCHOGRAPHY OF THE RIGHT
LUNG LOBES WITH 6m1 OF PROPYLIODONE SUSPENDED IN PEANUT OIL.

54

peanut oil present in the contrast media. Other areas of the lung and
bronchial tree were normal.

Cytologic findings are listed in Table 8 . Consistent with the radio-

graphic and post-mortem findings , significantly more inflammatory changes

were seen in the biopsies from the right lung than the left. There was no
significant difference between the right bronchial brush biopsies and the
bronchial wash cytologies in contrast to a significant difference between the

left bronchial brush biopsies and the bronchial washings .

The cytologic preparations from affected areas consisted primarily of
neutrophils. Many macrophages were seen as well as occasional mast cells
and eosinophils (Figure 19) . Bronchial epithelial cells were present in
fewer numbers in bronchial biopsies from normal dogs .

129.3

On day 0, the bronchogram revealed contrast media in all lobes of the
right lung and a small residual amount in the trachea.

On day 1 , the physical examination was characterized by crackles
ausculated over the right lung field . The body temperature was normal.
Radiographically, there were increased bronchial and interstitial densities
of the right lung . An alveolar pattern characterized by air bronchograms
was present in the right middle lobe .

By day 2, abnormal lung sounds were reduced in intensity.
Bronchial and interstitial densities were decreased in the right lung when
evaluated radiographically. The abnormal density in the right middle lobe
was reduced. The caudal part of the right cranial lobe showed evidence of

an alveolar pattern .

55

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n.

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‘3 a
Q E2 ti"

45 {:5 ?
_-_ ', I
3 _.

.9!

”733.»

.. E?

l

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34.
fi.

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i

FIGURE 19. CYTOLOGIC PREPARATION OF A BRONCHIAL BRUSH BIOPSY FROM LUNG
72 HOURS AFTER Oml OF PROPYLIODONE SUSPENDED IN PEANUT OIL WAS INJECTED
INTO ITS PRINCIPLE BRONCHUS. RED BLOOD CELLS(A), RUPTURED MACROPHAGES

(B), NEUTROPHIL (C), EOSINOPHIL (D) WERE IDENTIFIED IN THIS PREPARATION.
MODIFIED POLYCHROME METHYLENE BLUE STAIN; 72OX.

56

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57

On day 3, the lung sounds were normal. Bronchial and alveolar
densities in the right lung had further resolved, and the alveolar patterns
were no longer evident. Post-mortem examination revealed areas of consoli—
dation in the caudal part of the right cranial lobe, the right middle lobe, and
the cranial part of the right caudal lobes . In affected areas, there were histo-
logic changes similar to that described in dog 1 . Histologic examination of
the left lung revealed a neutrophilic exudate in some of the major airways,
while the lung parenchyma was normal.

The evaluation of the cytologic preparations are listed in Table 8.
There was a significant difference between the amount of inflammatory
change in the right and left bronchial trees as indicated by the bronchial
brush biopsies . There was a significant difference between the left

bronchial brushing and the bronchial washing , with the bronchial brush

biopsies from the left bronchial tree again containing fewer inflammatory
cells . No significant difference was present between the right bronchial
brush biopsies and the bronchial washings. On day 3, in the left bronchial
brushing , a moderate amount of inflammation was present, which correlated
with the histologic finding of a neutrOphilic exudate in the major airways of
the left lung.

9223

On day 0, the bronchogram revealed a substantial amount of contrast
media in the trachea. No abnormalities were seen radiographically or on

physical examination on day l .

58
On day 2, the dog had a temperature of 103.5°F , increased

respiratory rate, and mild dyspnea . An increase in interstitial and peri-
bronchial densities were noted radiographically throughout both lung
fields . The right middle lobe displayed an alveolar pattern characterized
by air bronchograms .

By day 3, the temperature returned to normal and the breathing
appeared normal. Radiographically, there was no change in the interstitial
or peribronchial densities from day 2 . There was reduced involvement of
the right middle lobe . Alveolar densities were evident in the right cranial
lobe. Post-mortem examination revealed consolidation of both lungs, the
right lung being more uniformly affected than the left. The right cranial
and middle lobes were the most severely affected. Histologically, the
appearance of the affected areas was as described in dog 1 . There were
more extensive changes in the right lung than in the left.

The evaluation of the cytologic preparations from this dog are listed
in Table 8 . No significant differences were seen between bronchial brush
biopsies from the left and right lungs . This reflected the bilateral involve-
ment as documented on the radiographic and post-mortem examinations .
There were no cells present in 3 of the cytologic preparations made from the
bronchial washings from this dog .

122a_4

On day 0, the bronchogram revealed that the right lung contained
contrast media, however, a substantial amount was also present in the

trachea .

59
On day l, a fever of 103°F was present. Radiographically, the

interstitial density of both lung fields was increased, and there was alveo-
larized contrast material in the caudal portions of the left cranial lobe. An
alveolar pattern was present in the right cranial lobe .

During bronchial catheterization, the catheter penetrated a bronchus
and the visceral pleura, and immediate dyspnea resulted. Radiography
indicated a left hemi-pneumothorax . No special therapy was instituted.

The dog was observed closely for the next 12 hours during which time the
dyspnea resolved .

On day 2, the temperature was normal. Wheezes were heard over the
dependent area of the right lung field . Radiographically, pneumothorax was
still present, but the amount of pleural air appeared to be reduced. An
overall increase in density was seen in both lung fields . Interpretation of
the increased density in the left lung field was difficult because of the
pneumothorax . The alveolar pattern of the right cranial lobe had resolved.

An alveolar pattern was now present in the right middle lobe . On day 3,

the wheezes were still present in the dependent areas of the right lung field.

The pneumothorax was still present as judged radiographically. An

increase in interstitial and bronchial densities was present. Areas of
alveolar density were seen in the right cranial and right middle lobes .

Post-mortem examination revealed consolidation of caudal portions of

the right cranial lobe, the right middle lobe, and the cranial portions of
the right caudal lobe . The hilar region of the lobes of the left lung was less
affected. A healing puncture wound on the lateral surface of the left

cranial lobe was identified. Histologically, the inflammatory change seen in

60

the previous dogs was present in the affected areas of the right lung .
Sections from the peripheral regions of the left lung were normal.

The evaluation of the cytologic preparations from this dog are shown
in Table 8 . No significant difference was seen between any of the biopsies

or washings. Five washings contained no cells. Three false negative wash-

ings were obtained from this dog .

The bronchial washings and nylon bronchial brushings from these 4
dogs were compared by the criteria described in Table 4. The 'results of
the evaluations of cytologic preparations obtained from bronchial brushings
and bronchial washings are shown in Table 9. There were significantly
more readable fields present in the bronchial brush biopsies as compared to
the bronchial washings . Less hemorrhage was present in the bronchial
washings. The nuclear and cytoplasmic detail was significantly better in

the bronchial brushings than in the bronchial washings .

Evaluation of Bronchial Brushings in Dogs With Clinical Disease

 

Listed in Tables 10 and 11 are the pertinent data from clinical patients
that underwent selective bronchial catheterization and bronchial brush
biopsy. The cytological findings obtained by bronchial brush biopsy were
evaluated in light of the other data available on these patients .

Data from dogs with confirmed neoplastic disease is shown in Table
10. Case 2 was primary lung neoplasia; cases 1, 3, 4, and 5 were

secondary neoplastic disease of the lung .

Neoplastic cells were not present in the bronchial brush biopsy from

the case with primary pulmonary neoplasia. The lack of neoplastic cells in

61

TABLE 9. EVALUATION OF CYTOLOGIC PREPARATIONS: BRONCHIAL
BRUSHINGS VS . BRONCHIAL WASHINGS

eadable Fields Hemorr e uclear Detail c Detail
ash- Brush- Wash- Brush Wash- Brush~ Wash- Brush-
Slide in in in in in in in

       

     

 

“340‘U‘ltwal—i

wNHi—IwNHNHwNwaHHwNHwNwt-awNwwHt—IwNu:
wwNwwuwNNwNwwNwNwwwr-IwuwNwwwNwwNw
wmwwwwwwwwwwwwwwwuwwwwwuwwmwmwuu
uNNwNNwwwNwwNwwwNwwHNwwwHNwwt-awmw
HHNHHHNHHNHHNHWHNHHNHNHNNNHNNHNN
mNNwNHWNNNwNNNwNNNwNNwwNNwNNNI—‘WN
HHNHHI—It—IHHNt—IHHHHHNNHHHHNHHHHNHHHN
HwNNwNwNwwNNwNNHNNwNNNmNNwNNwwNN

Mean .1a 2.63 .96b 2.5ID . . 2.28
Standard .88 . 54 . 18 . 71 . . . 58
Deviation

 

a,b,c,d
Significant Difference p < 0 . 05 .

62

 

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FIGURE 21. CYTOLOGIC PREPARATION FROM A BRONCHIAL BRUSH BIOPSY OF A
DOG WITH BRONCHITIS; DEGENERATIVE NEUTROPHILS (A) AND MACROPHAGES (B)
ARE PRIMARY CELL TYPES. MODIFIED POLYCHROME METHYLENE BLUE STAIN;
720x. '

72

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73

    

fi
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l- ‘
FIGURE 23. CYTOLOGIC PREPARATION FROM A BRONCHIAL BRUSH BIOPSY OF A 3
YEAR OLD GERMAN SHEPHERD DOG WITH PUIMDNIC INFILTRATION AND EOSINOPHILIA.

THE CELLS ARE PREDOMINANTLY EOSINOPHILS. MODIFIED POLYCHROME METHYLENE
BLUE STAIN; 720x.

74

the biOpsy from this case could have resulted from improper catheter
placement but post-mortem examination revealed no bronchi involved with
the tumor.

Two of the cases of secondary neoplasia had neoplastic cells present
in the biopsy samples .

One false negative biopsy was obtained in the cases of primary neo-.
plastic disease and. 2 false negative biopsies in those cases of metastatic
disease .

Table 11 displays the pertinent data from patients with inflammatory
disease . Evaluation of the bronchial brush biopsy in these cases is more
difficult than those with neoplastic disease because no histological sections
are available for a more definitive diagnosis . Fourteen cases of clinical
inflammatory disease were biopsied by the bronchial brush technique. All
14 of these cases had cytologies that were consistent with the clinical diag-
nosis determined by evaluation of the data from physical examination and
history, clinical pathology, radiography, and response to therapy. In 10
of the cases, the bronchial brush biopsy contributed to the diagnosis .
These cases included 1 through 3, 6 through 9, 11, 13, and 14. As would
be expected, all of these patients had disease problems which caused
exudation into the bronchial tree. In the other cases studied, case 4 had a
paralyzed vocal fold, and would not be expected to have any pulmonary
pathology unless the larynx was impaired enough to caused aspiration of

food and saliva. Case 5 had mild Dirofilaria immitis and would not be

 

expected to produce bronchial exudation unless the disease was severe.

Case 10 had interstitial pneumonia and one would not expect a bronchial

75

exudate unless it was severe. Capillaria aerophilia excretes its ova in the

 

bronchial tree and a bronchial exudation might be expected . Since neither
was found in case 12, it was considered to be a false negative result.

To summarize the results obtained in clinical patients undergoing
bronchial brush biopsy, 15 or 79% of the biopsies were consistent with the
final diagnosis. Twelve or 65% of the biopsies were of diagnostic value
because the cytological pattern suggested a particular disease entity. Four

false negatives were obtained .

DISCUSSION

Selective bronchial catheterization through the endotracheal tube does
not require removal of the endotracheal tube, therefore assisted ventilation
can be maintained throughout the procedure. No surgical scrub is
necessary, thus reducing the time needed for the procedure. idultiple
catheterizations are possible because the difficulty in passing the catheter
over the guide wire and through the cricothyroid membrane is avoided .
Manipulation of the patient under the fluoroscope is accomplished with less
difficulty. These observations suggest that the preferred method is through
the endotracheal tube rather than the cricothyroid membrane. A possible
exception would be the patient in which the clinician suspects that the
respiratory disease is caused by an infectious agent and other culture
attempts have either been unsuccessful or several different organisms were
present suggesting contamination by the upper airways . Catheterization
through the cricothyroid membrane, rather than an endotracheal tube,
would allow the upper airway to be bypassed and prevent contamination.
The organisms cultured from a specimen obtained in this manner should
more closely reflect of the flora of the lower respiratory tract .

It was clearly demonstrated that selective bronchial catheterization
allows direct access to the peripheral portions of the cranial lobes; this has

not previously been possible by other non-invasive diagnostic techniques in

76

77

the dog . The peripheral areas of other lobes were also accessible by this
technique, further broadening its diagnostic capabilities.

The nylon bronchial brushes were shown to more useful in the dog
than those constructed of stainless steel because they are easily manipu-
lated, cause less trauma at the biOpsy site, and the cytologic specimens
obtained were superior by the criteria used in this study.

An exception would be a patient with a mass present in an easily
accessible bronchial lumen . Use of a stainless steel brush in this situation
might allow collection of a large enough volume of tissue for histologic
examination. This type of lesion would most commonly be seen with a
bronchogenic carcinoma, which tends to be found in the hilar area and is
less disseminated than the bronchiolar alveolar carcinoma. Because the
incidence of primary pulmonary neoplasia is low - 3 . 4 per 10,000 dogs
(Cohen, e1 a_1_. , 1965) - and of the primary tumors, only 25% are of a type
other than the bronchiolar alveolar carcinoma (Jubb and Kennedy, 1970) ,
the number of cases where the stainless steel brush would be useful would
be too few to justify recommending its use.

Smearing'the specimens on the slides are shown to be superior to the

touch technique because more readable microscopic fields resulted. No
difference was seen in the amount of hemorrhage present on the slides, but
when the specimens were smeared, the red cells were dispersed and the

nucleated cells were more easily visualized; this adds support for choosing

this technique.
The cytologic preparations produced by bronchial brush biopsy were

shown to be more desirable than those obtained from bronchial washings.

78
It should be emphasized that 8 bronchial washings contained no cells for

examination, and in no case was this true of the bronchial brush specimens .
This would suggest that interpretable specimens will be more consistently
obtained by bronchial brush biopsy .

When cellular degeneration is seen in a bronchial brush biopsy
specimen, it is more probable that it is due to disease within the patient
than sampling artifact that can be associated with cytologic techniques .

This is substantiated by the nuclear and cytoplasmic detail being signifi-
cantly better in bronchial brushing specimens compared to bronchial
washing sepcimens. It follows that this technique should allow for more
critical evaluation of cells for neoplastic changes.

An attempt was made to create inflammatory disease in l lung of 4

dogs using the other lung as a control. In 2 of these dogs, radiographic
findings, post-mortem findings, and the cytologies from the bronchial
brushings revealed that inflammatory lung disease was present in both
lungs . This was probably due to aspiration into the left lung of contrast
media, which had been inadvertently placed in the trachea.

The radiographic and post-mortem examinations of the other 2 dogs

confirmed that pulmonary disease had been created only in the right lung .

The cytologies from the bronchial brushings were consistent with inflamma-
tory disease of only that right lung except for 3 specimens; 2 in dog 2 and 1
in dog 1 . The post-mortem examination of dog 2 confirmed the presence of

inflammatory exudate in the bronchial lumen with normal lung parenchyma .
This suggested that false positives can occur when airways from unaffected

areas contain aspirated exudate from other affected airways . It can be

79

concluded that the bronchial brush biopsy is a selective technique and

i that false positives are possible emphasizing the cytologic findings should
be evaluated in light of the other data available from a patient. No false
negatives were seen suggesting that if inflammatory disease was present,
it would be revealed by bronchial brush biopsy . This is in contrast to

3 false negatives that were obtained by bronchial washings .

Inflammatory cells in greater numbers than were present on control
samples were observed in the cytologic specimens from dog 3 twenty-four
.hours before clinical signs or radiographic examination revealed any evi-
dence of respiratory disease. This observation suggests that subclinical
respiratory disease in the dog might be revealed by cytologic techniques, as
is true in the horse (Beech, 1975) . This could be of value in patients with
a fever of undetermined origin or an unexplained leukocytosis . Bronchial
cytology may reveal low grade inflammatory respiratory disease in some of

these patients .

Selective bronchial catheterization and bronchial brush biopsy were
developed primarily as a means of confirming suspected cases of pulmonary
neoplasia in the human. During the period of this study, only 5 dogs with
pulmonary neoplasia were presented that could be confirmed by post-mortem
examination . One of these was primary pulmonary neoplasia and had no
neoplastic cells in the cytologic preparation obtained by bronchial brush
biopsy. This is not a large enough sample to compare to the results

reported in man .
The diagnostic accuracy of the technique in cases of metastatic

pulmonary disease in the dog was equal to that in the human; 50% of the

80

confirmed cases had neoplastic cells in the bronchial brush biopsy. The
diagnostic yield in metastatic disease cannot be expected to be as high as in

primary disease because metastatic sites usually start as tumor emboli

attached to the endothelium of capillary or lymph vessels (Schmidt, 1903).
From this beginning , the cells migrate to the perivascular interstitial tissue

where they grow by expansion and compress surrounding tissues (Janower

and Blennerharsett, 1971) . The tumors will not be detected by bronchial
brush biopsy until the eroded bronchus allows the cell to spread into the

bronchial tree . In contrast, in the primary lung tumor, the cell of origin is

likely to be in the bronchial wall, therefore the tumor would be accessible to
the bronchial brush at a much earlier time in the tumor's progression. The 3
false negatives obtained in the dogs with neoplastic disease were false
negatives in the sense that they did not reveal the lung pathology present
but were not true false negatives in that they probably did reveal the true

status of the bronchial wall at the time the biopsy was performed .

If selective catheterization and bronchial brush biopsy is to be a
useful diagnostic tool in the dog , it must be of value in the differential
diagnosis of inflammatory disease of the dog's respiratory system, since
the incidence of primary pulmonary neoplasia is low . In the series of 14
cases of inflammatory disease in this study, only 1 false negative was
present. The bronchial brush biopsy in this case may have reflected the
status of the bronchial wall and is a false negative only in the sense that
the disease process was not detected . The brush biopsy did not lead
directly to a diagnosis in the other 13 cases, but in all cases where an

abnormal bronchial exudate would be expected, the bronchial brush

81

biopsy revealed an abnormal exudate. The results of the technique in
evaluation of inflammatory disease in the dog are encouraging and have
established the basis for its continued investigation as a biopsy technique.

Also, its potential as a culture technique should be investigated.

Complications endangering the well-being of the patient were not
frequently encountered. One dog developed a pneumothorax as a result of
the bronchial catheter penetrating the pleura of the left cranial lobe. The
dog recovered with no specific therapy other than cage rest. No other

complications were observed .

The procedure is safe in the dog if a few precautions are adhered to.
First, the dog should be properly evaluated as an anesthetic risk. The
content of this evaluation will depend on the age of the animal and its
current medical problems . Second, the clinician should become familiar
with the bronchial anatomy of the dog to avoid unnecessary manipulation of
the catheter with possible trauma to the bronchial wall and prolonged anes-
thesia. Third, the clinician should become familiar with the instrumentation
prior to the procedure to avoid prolonged anesthesia and to insure that
adequate biopsies are collected. Fourth, the patient should be monitored
closely during the procedure to allow for immediate recognition of anesthetic
or other complications .

Selective bronchial catheterization and bronchial brush biopsy were
of diagnostic value in 63% of the patients in which they were employed. This
indicates that they should be considered as part of a diagnostic plan when

primary pulmonary neoplasia or inflammatory disease with bronchial exuda-

tion is suspected . Because a fluoroscope equipped with image intensification

82

is necessary to perform this procedure, it probably cannot be employed by
most general practices but should be a part of the capabilities of a referral
center accepting patients with suspected pulmonary disease. It is not
suggested that this technique replace other cytological techniques; its
advantage is that it allows the clinician to identify the site from which a
biopsy is taken . Therefore, collection of the biopsy from the diseased area

of the lung can be assured.

BIBLIOGRAPHY

 

BIBLIOGRAPHY

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Bean, W. J. ,Grahum, W. L. ,Jordan, R. B. , and Evenson, L. W.: Diagnosis
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Beech, J . : Cytology of Tracheobronchial Aspirates in Horses . Vet. Pathol. ,
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Bibbo, M. , Fennessy, J.J. , Lu, C . , Straus, F .H. , Variakogis, D . , and
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Dellman, Horst, Dieter: Respiratory System . In Textbook 91 Veterinary
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F ennessy, J .J .: A Technique for the Selective Catheterization of Segmental
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Hattori, S. , Matsuda, M. , Sugiyama, T. , Wada, A. , and Terazawa, T.:
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Metr‘as, H .: Une sonde pour le catheterisme des bronchus du lobe
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