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4 Comparison ofEndobronchial Ultrasonography Images andResected Specimens

Fig. 4.1 Excised lung. (a) Collapsed excised lung. The lesion could be

identied after ination from its position relative to the pattern of distribution of patchy anthracosis on the visceral pleura. (b) Inated-xed

4.1.2 Make Every Eort toRestore theLung toIts InVivo State

Prepare the inated-xed lung by formalin injection. In some institutions, injection is carried out through the bronchial stump, but ination is easier if injection is performed from the pleural side. If the bronchial stump is open, clamping it with Pean forceps or another hemostatic clamp or suturing it closed prevents formalin leakage and speeds up ination. Even if an incision has been made to take a sample from the fresh specimen, given sufcient time, good ination can be achieved. Make a small hole in a plastic intravenous (IV) drip bag to hold the formalin, hook up several parallel IV routes, and attach a pink (18G) needle to the end of each route (Fig.4.2, left).

Make sure the IV clamp is closed. Fill the IV bag with formalin, and hang it from a suitable hook. In our hospital, we use a laundry pole hanger and a hook made from a wire hanger. The lling pressure is determined by the difference in height between the IV bag and the specimen, but as Spencer’s Pathology of the Lung states that this should be 2–3ft [1], there is no need to worry too much about the height in regular lung cancer diagnosis. Place the excised lung inside a large clear plastic bag, insert the pink needle via the pleural surface at a point away from the lesion, and open the clamp (Fig.4.2). If the ow rate is poor, insert the pink needles slightly less deeply, in which case, to begin with, each of the secondary lobules visible on the surface of the lung can be seen to swell up one by one. Gather up the top of the plastic bag, being careful not to dislodge the pink needles, and use a large (st-sized) plastic clip to clip it to a vertical pole or simi-

lung. The arrow shows the estimated position of the mass. This is a different patient from the case illustrated in Fig. gures. It is the excised lung of the case illustrated in Fig. subsequent gures

4.2 and subsequent

4.17 and

lar object to minimize the irritating smell of formalin. The plastic bag and its contents can be placed in a large kidney basin or similar container to keep it stable. Once the lung has inated sufciently, remove the routes without contaminating your hands, tie up the plastic bag, and store it as it is. Some formalin will always leak inside the plastic bag, and if the amount is small, more must be added as necessary to immerse the entire specimen. In our hospital, we hang up the plastic bag and its contents for storage, placing a large container underneath so that, even if it falls, the formalin will not contaminate a wide area. The storage location and equipment used should be adapted as required.

4.2 Cutting Out

4.2.1 Overall Observation andPhotography

If an air extraction system is not available, use of a formalin mask is essential. Take another look at the bronchial stump, and envision the course of the involved bronchus or bronchi within the excised lung. If you are unsure, check whether or not a particular route leads to the lesion by carefully inserting a surgical probe while you envision the direction of branching. In some cases, it may be helpful to make a small longitudinal incision in the lobe/segment bronchial wall where the involved bronchus is located (Fig.4.3). For peripheral lesions, unless the bronchial stump is destroyed, there may be little effect on subsequent pathological testing.

EBUS for resected

4.2 Cutting Out

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145

specimen

air pump

Pathological

correlation

with EBUS images

after formalin

-inflated -

fixed Lung

air inflation

formalin inflation

Fig. 4.2 Air ination and formalin ination. Left: formalin injection

set using an IV bag and routes. Top center: after ination with an air pump, the specimen can be examined by ultrasound. Top right: airinated lung. The pink needle is inserted via the pleural surface. When

performing formalin xation after air ination, care must be taken to collapse the lung again before the formalin is injected or xation will be uneven. Bottom right: formalin-inated-xed lung. Sufcient formalin ination and xation was complete on the following day

cii

B3b

1+2

Fig. 4.3 Identication of bronchial branching morphology and cutting

out. (a) The bronchial stump was opened, and the bronchial morphology was reidentied. Left B

b ran down straight in front. The initial cut

was made across B

b at right angles, leaving the hilar region. (b) After the hilar region had been removed, the specimen was divided craniocaudally into three equal parts

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4 Comparison ofEndobronchial Ultrasonography Images andResected Specimens

4.2.2 Make anIncision

Make an incision perpendicular to the bronchus that is believed to have been involved, that is, the bronchus through which the ultrasound probe was passed (Fig.4.4). If the staple line at the interlobular margin coincides with the extension of the incision, the knife will immediately stop cutting; in this case, resect the staple line with scissors before using the knife (in specimens obtained from partial resection by videoassisted thoracoscopic surgery, stapling may have crushed the main lesion, in which case resect and preserve the staple line before xation [2]). To slice it properly, place the initial cut face down on a stand, and use the thickness of the grip of a special knife to produce slices of equal thickness. When cutting slices perpendicular to the airway, there is no chance of destroying areas of pleural invasion, but if incisions are being made parallel to the axis of the involved bronchus in order to expose its length, this may involve incising the area of greatest pleural invasion on gross examination. When I started surgical training, I was told to avoid this [ proper to avoid it to enable accurate pathological diagnosis.

3], and in fact it is only

4.2.3 Spread OuttheCut Slices

oftheSpecimen andPhotograph theCut Surfaces (Fig.4.5)

If the involved bronchus runs in the direction of the long axis of the body, the cut surfaces will coincide with the horizontal plane on computed tomography (CT), making the connections between the target vessels relatively easy to identify. However, this is frequently not the case, making it difcult to identify the target vessels. In either event, the cut surfaces must be carefully lined up in the right order. If they cannot easily be explained later by the doctor who photographed them, no one will be able to understand them. An overall view and close-up photographs are generally taken, but the opposite side (reverse surface) should also be recorded. Cut surfaces of interest should be photographed not only from the front but also from various different angles and from the side to give a threedimensional picture. If a structure could be either a blood vessel or a bronchus, this can be distinguished by its hardness when touched with forceps or a nger. Some things can only be learned by taking part in the cutting-out process.

Apical region

of the lung

Center

part

Hilar region

of the lung

Lingula

segment

Peripheral

part

Fig. 4.4 All parts of the left upper lobe. The central portion that had just been divided into three equal parts was cut into six slices from the center

to the periphery, and these are laid out to reect their anatomical orientation

4.3 Comparison

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B3b

147

cii

B3ci

Fig. 4.5 Gross observation of the involved bronchus and lesion. The bronchi and vessels are carefully laid out to enable their courses to be easily

understood. This shows the courses of left B

4.2.4 Reconstruction oftheSpecimen

b, B3ci, and B3cii and how they spread out. The yellow arrow indicates the lesion

passed), and the tumor and the biopsy site are identied. If only a short time has elapsed from biopsy to excision, the

Peripheral lesions are usually cut into slices by two or three incisions. Even if they become somewhat deformed during photography as the formalin drains out, they regain their shape when reimmersed in more formalin, making reconstruction simple. However, if they have been cut into more

scar can be identied under the microscope. Whether this change is identied macroscopically or not, depending on the case, can be easily observed. This is because the images are imported, so there is no time limit on viewing them. A typical case (Case 1) is described below.

slices than this, reconstruction may be difcult in the pathology department, and the former shape of the lung must be carefully reproduced in an orderly fashion. Needle sutures can be placed at three or so points where no lesion is thought to be present (Fig. 4.6). Findings must be described in as much detail as possible on the pathology slip, and, if necessary, these may be submitted with representative photographs attached.

Case 1. Male in 70s. Chief Complaint: Left Lung Nodule

Medical history: In October 2009, the patient underwent low anterior resection for advanced rectal carcinoma. In 2012, a nodule was identied in the left upper lobe, and this was kept under observation. In December 2013, it was seen to have grown, and bronchoscopy was performed, but since it was inconclusive, he underwent EBUS-GS in our hospital in late January 2014. Rectal carcinoma metastasis was diagnosed, and he underwent upper left lobectomy in the hospital where

4.3 Comparison

he was previously treated.

Imaging ndings: A small nodule measuring 13mm × 9mm This task can basically only be carried out on the computer screen. It involves comparing ndings and structures of interest on EBUS images to see how well they correspond with macrophotographs of the specimen. In addition to the cancer tissue, the position of accompanying blood vessels, the positional relationships between the involved bronchus (the branch through which the probe is believed to have

directly involved left B3b, and wide A3b also entered the nodule, posing a high risk of hemorrhage (Fig.4.7). The pattern was small S

1+2

c and large S3.

Bronchoscopy ndings: A thin bronchoscope with a

4-mm-diameter tip was used to examine the bronchi as far as the opening of left B3b. Edematous stenosis was present, and an ultrasound probe was inserted (Fig.4.8).

148

4 Comparison ofEndobronchial Ultrasonography Images andResected Specimens

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Fig. 4.6 Reconstructed specimen. If a specimen has been divided into a large number of parts, needle sutures can be used

1+2

B3b＋B3c

aii

B3a

b＋A3c

Fig. 4.7 CT ndings. HRCT images are arranged from left to right in

order from caudal to cranial. The superior segment of the left upper lobe shows a bifurcation pattern between B

1+2

and B3. From B3, B3a rst

cii

tumor

branches off laterally, with the remaining B running laterally and B

c running anteriorly. B3b immediately reaches

tumor

A3b

b+B3c dividing into B3b

the lesion. Immediately cranially, however, the wide A artery is directly involved

b pulmonary

4.3 Comparison

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149

b+B3c

1+2

B3c

Fig. 4.8 Bronchoscopy ndings. The superior segment of the left upper lobe shows a bifurcation pattern between B

b at its opening, and no observation of deeper regions is possible

EBUS ndings: A pulsing pulmonary artery (PA) ran

alongside B3b just before the lesion (Fig.4.9: 3-A). In the

4.3.1 Analysis ofHistological Presentation andCorrelation withTechniques

cii

proximal part of the tumor interior, part of the tumor could be seen via the PA, but the lesion was not clearly visible in the vicinity of the bronchus containing the probe (Fig.4.9: 3-B). Since the pulsing PA was also visible inside the tumor in the lesion around the bronchus (Fig.4.9: 3-C), a biopsy was taken by angling the bronchoscope away from the PA using its up/down angle (Fig.4.9).

Pathology results: The histological type was similar to that of rectal carcinoma, and lung metastasis was diagnosed.

Comparison: Starting with Fig.4.9, orthogonal multiplanar reconstruction (MPR) images of left B3b generated from CT images were consistent with the view from the bronchoscope. In this patient, they corresponded to the sagittal view from the median side. B3b was accompanied by a pulmonary artery on the cranial side and was obstructed partway down. The corresponding images isolated by EBUS, the corresponding cut surfaces from the xed specimen, and the corresponding hematoxylin-eosin (HE)-stained macro images are shown in each row.

The bifurcation peripheral to left B3b could not be observed bronchoscopically, but B3bi and B3bii could be seen on the cut surface of the tissue. In Fig.4.10, the left branch containing mucus and the right branch containing an organized substance corresponded to B3bi and B3bii, respectively. Although the stenosis of B3b prevented the observation of more peripheral bronchi in this case, a positional comparison of the EBUS images and tissue ndings and the biopsy artifacts in tissues described below suggested that the probe must have entered the right branch (B3bii). In Fig.4.11, the tissue structures visible in each slice are labeled “Tumor,” “PA,” “Br1,” “Br2,” and “Mucus Lake” for the purpose of comparison. High-power observations conrmed the absence of cancer cells in the airways of both Br1 and Br2 at the proximal level of the tumor in Fig.4.11. In the central part of the tumor in Fig.4.12, Br2 was located on the tumor margin, but there was no tumor exposure in the lumen. However, Br1 had disappeared. For ease of reference, we divided the tumor into three parts, following the direction of

1+2

and B3. There is stenosis of

1−A

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BCDEA

2−A

3−A

b＋B3c

2−B 2−C 2−D2−E

aii

3−B

Cart

4−A4−C4−B

5−

Br2

Br1

Cart

Br2

accompanying

artery

cii

3−C

3−D

4−D

Br2

Muc

5−B

Br1

Br2

Muc

−C

Br2

Muc

Fig. 4.9 Comparison of CT, EBUS, macrophotographs of the gross

specimen, and HE-stained images. Row 1 (1-A): CT axial view, laterally reversed to facilitate the imaging of left B from left B MPR images in the plane orthogonal to left B MPR images was adjusted so that the left lateral side was viewed from the left hilar region. Row 2 (2-A, B, C, D, E): MPR images (in this case uniformly in sagittal view, with the ventral side on the right and the dorsal side on the left of each image). Images (A–E) in Rows 1 and 2

. The area indicated by the red arrows was used to produce

b branching laterally

b, and the direction of the

correspond approximately with each other. In 2-C in Row 2, the pulmonary artery runs next to B to be the blood vessel. In 2-E, the vessels spread out peripherally. Row 3 (3-A, B, C, D): EBUS images. Row 4 (4-A, B, C, D): macrophotographs of the gross specimen. Row 5 (5-A, B, C): macrophotographs of the HE-stained specimen. In Rows 2–5, the red arrows indicate approximate correspondence. PA pulmonary artery, PV pulmonary vein, Tu tumor, Cart cartilage of the bronchus, Br bronchus, Muc mucus lake of peripheral lung

b. In 2-D, most of the solid lesion is believed

4.3 Comparison

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151

2mm

Fig. 4.10 Bronchial lumina. The left bronchus contains mucus, and the right shows organization with squamous metaplasia. The left and right

bronchi could be distinguished by their contents

Fig. 4.11 Proximal part of

the tumor. The bronchus exhibiting organization is labeled Br1 and that lled with mucus Br2. The accompanying artery is labeled PA and the collection of mucus in the peripheral lung as “Mucus Lake.”

Tumor

2 mm

Br2

Mucus Lake

Br1

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Fig. 4.12 Central part of the

tumor. Br2, which contained mucus in the lumen, is still present, but Br1, which showed organization, has disappeared. Magnied images show no tumor exposure of the Br2 lumen. The tumor is divided into three parts according to their position relative to the PA, called Tumors A, B, and C

4 Comparison ofEndobronchial Ultrasonography Images andResected Specimens

Tumor A

Tumor C

2 mm

the PA, and called them Tumors A, B, and C. Figure4.13 corresponds to the peripheral side of the main lesion. As shown in Fig.4.14, on close observation of Tumor B, which seemed to be on the line of extension of Br1, the cancer ducts were split and broken, and this was a biopsy artifact. An examination of the biopsy tissue indicated that the specimen, which included an alveolar area, was sampled from a region away from the PA, as shown in Fig.4.15. The biopsy was performed by angling the bronchoscope away from the PA.These ndings and others suggested that the biopsy was performed properly by entering Br1 (B3bii in this case) and using a direction that avoided the PA. As can be seen by comparison with Fig.4.16, EBUS may enable the detailed structure of the tumor interior to be distinguished. In Figs.4.10 and 4.11, Br2 contained only mucus, but organization with squamous metaplasia was visible in Br1. If this is an expression of the difference in the time elapsed since the development of airway stenosis or occlusion by the tumor, it is extremely interesting.

Identication of the bronchi involved in pulmonary peripheral lesions is the most important and interesting preparatory task in bronchoscopy. The bronchial branching morphology from the center to the periphery is inferred/predicted from CT, and a hand-drawn branching map is completed. The numbers and branching directions of the segmental and subseg-

Br2

Tumor B

Br1 ?

Mucus Lake

mental bronchi are anatomically consistent, but the smaller branches vary widely between individuals. Of course, this means that even the routes of involved bronchi differ from person to person. For this reason, it is worth mapping the branches. Having the computer draw virtual bronchoscopy images for use in a navigation system is useful, but it may not be possible for the ne peripheral bronchi to be drawn automatically, and if they have to be produced manually in the navigation system, this is the same as drawing them visually from CT images. Is it still unclear whether the branch map of the peripheral branches that could not be observed bronchoscopically is correct? The easiest way to answer this question is to cut along the airways and open them up when investigating the resected specimen.

Do only bronchi that are endoscopically observable constitute the landmarks on the way to the lesion? When a ne ultrasound probe is used, the outer surface of the probe usually adheres to the bronchial wall in areas peripheral to the subsegmental bronchi, enabling the visualization of structures on the other side of the wall [4]. Not only the bronchi

but also their accompanying pulmonary arteries may provide extremely useful landmarks on the way to the lesion.

In the next case, the specimen was cut out along the airway axis to identify peripheral airway branching that could not be observed by bronchoscopy.

4.3 Comparison

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Fig. 4.13 Peripheral part of

the tumor. The PA has bifurcated, and part of Tumor C is still visible in the cut surface

2mm

Br2

TumorC

153

MucusLake

Fig. 4.14 Magnication of

Tumor B.The ductal structure of the tumor is split, and this was considered to be a biopsy artifact. It affects the approximate area indicated by the broken yellow line

Br2

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