Histopathology 2007, 50, 232–242. DOI: 10.1111/j.1365-2559.2006.02561.

Whole-specimen histopathology: a method to produce

whole-mount breast serial sections for 3-D digital
histopathology imaging
G M Clarke,1,2 S Eidt,1 L Sun,1 G Mawdsley,1 J T Zubovits3 & M J Yaffe1,2
1
Imaging Research, Sunnybrook and Women’s College Health Sciences Centre, 2Department of Medical Biophysics,
University of Toronto and 3Department of Pathology, Sunnybrook and Women’s College Health Sciences Centre, Toronto,
Ontario, Canada

Date of submission 1 November 2005

Accepted for publication 22 March 2006

Clarke G M, Eidt S, Sun L, Mawdsley G, Zubovits J T & Yaffe M J

(2007) Histopathology 50, 232–242
Whole-specimen histopathology: a method to produce whole-mount breast serial sections
for 3-D digital histopathology imaging

Aims: To develop a method for preparing diagnostic- schedule and extended paraffin infiltration. Whole-
quality, whole-mount serial sections of breast speci- mount serial breast sections are produced with
mens while preserving 3-D conformation. This required features of equal or superior quality to that which
supporting the fresh specimen prior to breadloafing and can be achieved using conventional methods. The
refining the conventional tissue processing method. method is compatible with some immunohistochem-
The overall goal is to use digital images of whole- ical stains but requires further optimization for
specimen histopathology to improve the estimation of others.
extent of disease. Conclusions: The technique is currently suitable for
Methods and results: To maintain a 3-D conformation, research applications. With the reduction in processing
the specimen is suspended in 3.5% agar at 55C. time achievable with microwave-assisted processing,
The block is sliced at 5-mm intervals. Sectioning is there is the potential for its use as a routine clinical
performed after extended fixation in 4% formaldehyde method. This tool may improve the accuracy of margin
from paraformaldehyde in 0.1 m Millonig’s buffer, estimates and identification of multifocality in breast
followed by paraffin processing using a non-routine cancer; further evaluation is necessary.
Keywords: 3-D imaging, breast histopathology, digital imaging, whole-mount section
Abbreviations: DCIS, ductal carcinoma in situ; NBF, neutral buffered fomalin; PFA, paraformaldehyde

tomy. The goal of lumpectomy is the excision of only

Introduction
t h e cl i n i c a l p r o b le m
The majority of breast cancers diagnosed today are
early stage, owing to advances in mammographic
technology and more widespread implementation of
screening programmes.1,2 This frequently allows con-
servative treatment (lumpectomy) to be considered,
offering improved cosmesis compared with mastec-
Address for correspondence: Gina M Clarke MSc, Sunnybrook and
Women’s College Health Sciences Centre, 2075 Bayview Avenue,
Room S6-35, Toronto, Ontario, Canada. e-mail: gina.clarke@swri.ca
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Limited.
the lesion plus a margin of normal tissue.3 It is often
difficult, however, to define the extent of early-stage
cancer accurately. Preoperative localization with mam-
mographic guidance can markedly underestimate the
size of small masses or ductal carcinoma in situ (DCIS),
or fail to detect certain lesions, such as infiltrating
lobular carcinoma.4–6
Margin status is the most significant indicator of the
risk of relapse following lumpectomy; the risk of local
recurrence is more than doubled by a close or involved
margin, with or without radiation therapy.7–9 Some
studies show that multifocality is also an indicator of
poor prognosis, either independently or by association

with other factors.10–13 It is therefore important to
identify margin status and multifocality accurately for
planning of secondary treatments.
There are two limitations of current histopatholog-
ical methods that can impair accurate estimates of
margins and multifocality: the specimen is vastly
undersampled and the flattening of the flaccid, fatty
specimen immediately after excision distorts the true,
3-D distribution of disease. The unsupported specimen
tends to collapse in the posterior-anterior (PA) dimen-
sion and consequently elongate in the mediolateral
(ML) and superior-inferior (SI) dimensions. For most
lumpectomies, the 3-D disease is represented on 10–40
standard slides, and this may represent only 0.007–
0.02% of the total tissue volume.* Undersampling has
been implicated in missed diagnoses14,15 and can also
be responsible for failure to identify the presence of
a close or involved margin. The total tumour area
may be underestimated due to undersampling and the
presence of multiple disease foci may be undetected. It
may also be the case that unifocal disease appears to be
multifocal due to the way it is sampled. The loss of 3-D
conformation and consequent distortion can produce
the appearance of artificially clear or involved margins
and inaccurate measurements of the distances between
foci or centres, which define multifocal and multi-
centric disease.
3 -dwh ol e - sp eci m e n brea st h is topa thol ogy:
a no v e l a p p r o a c h u s i n g d i g i t a l i ma g i n g
In our method we process an entire lumpectomy into
whole-mount serial sections as it is maintained in a 3-D
conformation. This markedly increases sampling and
can reduce conformational change. In our approach,
the total tissue volume available for evaluation is about
a factor of 30 larger than in conventional methods.
This assumes that a 50 · 50 · 50 mm3 lumpectomy is
processed into 100 whole-mount serial sections versus
35 conventional-sized (15 · 15 mm2) slides. To render
this vast amount of data feasible to analyse, we ulti-
mately apply techniques from digital imaging science
and computer display to the slide set.
The slide set is produced by first suspending the fresh
specimen in a gel to reduce slumping and to support
‘breadloafing’ into 5-mm uniformly thick serial slices.
The buoyancy of the gel reduces specimen flattening
*One standard slide contains a volume of approximately 0.9 mm3 of
tissue ( 15 mm · 15 mm · 4 lm). For a lumpectomy of dimen-
sions 50 mm · 50 mm · 50 mm (1.25 · 105 mm3 total tissue
volume) about ten slides will be taken for a mass lesion, and an
average of 35 for DCIS or more extensive disease.
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
Whole-specimen breast histology 233
and thus may help to reduce distortion of the relation-
ships between tumour and margin, although this does
not necessarily recover the ‘original’ 3-D conformation.
The gel around each section is also important as an
external landmark for realigning the serial section
images. The slices are paraffin-processed, using a
modified protocol, serially sectioned in whole-mount,
and stained.
We have developed methods to address the chal-
lenges of specimen preparation and tissue processing
involved in producing the set of serial, whole-mount
sections required for 3-D imaging. Techniques devel-
oped for computer imaging and display, and a reader
study to evaluate the adequacy of the image for
detection of malignancy, are detailed elsewhere.16
research context and c hallenges for
h i s t o p a t h o l og i c a l m e t h o d s
Whole-specimen imaging of breast at cellular resolu-
tion has not been previously reported, although some
individual aspects of the problem have been addressed.
Using analogue methods, others have demonstrated
3-D features of breast pathology as well as normal
anatomy through macroscopic imaging of subgross,
whole-organ sections,17–19 microscopic imaging of
‘whole-mount’ sections assembled from small serial
sections20 and stereoscopic ‘3-D’ imaging from non-
serial whole-mount sections.21,22 Digital reconstruc-
tion has demonstrated very limited volumes from serial
sections using conventional histopathological meth-
ods,18,23 or more extensive volumes at subgross
resolution.24 Conformational distortions have been
controlled for wedge-shaped specimens only, also in
the context of routine, small-format histology.25
Hydrocolloid gels have been used in other histological
applications: agar to orientate small biopsy specimens,
and gelatin to support whole organ, subgross serial
sectioning.26,27
Specimen preparation and processing methods must
overcome two challenges which are especially acute for
fatty breast tissue. The gel must possess a very specific
set of properties. It must have excellent adhesion to the
specimen and rigidity to support thin breadloafing. It
must also be compatible with the solvents and physical
conditions of the tissue-processing protocol to support
subsequent sectioning and also provide a convenient
external reference ‘landmark’ for realigning the serial
images. However, many water-based gels adhere
poorly to fat, and the gel may become brittle after
processing. Second, the requirement for diagnostic-
quality, whole-mount serial sections imposes stringent
specifications on the tissue-processing protocol.
234 G M Clarke et al.
Figure 1. Accessories made for whole-mount specimen preparation. a, Two-litre ‘breadloafing’ box mechanically stabilizes the gel-tissue block
for cutting serial slices of uniform, adjustable thickness (4–5 mm typically) with guides for a brain knife. b, Porous-shelved fixation racks for the
120 · 170 · 50 mm slices.
The challenges of processing and sectioning fatty
tissue are well known. Methods that might produce
individual whole-mount sections or small serial sec-
tions are not reliable for serial whole-mount sections.
Section rejection often occurs due to tearing or
crumbling of the fatty tissue, which is not cross-linked
by formaldehyde. This can prohibitively reduce the
number of whole-mount serial sections available for
3-D imaging. On the other hand, non-routine methods
that achieve superior fixation or infiltration of fat to
support whole-mount, serial sectioning can produce
unacceptable artefacts due to over-drying, or render
the tissue unsuitable for immunohistochemical stain-
ing due to destruction of antigenic epitopes. Both
challenges are further compounded by the sheer
volume of fat in the whole specimen, as well as by
the larger area of the fragile section.
Materials and methods
Breast tissue specimens were received from the Depart-
ment of Pathology at Sunnybrook and Women’s
College Health Sciences Centre from 2001 to 2005
under Institutional Review Board approval. Methods
were developed using tissues from 70 reduction mam-
moplasty cases and further evaluated using slices from
12 mastectomy specimens and three lumpectomy
specimens.
h i s t o p a t h o l og i c a l m e t h o d s
Gel specimen preparation
Accessories specially constructed for specimen prepar-
ation include a 2-l ‘breadloafing’ box to support and
guide thin, accurate slicing of the gel-tissue block and
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
racks to hold the slices during fixation. These are
illustrated in Figure 1. In addition to agar and gelatin,
the following gels were evaluated: deacetylated chito-
san chloride (FMC Corp., Princeton, NJ, USA), pectin,
albumin, carageenan, HistoGelTM (Richard-Allan Scien-
tific, Kalamazoo, MO, USA) and HistoMerTM (Vibratome,
St Louis, MO, USA). The gels were prepared as detailed
in Table 1 and a tissue specimen was suspended in
each as it set. In some of the cases where polysaccha-
ride gels were used, the specimen was either coated
with a polypeptide ‘adhesive’ gel before it was suspen-
ded, or the adhesive was added to the polysaccharide
gel preparation. After setting, the tissue-gel block was
cut sagitally into 4–5 mm thick slices and the adhesion
and rigidity were evaluated visually. If these were
adequate, a small sample of the gel was subjected to
paraffin processing (‘Routine breast’ in Table 2) after
fixation in 10% neutral-buffered formalin (NBF) to
establish compatibility. The average tissue specimen
sliced in this experiment was 50 · 80 · 40 mm.
Tissue processing
In routine practice at our institution, 7 mm thick
breast slices (without gel) are fixed in 10% NBF for
24 h and small samples (average 15 · 25 · 3 mm)
are processed using the ‘Routine breast’ schedule in
Table 2. Four promising alternative schedules (‘A’,
‘B’, ‘C’ and ‘D’*) were identified in an attempt to satisfy
the requirements of whole-mount sectioning.
*Schedules A and B are from P. Hyam, Leica Microsystems, Toronto,
Canada (source: Department of Pathology, Mount Sinai Hospital,
Toronto, Canada) and Schedule C from Department of Pathology,
Hospital for Sick Children, Toronto, Canada. Schedule D is adapted
from B. Noren and T. Tot, Department of Pathology and Clinical
Cytology, Central Hospital, Falun, Sweden.
 Whole-specimen breast histology 235

Table 1. Gel and adhesive

Tp Ts
formulations which were
Gel preparation (aqueous solutions) (C) (C)
compared: reagents,
preparation temperature Polysaccharide 2%, 3.5% agar; with or without 0.01 M NaCl 85 45–55
(Tp) and the suspending
temperature (Ts) to which 1.5% chitosan in 3% acetic acid, with or without
gel is cooled when tissue is 0.01 M KCl
added to or suspended in it
24% pectin 85 N⁄A
(RT ¼ room temperature)
0.7–1.5% carageenan, with or without 0.01 M KCl 85 N⁄A
TM
100% HistoGel 85 35

50% HistoMerTM RT RT

Polypeptide 5% gelatin 85 45–55

5% albumin 85 N⁄A

Mixture 0.015–3% albumin in 3.5% agar 85 45

0.4% deacetylated chitosan chloride in 3.5% agar 85 45

1–1.5% gelatin in 2% agar 85 45–55

Table 2. Paraffin processing schedules: ‘Routine breast’ is used for patient care at Sunnybrook and Women¢ s College Health
Sciences Centre with conventional, small samples, and Schedules A, B, C, D are compared for large slices
Schedule Routine breast A B C D

Fixation 1h 0.5 h

Dehydration (EtOH) 70%, 1 h 50%, 24 h 50%, 12 h 70%, 2 h 70%, 1.5 h

80%, 1 h 70%, 24 h 70%, 12 h 80%, 8 h 95%, 5 h

95%, 1 h 100%, 24 h 95%, 12 h 95%, 8 h 95%, 7 h

100%, 1.5 h 100%, 24 h 100%, 12 h 100%, 8 h 95%, 6 h

100%, 1.5 h 100%, 24 h 100%, 12 h 100%, 8 h 100%, 4 h

100%, 1 h 100%, 12 h 100%, 8 h 100%, 3 h

100%, 12 h 100%, 8 h 100%, 2 h

Clearing (xylene) 2h 24 h 12 h 8h 2.5 h

1.5 h (toluene) 24 h 17 h 8h 2.5 h

Infiltration (paraffin, 60C) 1h 24 h 19 h 8h 4h

1h 24 h 19.5 h 8h 4h

1h 22 h 8h 4h

8h

Pressure ⁄ vacuum Infiltration only No No No Infiltration only

Heat Yes, 40C No No Yes, 37C No

 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
236 G M Clarke et al.
Figure 2. Manual ‘H&E’ staining station for 120 · 170 mm glass
slides (Brain Research Laboratories, Newton, MA, USA), gelatin-
alum-coated to improve adhesion, held in Teflon-coated racks
(Alltech Associates Inc., Deerfield, IL, USA). Staining jars are glass
construction blocks with sawn-off tops.
Normal breast tissue slices (30–70 · 40–90 ·
5 mm) were fixed in 10% NBF or 4% formaldehyde
made from paraformaldehyde (PFA) in 0.1 m Millonig’s
buffer with mild agitation in a 15–20 : 1 solvent-to-
tissue volume ratio. Two fixation times were compared:
7 days and 10–14 days. Fixed slices then underwent
automated processing according to one of the schedules
A–D in Table 2. Tissue from each schedule was
compared for adequate processing, based on empirical
features such as residual xylene odour and ease of
sectioning. For the most satisfactory schedule, tissue
was subjected to extended paraffin infiltration passively
in a 60 C oven and the optimum infiltration time was
determined by removing a slice from the oven daily
over a 15-day period and embedding and sectioning it
to measure the number of viable sections out of a
contiguous series of 10. This experiment was performed
for five specimens to estimate errors.
Sectioning was performed using a Leica SM2500
motorized sliding microtome with a disposable knife
blade, and with either a 120 · 80 mm or 160 ·
120 mm baseplate. Section thickness ranged from
4 to 10 lm. A station assembled for manual haema-
toxylin and eosin (H&E) staining of the whole-mount
sections is shown in Figure 2.
demonstra tion o f t he opt ima l meth o d
Compatibility with immumohistochemical staining
We verified compatibility of the optimal processing
schedule with the following immunohistochemical
stains: oestrogen receptor (6F11 clone; Novocastra
Laboratories, Newcastle upon Tyne, UK), progesterone
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
receptor (16 clone; Novocastra), myosin smooth mus-
cle (SMMS-1 clone; BioGenex, Carlsbad, CA, USA),
E-cadherin (36 clone; BD Biosciences Transduction
Laboratories, San Jose, CA, USA) and human epidermal
growth factor (Her2 ⁄ Neu) (Tab250 clone, Invitrogen
Corp., Carlsbad, CA, USA; CB11 clone, Novocastra).
Fragments of whole-mount sections from five mastec-
tomy specimens were mounted onto small slides and
treated with a routine, automated staining protocol.
The quality of staining (preservation of cellular detail
and absence of non-specific staining) was compared
with the ‘gold standard’ of conventional slides used for
patient care, taken from comparable tissue from the
same specimen and treated with the same staining
protocol. A whole-mount section was also stained
manually and in its entirety to demonstrate staining for
Her2 ⁄ Neu (Tab250) following protease digestion.
Margins and multifocality in whole-mount: demonstration
of 2-D features with digital imaging
Two whole specimens were prepared and processed
using the optimal methods: a lumpectomy with ductal
carcinoma not otherwise specified (NOS), with a close
margin, and a mastectomy with extensive infiltrating
lobular carcinoma. Taking whole-mount sections as
a ‘gold standard’, we compared the illustration of
margins and multifocality with the set of conven-
tional slides prepared for patient care from the
adjacent slice.
Results and discussion
The gel preparations listed in Table 1 are shown in
Table 3 with an indication of whether they were
observed to provide adequate support for slicing,
adhesion to tissue and compatibility with paraffin
processing (if assessed). The relative ranking of fixation
and the tissue processing methods in Table 2 is shown
in Table 4, and it is seen that schedule D gave the most
satisfactory performance. The contiguous serial section
yield is shown in Figure 3 as a function of extended
infiltration time following fixation in 4% PFA for 10–
14 days, processing with schedule D, and sectioning at
4 lm thickness.
Immunohistochemical staining obtained from tis-
sue processed using schedule D versus routinely
processed tissue from the same specimen is shown
in Figure 4. Figures 5 and 6 compare the depiction of
2-D features in a whole-mount section with the
conventional slides prepared for patient care from the
adjacent slice, for a case of duct carcinoma NOS with
a close margin and for multifocal infiltrating lobular
carcinoma.
 Whole-specimen breast histology 237

Table 3. Summary of the results for specimen preparation gels in Table 1 for properties: (I) rigidity, (II) tissue adhesion to
support thin breadloafing, and (III) compatibility with paraffin processing
Gel preparation I II III

Polysaccharide 2%, 3.5% agar; with or without 0.01 M NaCl

Ts ¼ 55C Y Y Y

Ts ¼ 44C Y N Y

1.5% chitosan in 3% acetic acid N N⁄A N⁄A

With 1% KCl Y N⁄A N⁄A

24% pectin N N⁄A N⁄A

0.7–1.5% carageenan, with or without 0.01 M KCl Y N⁄A N⁄A

100% HistoGelTM Y N Y
TM
50% HistoMer Y N Y

Polypeptide 5% gelatin Y Y N

5% albumin N N⁄A N⁄A

Mixture 0.015–3% albumin in 3.5% agar Y N Y

0.4% deacetylated chitosan chloride in 3.5% agar Y N N⁄A

1–1.5% gelatin in 2.5% agar Y Y N

Adequacy is indicated by Y (yes) or N (no).

Table 4. Relative ranking of fixatives, fixation times and 10

automated processing schedules
Processing
8
Fixative Fixation time schedule
Section yield (#/10)

10% 4%
NBF PFA 7 days 10–14 days A B C D 6

Rank 2 1 2 1 3 2 2 1
4

2
de v el o p m en t of me t h o ds
Gel specimen preparation
0
The 3.5% agar gel at Ts ¼ 55C at the time when the 2 4 6 8 10 12 14
tissue is added showed the best performance. Large Number of days infiltration
tissue specimens did not adhere to agar when added
at lower temperatures. The temperature dependence of Figure 3. The optimal number of days for extended paraffin infiltra-
tion: the number of viable, 4 lm thick sections from a contiguous
adhesion is likely a coagulation effect.28 Specimen size series of 10 is shown for each of 1–15 days of extended infiltration
is also a critical factor in adhesion. For example, time. Each point represents an average taken over five specimens and
adhesion was adequate with 2.5% agar at tempera- the mean error is shown.
tures as low as 35C for standard-sized samples.
HistoGelTM showed no advantage over agar at Ts ¼ 25C. However, gelatin becomes brittle and atrophic
35C, so its use at Ts ¼ 55C was not explored. Gelatin following dehydration, as do other yolky or collagen-
tissue adhesion was excellent at temperatures as low as rich materials, and cannot be sectioned at any
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
238 G M Clarke et al.

a b 0 5 10 15 (mm)

c d

e f

Figure 4. Immunohistochemical staining of whole-mount processed

tissue compared with conventionally processed, for: a,b, oestrogen
a
receptor, c,d, progesterone receptor, and e,f, Her2 ⁄ Neu Tab250
clone. Whole-mount-processed tissue fragments were mounted 0 5 10 (mm)

on a conventional-sized slide for automated staining using routine

methods.

concentration.29 This may be due to the glass-trans-

ition point characteristic of this polypeptide.30 Albumin
did not appear to improve tissue adhesion to agar. 0 5 10 (mm)

A number of preparations failed to produce a viable b c

gel as prepared: pectin and albumin did not set,
carageenan was dissolved by formaldehyde and chito-
san in acetic acid with KCl, HistoMerTM and albumen
in agar all dissolved inadequately, thus preventing
adequate sectioning thinner than 10 or 20 lm.
Deacetylated chitosan chloride complexed with the
agar into a soft, mushy block as prepared. Thus, the
cationic polysaccharides, which might have adhered to
negatively charged tissue, were not successful.
The gel selected for use in our work has some
practical drawbacks in that after placing the specimen
in the gel it is necessary to wait for the gel to cool from
55C to the setting temperature of approximately 25C
Figure 5. Comparison of superior (S), posterior (P) and inferior (I)
margins using (a) a whole-mount section, and (b,c) the set of
conventional sections used in the surgical pathology evaluation
sampled from the adjacent 5 mm thick tissue slice. Images have been
downsampled for display in entirety; however, the inset in (a) shows a
1 · 1 mm area from the mass at a higher resolution. The margins in
each section are orientated as follows: top (S), right (P), bottom (I),
left (A).
before proceeding to the next step. The sensitivity of
adhesion to cooling time demands that the preparation
of the gel be precisely coordinated with the completion
of surgery, and this can be difficult. As an alternative, a

Figure 6. Comparison of multifocal disease in (a) a whole-mount section, and (b–e) the set of conventional sections used in the surgical
pathology evaluation, sampled from the adjacent 5 mm thick tissue slice. These show: b, nipple and areola; c, mass, including anteriorly located
skin; d, mass including posterior margin; and e, remainder of mass. Images have been downsampled for display in entirety; however, the
inset in (a) shows a 1 · 1 mm area including skin at a higher resolution. The medial (M), lateral (L), anterior (A) and posterior (P) aspects on the
whole-mount section correspond to the bottom, top, left and right sides respectively.

 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
 Whole-specimen breast histology 239

a b

gel which might adhere to tissue at lower temperatures tissue adhesion is that this gel preparation does not
over a wider range, or which polymerizes based on pH adhere to skin or margin ink that may be present
instead, might be explored. Another limitation of agar– on the specimen surface. Skin, and ink, which may
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
240 G M Clarke et al.

contain glutaraldehyde or other solvents, may inhibit a

coagulation-based adhesion. However, the gel itself is a
useful alternative to inking for labelling margins. It can
also assist in aligning serial section images by providing
a visible external reference.
Various other methods for producing external land-
marks to assist in realigning the serial images were
deemed non-feasible. These include dyes, pigments and
fibres inserted into the fresh tissue-gel block using
syringes, perpendicular to the plane of sectioning.
These did not provide reliable alignment for image
processing methods because of extensive microtear
artefact in fat. Moreover, dyes and pigments were
dissolved with the fat in paraffin processing.

Tissue processing
The quality of formaldehyde fixation is critical if
excellent sectioning is to be achieved.31,32 Fatty breast
tissue is routinely under-fixed in clinical work and this
degrades the quality of sectioning. The extended
fixation time of 10–14 days ensures that fixation is 7 mm
completed by the action of formaldehyde rather than
by ethanol during processing. The morphology in our b
sections was found to be appreciably better with 4%
formaldehyde made from PFA compared with 10%
NBF. In addition to containing methanol, 10% NBF
has a higher concentration of formic acid than 4%
formaldehyde made from PFA, and this can degrade
morphology.33 Since this improved morphology
results from superior fixation,34 tissues fixed in PFA
may provide greater ease in whole-mount serial
sectioning, although this was not rigorously tested.
Blocks from tissue processed using schedules A–C were
very difficult or impossible to section. These blocks may
have been subject to too rapid or too prolonged
dehydration to allow tissue integrity to be maintained.
The yield of high-quality, 4 lm thick serial sections is 200 µm
maximized with an extended paraffin infiltration time of
approximately 7 days following processing. The matrix
of interstitial support created by prolonged infiltration is Figure 7. Whole-mount section from a breast reduction specimen,
following gel preparation and microwave-assisted fixation,
essential for whole-mount serial sectioning. Beyond
processing and infiltration. a, Architecture in whole-mount
the optimal time, section yield may decrease due to (the gel surrounding the tissue is faintly stained by haematoxylin).
over-drying. b, Morphology in a selected area of tissue. This protocol reduces
This technique is currently a research tool, which total time from fresh specimen to slide from about 21 days to
lends itself to many applications in studying the basic 2–3 days.
pathology of breast cancer in three dimensions. There is
potential for future work to achieve a reduction in time and infiltration time from about 21 days to 2–3 days.
from the fresh specimen to the paraffin block and hence Figure 7 shows a whole-mount section from a breast
improve the feasibility of this technique in clinical use. A reduction specimen produced using this protocol. The
reduction in infiltration time was not achieved with the ability to overcome the challenges imposed by fatty
use of a vacuum oven. However, in preliminary experi- tissues is evident. The morphology is excellent and a yield
ments a microwave-based protocol has been developed of 10 ⁄ 10 contiguous serial sections, at 4 lm thickness,
which successfully reduces the total fixation, processing was achieved with this method.
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.

Initially in this investigation we explored whole-
mount, frozen serial sectioning as a method of reducing
processing time, as well as shrinkage, which is desir-
able in 3-D applications. We evaluated various approa-
ches to minimizing ice crystal and chatter artefact.
These included infiltration with a sucrose-based cryo-
protectant, flash-freezing in a hexane-dry ice slurry and
the use of a ultraviolet-curing adhesive tape to transfer
a section from the blockface to a slide. We found these
methods to be non-feasible and abandoned them at an
early stage in this project.
evaluation of methods
The optimal processing method is generally compatible
with immunohistochemistry, although some optimi-
zations are required. Good results with staining for
ostrogen receptor, progesterone receptor and Tab250
are demonstrated in Figure 4. Good results were also
obtained for myosin smooth muscle staining. Further
optimizations, such as more aggressive antigen retrieval,
may be required for CB11 and E-cadherin staining. CB11
staining showed a slight increase in non-specific uptake,
and E-cadherin showed clear but weak membranous
staining. The epitopes for these antibodies may be more
sensitive to the crosslinking or ‘masking’ effect of
extended formaldehyde fixation.34 The method of epi-
tope retrieval influences the feasibility of whole-mount
immunostaining. Large sections adhered very poorly to
coated slides during heat-induced epitope retrieval.
However, whole-mount staining for Her2 ⁄ Neu Tab250,
following protease digestion, was successful.
Two-dimensional relationships that are difficult to
synthesize or not available using conventional methods
can be readily illustrated in whole-mount histopathol-
ogy. Measurements of all four margins are readily
available in Figure 5a, which also eliminates any
ambiguity in locating the closest margin for each.
The anterior margin is too far from the tumour to
be demonstrated on the conventional, small slide
(Figure 5b,c). Actual measurements of margins are
not exactly comparable, as whole-mount and small
sections were taken from adjacent tissues. The whole-
mount section in Figure 6a contains more tumour
than that which is grossly appreciable in the set
of conventional slides. Furthermore, secondary foci,
which are not represented in the standard sections
(Figure 6b–e) are clearly seen. The whole-mount pres-
entation appears to better convey information on the
full extent of disease and its orientation compared with
the conventional piece-wise presentation.
Using the typical values for conventional sampling
and whole-specimen sampling noted above (and
 2007 The Authors. Journal compilation  2007 Blackwell Publishing Ltd, Histopathology, 50, 232–242.
Whole-specimen breast histology 241
assuming a circular section area), the 3-D method
presents a total sampled margin length that is
increased by a factor of 10 and an increase in total
tissue area increased by a factor of 30 over conven-
tional methods. The relationship between this
increased quantity of information and the potentially
increased quality of information (i.e. more accurate
estimates of margins or multifocality) will be the
subject of a future study.
The use of the gel may also better preserve three-
dimensional relationships. Its buoyancy may help
reduce slumping. Some preliminary data that com-
pare specimen measurements with and without the
gel suggest that at least part of this distortion and
elongation can be eliminated by use of the gel.
We have developed a method for producing diag-
nostic-quality, whole-mount serial sections using
whole lumpectomies maintained in a 3-D conforma-
tion, which is compatible with most routine breast
immunohistochemical stains. The entire specimen
is suspended in a 3.5% agar gel at 55C, fixed in
4% formaldehyde made from paraformaldehyde for
10–14 days, processed using Schedule D and paraffin-
infiltrated in a hot-air oven for 7 days. The method
has the potential to provide superior demonstration of
2-D features compared with conventional methods
and produces the serial slide set required for
whole-specimen, 3-D digital imaging. This is a poten-
tially powerful tool for research which can be further
developed for use in addressing clinically oriented
problems such as margin status and multifocality.
Acknowledgements
This work was funded by the Canada Foundation
for Innovation, Ontario Research and Development
Challenge Fund, and the den Haag Foundation. The
authors wish to acknowledge the following for their
invaluable contributions to this work: Mr I. Cooper,
Ms M. Ge, Dr C. Holloway, Mr P. Hyam, Mr T. Morgan,
Ms A. M. Moskaluk, Ms B. Noren, Mr C. Peressotti,
Mr M. Pozzobon, Ms R. Rossetti and Dr T. Tot.
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