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The multitumor ("sausage")
tissue block was introduced by Hector Battifora (1986) as a novel method
of testing monoclonal antibodies for immunohistochemistry (Battifora, 1986;
Battifora and Mehta, 1990; Enghardt et al, 1995; Miller et al, 1993; Press
et al, 1994). Other applications have included the assessment of molecular
genetic probes for fluorescence in situ hybridization (Press et al, 1997)
and quality control in immunohistochemistry. However, their preparation
is cumbersome, timeconsuming, and limited by the technical difficulty of
maintaining the prearranged specimen distribution during experimental procedures.
We now introduce a multichambered ("honeycomb") tissue mold that
simplifies and improves the technique of embedding many tissue samples in
a single paraffinized specimen block. The honeycomb dividers were prepared
with plastowax and placed in conventional, metal tissueembedding molds to
provide multiple, equalsized spaces for small tissue specimens. The molds
were embedded using paraplast embedding medium at 58 to 60°C. Tissue
processing, sectioning, histologic and immunohistochemical staining, and
fluorescence in situ hybridization were performed using routine methods.
Individual tissue specimens in these multispecimen tissue blocks remained
in their assigned locations throughout all procedures. This approach not
only conserves the amounts of reagent and technician time required, but
it also subjects a large number of tissue samples to the same preparation
conditions.
Multitumor blocks facilitate comparative studies of various antibodies and
quality assessment by providing a reliable means of monitoring variations
in antibody sensitivity and specificity, both within and among different
laboratories. Battifora and Mehta (1990) refined the multitissue block with
the introduction of the "checkerboard" block technique, which
allows tissues to be assigned to specified positions within the block. We
have used this approach to evaluate theimmunoreactivity of estrogen receptor,
progesterone receptor, p53 (Chen CW and Press MF, unpublished observations),
and HER2/neu antibodies (Press et al, 1994). Other reagents, such as molecular
probes, have also been assessed using this method (Press et al, 1997). Although
the checkerboard technique is an important improvement in the multispecimen
method, production of these blocks is technically difficult, timeconsuming,
and potentially expensive, requiring the use of agar and specially designed
molds.
Largescale molecular epidemiology investigations of breast cancer have necessitated
more efficient methods of preparing large numbers of tissue block samples
to be linked to a computer database for analysis of results. Using honeycomb
tissue dividers has addressed and solved this problem. These dividers consisted
of a series of parallel plates that intersected at right angles; they were
prepared from plastowax using a rubber mold (Fig. 1A) designed for this
specific purpose by a local manufacturer (Gina's Jewelry, Los Angeles, California).
After the plastowax melted, it was poured into the mold under pressure and
cooled to room temperature. The divider was then removed from the mold (Fig.
1 B). The honeycomb dividers were designed to fit into conventional, metal
tissueembedding molds (3.0 x 2.5 x 1.5 cm) (Miles Scientific, Naperville,
lllinois).
Because plastowax and paraplast are chemically and physically similar, other
than melting point (71 versus 58°C), the multichamber divider does not
interfere with tissue sectioning. Moreover, the higher melting point of
plastowax allows the divider to remain intact during routine tissue processing
at 58 to 60°C. This facilitates tissue preparation and processing of
the multitissue blocks by routine embedding techniques using commercially
available supplies (Fig.1, C and D).
In our application of the technique, tissue specimens were selected from
previously embedded archival tissue blocks and removed by gradual melting
of the paraplast embedding medium. An approximately 2.0mm wide, rodshaped
tissue segment was trimmed from one edge of the specimen, so as to include
a sampling of tumor histopathology, and placed in individually labeled microfuge
tubes for later assembly in multitumor blocks. Each tissue sample loaded
into the honeycomb chambers had a previously assigned row and column, allowing
for easy correlation of the specimen with its source. To further aid identification,
the specimen in Row 1/Column 1 was labeled with India ink; tissue samples
in subsequent, alternate rows were labeled with red, green, or blue ink.
Tissues were then warmed and submerged for several minutes in paraplast
embedding medium at 58 to 60°C. The multitissue block was then removed
from the tissue processor and cooled to room temperature.
The paraplast and plastowax did not separate from one another at any stage
of tissue processing, sectioning, or staining. After cooling to room temperature,
the two waxes solidified into a homogeneous unit. The resulting tissue block
was routinely processed in a manner similar to that required by a singlespecimen
paraffin block. Tissue sections, 3 to 6mm in thickness, were prepared routinely
with histology microtomes and mounted on standard histology glass slides.
As was expected, both paraplast and plastowax were dissolved by xylene during
processing of tissue sections. Tissue sectioning and histologic and immunohistochemical
staining were performed using routine methods (Press et al, 1994, 1997).
Immunohistochemical staining with a variety of antibodies (eg, estrogen
receptor, progesterone receptor, HER2/neu oncoprotein, p53 tumor suppressor
protein) provided highquality results similar to those obtained with single
specimen sections from the same cases.
In conclusion, the use of tissue blocks containing multiple specimens is
costeffective and conserves reagents and technician time, making the method
well suited to conducting largescale research studies using immunohistochemical
methods. The addition of the honeycomb tissue divider is yet another improvement
in the multitissue block method and should contribute to the efficacy and
utilization of this method in future laboratory investigations.
Acknowledgements
We gratefully acknowledge Michelle MacVeigh and Armen Babirian for excellent
technical assistance and Ivonne Villalobos for assistance in preparation
of the manuscript.
References
Battifora H (1986). The multitumor (sausage) tissue block: Novel method
for immunohistochemical antibody testing. Lab Invest 55:244- 248.
Battifora H and Mehta P (1990). The checkerboard tissue block: An improved
multitissue control block. Lab Invest 63:722-724.
Enghardt HM, Aghassi BN, Bond JC, and Elson MD (1995). A simplified multitissue
control block. J Histotechnol 18:51-55.
Miller RT (1 993). Multitumor "sandwich" blocks in immunohistochemistry:
Simplified method of preparation and practical uses. Appl Immunohistochem
1:156-159.
Press MF, Bernstein L, Thomas PA, Meisner LF, Zhou JY, Ma Y, Hung G, Robinson
RA, Harris C, ElNagger A, Slamon DJ, Peyrot M, Ross J, Phillips R, Wolman
SR, and Flom KJ (1997). HER2/neu gene amplification by fluorescence in situ
hybridization: Evaluation of archival specimens and utility as a marker
of poor prognosis in nodenegative invasive breast carcinomas. J Clin Oncol
15:2894-2904.
Press MF, Hung G, Godolphin W, and Slamon DJ (1994). Sensitivity of HER2/neu
antibodies in archival tissue samples: Potential source of error in immunohistochemical
studies of oncogene expression. Cancer Res 54:2771-2777.
Received April 23, 1997.
Affiliations: Department of Pathology (KP, MFP) and Norris Comprehensive
Cancer Center (MFP), University of Southern California School of Medicine,
Los Angeles, California.
Supported in part by the National Institute of Child Health and Human Development
(N01HD33175), National Cancer Institute (CA48780), United States Army Medical
Research and Material Command (DAMD1794J4290), the University of Southern
California (USC) Breast Cancer Research Program, and the University of California
Los Angeles/USC Breast Tumor Bank (DAMD1794J4234).
Address reprint requests to: Dr M. F. Press, NOR5410 (Mailstop No. 73),
Norris Comprehensive Cancer Center, USC School of Medicine, 1441 Eastlake
Avenue, Los Angeles, California 90033. Fax: (213) 7640122. |
