research-article2016
JHCXXX10.1369/0022155416651065Cell and Tissue Display: A New Microscopy ToolTheodosakis et al.
Article Journal of Histochemistry & Cytochemistry 2016, Vol. 64(7) 403–411 © 2016 The Histochemical Society Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1369/0022155416651065 jhc.sagepub.com
Cell and Tissue Display: An Alternative Multipurpose Tool for Microscopy Nicholas Theodosakis, Goran Micevic, Marcus W. Bosenberg, and Nemanja Rodić
Department of Pathology (NT, GM, MWB) and Division of Dermatopathology, Department of Dermatology (NT, GM, MWB, NR), Yale University, New Haven, Connecticut
Summary We developed a method, termed Cell and Tissue Display (CTD), for embedding 16 or more different tissue samples in multicompartment agarose blocks. The CTD-generated blocks allow uniform multiplexing of cell lines and small tissue fragments within a single histologic block. The distribution of individual cells within the CTD blocks is improved, likely because the individual agarose compartments are small and uniform. The composition of each CTD block can be customized based on intended use. Some potential uses of CTD histologic blocks include improved sectioning of small tissue fragments, such as needle biopsy specimens or punch biopsies; multiplexing of tissue fragments within a single block; and the generation of control slides for laboratory proficiency testing. (J Histochem Cytochem 64:403–411, 2016) Keywords Cell and Tissue Display, histologic block, histology
Introduction A complex need for detailed histologic and molecular analysis in clinical and research biomedicine has resulted in the development of a wide range of assays, such as in situ hybridization, immunofluorescence, and immunohistochemistry.1–3 Reliable preservation of both normal and tumor tissue continues to be integral to the proper use of these assays. One economical and rapid method in the clinical setting is to simply place multiple tissue specimens into a single histologic block. In research investigations, where hypocellular specimens are often a major limitation, investigators frequently turn to adhering cells to slides by culturing cells in vitro or by manually spreading cells on slides.4 Histologic block-making has undergone iterative improvement over the last several decades. The first hallmark improvement was devised in 1986, when Dr. Hector Battifora developed a “sausage” histologic block method, which entailed the arrangement of multiple approximately 1 mm thick tissue fragments obtained from different specimens in a segment of
small intestine.3 This method marked a significant technological advancement, allowing for multiple tissue specimens to be easily evaluated under identical processing conditions. However, the individual tissue fragments could not be identified with certainty. In 1990, Dr. Battifora modified his original method and termed his new technique the “checkerboard” tissue block. This iteration allowed even distribution and identification of multiple tissue specimens in one block.5 The next major technological advancement was the development of tissue microarray (TMA) technology by Kononen et al.6 TMA allowed for the identical configuration of hundreds of tissue cores within a block. However, technical limitations persisted despite near-universal application of TMA in research settings. Most notably, tissue loss was detected in 10% to 30% of assembled Received for publication October 22, 2015; accepted April 18, 2016. Corresponding Author: Nemanja Rodić, Yale Dermatopathology Laboratory, 15 York Street, LMP5031, P.O. Box 208059, New Haven, CT 06520-8059, USA. E-mail: [email protected]
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404 cores, resulting either from loss during cutting or from the transfer of array sections from archival blocks.7 The most common approach for correcting uneven cell display of tissue fragments on slides was to create multiple cores of an identical case. Similarly, the most common approach used to correct uneven cell displays of cell lines on slides was to massively increase the input, often by increasing the number of cells submitted for the construction of the histologic block. While this may be readily achieved for untreated established cancer cell lines, cells grown in culture are often treated with reagents that cause apoptosis or inhibit cancer cell growth. In those instances, it becomes difficult to procure sufficient numbers of lesional cells to generate blocks. Other researchers have previously pioneered the incorporation of cell lines in TMAs8,9 and agarose blocks10,11 or molds12 to increase yield from paucicellular specimens. Herein, we expand on this knowledge and describe a novel method, Cell and Tissue Display (CTD), which achieves both improved cellular homogeneity of the x–y planes of a z-axis and also permits rapid multiplexing and uniform arrangement of both tissues and cell lines in one block.
Materials and Methods Cell Culture Cell lines were provided as a gift by Dr. Ruth Halaban (Yale University, New Haven, CT).13 Surgical melanoma resections were used to generate the lines. Specimens were collected with patients’ informed consent in accordance with the Health Insurance Portability and Accountability Act (HIPAA) under a Human Investigations Committee protocol. All melanoma cell lines (YUVON, YUGASP, YUKOLI, YUHEF, YUROB, YUKSI, YUSIK, YUTIKA, YURIF, WM1346, and YUGEN8) were grown routinely in Opti-MEM® (Invitrogen, Carlsbad, CA) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum, and maintained in a 37C incubator with 5% CO2.
Human Normal Tissue Procurement Following review, written permission was obtained from the director of autopsy services at Johns Hopkins Hospital to procure fresh normal skin tissue from routine adult autopsies. Apart from designating the tissue of origin for each specimen, no additional identifiers were recorded.
Animal Tissue Preparation All mice were bred on a C57BL/6 inbred genetic background. All experiments involving animals were
Theodosakis et al. reviewed and approved by the Yale Institutional Animal Care and Use Committee. The mice were euthanized according to the Yale University animal protocol. Tissue was harvested and freshly embedded in agarose mold. CTD agarose blocks were fixed in 1% neutral buffer formalin solution for ~1 hr prior to submitting the blocks for routine processing at the Orthopaedic Histology and Histomorphometry Laboratory at the Yale School of Medicine.
Histologic Block Construction Traditional histologic blocks used as controls were prepared according to previously published protocols.14-16 Cytologic blocks were prepared routinely by the Cytology Service at the Yale School of Medicine using standard Cellient™ automated cell block technology (Hologic, Inc., Bedford, MA). The CTD histologic block procurement method was performed as follows. First, 3 g of standard melting point agarose (UltraPure Agarose, Invitrogen, Inc.) powder was dissolved in 100 ml of 1× phosphate-buffered saline (PBS) solution by heating in a standard microwave for 60 to 90 sec. Molten agarose solution was poured into an inverted pipette container lid from a BioDOT Universal Fit pipette tips container (DOT Scientific, Inc., Burton, MI). Next, either a MicroAmp Optical 96-well reaction plate or a 384-well reaction plate (Life Technologies, Inc., Carlsbad, CA) was placed onto the molten agarose solution. The mixing step, in which cells/tissue are mixed with molten agarose, is critical to perform prior to deposition into the agarose mold to prevent any shrinkage artifact. The preparation was then left at room temperature for 30 min to allow the agarose to solidify. Afterward, the container lid was removed, and the agarose mold was carefully extricated from the plastic lid. The agarose mold was then cut and trimmed to fit into a closed anatomic pathology cassette. Excess agarose mold may be placed in 1 × PBS solution and stored in a refrigerator at 4C for at least 1 month. Cells were routinely retrieved from cell culture plates,17 then fixed in 1% buffered formalin solution, optimally at a concentration of 1 × 106 cells/ml. Next, 50 ml aliquots were removed and placed into new microcentrifuge tubes. The cells were allowed to settle out of suspension on the benchtop for 30 to 60 min. After the cells have settled, supernatant solvent was carefully removed with a pipette. The remaining cells were resuspended in approximately 50 µl of 1% molten agarose solution and injected into the agarose spots or wells created by the bottoms of the 96- or 348-well reaction plates. The agarose mold was then quickly placed at the corner of an inverted Corning Costar® cell culture
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Cell and Tissue Display: A New Microscopy Tool Cell and Tissue Display: A New Microscopy Tool plate lid (Sigma-Aldrich, Inc., St. Louis, MO) and spun for 1 to 3 min at 1500 rpm using a Beckman microplate carrier 270-453790-B and G.H. 3.7 Rotor/Centrifuge (Beckman, Inc., Brea, CA) to achieve better cellular homogeneity. For tissue fragment processing, each fragment was placed in a spot or well and covered with molten agarose solution. Cells were enumerated using Countess® Cell Counting Chamber Slides (Life Technologies, Inc., Carlsbad, CA). The agarose blocks were first allowed to rest on the benchtop for 30 min. They were then placed in 1% buffered formalin solution and taken to the histology laboratory for paraffin embedding. Histoprocessing was performed routinely using a standard vacuum infiltration processor (Shandon Excelsior ES; Thermo Fisher Scientific, Inc., Waltham, MA) at the Orthopaedic Histology and Histomorphometry Laboratory, Yale School of Medicine. We used Paraplast Plus® embedding media (supplier no. 39502004; VWR International, Inc., Radnor, PA) for embedding. The paraffin-embedded CTD histologic blocks were stored routinely at room temperature. At the time of publication of this report (1 year following the original CTD histologic block constructions), we examined the CTD histologic blocks for gross evidence of shrinkage. We detected no shrinkage between specimen and agarose matrix, nor between agarose matrix and surrounding paraffin. Deeper step sections (cut nos. 30, 40, 50, 60, 70, 80, 90, and 100) into CTD histologic blocks were cut routinely, and both unstained sections and hematoxylin and eosin (H&E) stained slides were produced. By cut no. 70, we had cut through two spots or wells so that 10 out of 12 spots/wells were visualized. By cut no. 100, we cut through an additional one spot/well, so that nine out of 12 were visualized. As CTD entails working with molten agarose, experimenters should use laboratory coats, gloves, silicone insulation sandwiched gloves, and safety goggles. Rarely, molten agarose solution can become superheated and violently boil over immediately after mechanical agitation, such as immediately after lifting the beaker from the microwave.
was obtained from Life Technologies, Inc. (Grand Island, NY). Monoclonal mouse anti-human primary antibodies against microphthalmia-associated transcription factor (MITF; product no. M362129-2) at a dilution of 1:100, A1/A3 (product no. M351529-2) at a dilution of 1:50, polyclonal rabbit anti-human primary antibodies against S-100 (product no. Z031129-2) at a dilution of 1:400, and cytokeratin (CK116; product no. Z062201-2) at a dilution of 1:500 were used according to the manufacturer’s instructions (Dako North America, Inc.). Secondary anti-mouse antibodies conjugated to horseradish peroxidase (HRP; catalog no. M32207) at a 1:2000 dilution (Thermo Fisher Scientific, Inc.), anti-rabbit antibodies conjugated to fluorescein isothiocyanate (FITC; catalog no. F9887) at a dilution of 1:200 (Sigma-Aldrich Inc.), and anti-rabbit antibodies conjugated to Cy3 (catalog no. C2306) at a dilution of 1:200 (Sigma-Aldrich, Inc.) were used according to the manufacturer’s instructions. All antibodies incubations were performed overnight at 4C in the humid chamber.
Antigen Detection
We have developed an alternative approach, termed CTD, to embed cells and/or tissue fragments in small potential spaces, herein termed spots or wells. To begin to create the agarose mold, we started by pouring molten agarose into a plastic receptacle. We then placed empty 96- or 384-well reaction plates over the molten agar to create spots/wells within the agarose (Fig. 1F). Following solidification of the agarose, cells or tissue fragments were placed in the spots/wells (Fig. 1G). Agarose molds were cut to fit standard histology cassettes (Fig. 1H). The specimens were then
Both immunohistochemistry and immunofluorescence were performed by the Yale Dermatopathology Laboratory. Briefly, antigens were first unmasked in 10 mM sodium citrate buffer (pH 6.0) antigen-retrieval solution using PT Link Module Plus (Dako North America, Inc., Carpinteria, CA). Tissues slides were then processed routinely on Dako Autostainer Plus (Dako North America, Inc.). DAPI (4′,6-diamidino2-phenylindole, dihydrochloride; product no. D1306)
Image Analysis Representative digital photomicrographs (400× final magnification) of tissue spots/wells were captured using laboratory microscopes (BX41 microscope with a DP26 camera, Olympus America, Inc., Melville, NY) equipped with imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI). We then used ImageJ analysis software (version 1.49) to measure cellular homogeneity within each photomicrograph. Shapes within each photomicrograph of >100 square pixels with a roundness coefficient between 0.2 and 1 were counted as cells. The contrast threshold was adjusted manually to account for background on each image. Once the cells were defined, a nearest neighbor distance (NND) calculator plugin for ImageJ was used to calculate the NND value for each particle in the field, according to described specifications.16
Results
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Figure 1. Key features of Cell and Tissue Display (CTD) method. (A–F) Pictorial representation of agarose mold preparation from molten agarose (first two diagrams). (G, H) Cells and/or tissue fragments can be deposited into spots/wells (with or without subsequent centrifugation) and submitted for routine histologic block-making.
delivered to the histopathology laboratory for routine embedding and slide preparation. We compared the performance characteristics of slides created from CTD cell line blocks to three commonly used methods in a series of qualitative (Figs. 2 and 3) and quantitative (Fig. 4) tests.14-16 Two of these
three methods are traditional histologic techniques used to prepare histologic blocks in the research setting at Johns Hopkins Hospital and the Yale School of Medicine. The third cytologic block method is a standard technique to procure cytologic specimens for routine clinical evaluation.
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Figure 2. Additional qualitative features of histologic sections made by Cell and Tissue Display (CTD) method. (A–C) Representative photographs from histologic blocks made with traditional methods; provided to illustrate comparable inhomogeneity of visible cellular aggregates and clusters in those preparations. (D–I) Representative images showing hematoxylin and eosin (H&E) stained sections from histologic blocks made by CTD methodology. (D) Image of CTD slide multiplexed with 16 murine tissue fragments. (E) Photographs of CTD slide multiplexed with 11 human melanoma cell lines and one normal human skin tissue fragment. Representative photomicrographs of CTD spots/wells containing (F) murine colon; (G) normal murine brain; (H) low-cellularity melanoma cell line, YURIF; and (I) high-cellularity melanoma cell line, YUGASP. Final magnification, parts (F–I), 200×; white scale bar in (A, D) denotes 5 mm; black scale bar in (F) denotes 250 µm.
Both the manual inspections and the microscopic evaluations of the CTD sections showed that they can be multiplexed and tiled in consecutive uniform rows and columns, similar to the slides made by TMA technology. Either cells or tissues can be arranged in spots or wells (Fig. 2D–I), a feature which represents a technological improvement from the other three common methods of histologic block-making (Fig. 2A–C). In addition, mixed cultured cell and tissue fragments can be multiplexed within the same CTD block (Fig. 2E, 2G–I). Next, we generated another CTD block that contained normal murine tissues procured from routine animal necropsy (Fig. 2D, F). Within a single histologic block, we arranged 16 unique tissue fragments and achieved full cross-sections of all 16 tissue fragments on a single slide. Upon microscopic evaluation of the slides made with the CTD method, we noted a marked qualitative improvement in cell homogeneity (Fig. 3A, lower panel) relative to the other three methods (Fig. 3A, upper panel). Importantly, cells procured by CTD maintained appropriate epitope antigenicity by immunohistochemistry and immunofluorescence (Fig. 3B). Upon further optimizing the protocol to include an additional centrifugation step
prior to agarose-based mold solidification, we observed further enhancement in microscopic homogeneity. To quantify this effect, we applied ImageJ software to representative photomicrographs and evaluated a correction value, NND, which represents the intercellular distances between individual cells. We measured hundreds of NND values within a given field of view. The scores were then reported as relative coefficients of variation (CVs) for NND values (Fig. 4). Finally, we determined the cell count and distribution in deeper sections to measure homogeneity through various levels of the z-axis. We focused our analysis of cellularity and distribution on three representative cell lines: a low-cellularity cell line, YURIF; moderate-cellularity cell line, YUKSI; and high-cellularity cell line, YUVON (Fig. 5A). By applying ImageJ analysis on representative photomicrographs, we determined that the cellularity of all three cell lines remained relatively uniform (Fig. 5B). On an early cut (cut no. 10), we detected 33 YURIF cells; on an intermediate cut (cut no. 50), we detected 46 YURIF cells; and on a deep cut (cut no. 100), we detected 34 YURIF cells. The number of YUKSI cells was 154, 99, and 113 on cuts 10, 50, and 100, respectively. The number of
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Figure 3. Homogeneity and epitope antigenicity are preserved by the Cell and Tissue Display (CTD) method. (A) Representative photomicrographs showing YUGEN8 cells procured via traditional methods (upper panels), compared with cells procured via CTD (lower panels, low to moderate to high cellular density for comparison). Final magnification, part (A), 200×; black scale bar in (A) denotes 250 µm. (B) Representative photomicrographs showing appropriate epitope detection following CTD histologic block-making with immunohistochemistry (upper panel) and immunofluorescence (lower panel). Final magnification, part (B), 400×; black and white scale bars denote 60 µm. Abbreviations: H&E, hematoxylin and eosin; DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride.
Cell and Tissue Display: A New Microscopy Tool Cell and Tissue Display: A New Microscopy Tool
Figure 4. Improved YUGEN8 cellular homogeneity in x- and y-planes using Cell and Tissue Display (CTD) methodology. The y-axis denoted coefficient of variation (CV) for value nearest neighbor distance (NND), which represents the intercellular distances among individual cells within a given field of view. Variegated and comparably higher CV in histologic blocks procured using traditional methods (traditional methods 1 and 2, and the cytologic block method) compared with several iterations of CTD histologic block preparations.
YUVON cells was 530, 605, and 541 on cuts 10, 50, and 100, respectively. To evaluate the distribution of cells, we determined number of cells and their corresponding NNDs. Both values remained largely unchanged among an early cut (cut no. 10) and later serial cut (cuts no. 50 and 100; Fig. 5C). YURIF cells retained similar distribution (NND ± CV) on an early cut (149 ± 49), on an intermediate cut (157 ± 43), and on a deep cut (174 ± 52) as well. NND and CV values for YUKSI and YUVON cell lines were 91 ± 31 (cut no. 10), 107 ± 42 (cut no. 50), 103 ± 38 (cut no. 100), and 49 ± 24 (cut no. 10), 43 ± 21 (cut no. 50), 52 ± 22 (cut no. 100), respectively. Taken together, our findings support the hypothesis that microscopic homogeneity in the z-plane of sectioning is reliably achieved with CTD. This feature of our method supports its use in the clinical setting for making histologic blocks using markedly hypocellular specimens, such as buccal swabs.18
Discussion The potential applications of CTD for rapidly generating reliable testing controls in the clinical setting are numerous. The need for reliable positive and negative controls in laboratory medicine and clinical pathology necessitates dependable reproducibility from test to test. The marked homogeneity of tissue embedded using CTD from section to section, as well as long-term preservation of antigenicity mark it as ideal for this purpose. We
409 also believe that coupling of CTD to cell-sorting tools such as flow cytometry could be used to construct control blocks containing cell lines both enriched and homogeneous for markers of interest, further improving both reproducibility and utility for quality control. Furthermore, the ability to mix purified cell lines expressing different markers would provide a potential test modality to verify the specificity of new antibodies or staining techniques for clinical use in a controlled manner. Analytic sensitivity may be measured in an analogous fashion by mixing purified cells with variable expressivity of important markers to determine detection thresholds. The biological homogeneity offered by cell lines may also allow better evaluation of the effects of different pre-analytical variables, with controlled manipulation of characteristics such as fixative and length of fixation, yielding improved characterization of false-detection rates. While validation work would have to be done, given that cell lines often express proteins in non-physiologic ranges, comparison with marker-expressing tissue samples may be of use in certain settings. CTD has the additional advantage of allowing multiple tissue fragments to be tiled directly from the grossing station (in clinical investigations) or the benchtop (in research applications), whereas histologic blocks must be retrieved to create TMAs. Alternatively, if there is a collection of fixed tissue fragments, they can simply be tiled into CTD spots/wells, and the agarose block can be submitted for histopathologic processing. We termed this protocol a prospective, as opposed to retrospective, construction. CTD can also easily be incorporated into a TMA construction by coring the foci of interest. Using the principles presented herein, any clinical laboratory can construct in-house CTDs that are tailored to their own practice. CTDs can be constructed centrally, then distributed to the practicing clinical laboratories to help improve quality control. Alternatively, the CTD method may prove useful as an alternative platform for initiatives toward synchronization of immunohistochemical staining protocols.19 With regard to guaranteeing quality from the CTD protocol, we have noted only a handful of minor issues. First, we rarely observed small (approximately 100– 200 µm2 areas, best seen in Fig. 3, lower panel, center of middle image) cell-free regions, which may be due to artifactual introduction of air bubbles during molten agarose pipetting. This feature indicates that while CTD signifies an improvement in histologic block-making, it is not a perfect method. Second, we rarely observed tissue folding during the microtome-cutting step of the CTD-made blocks. While tissue folding can occur during the routine processing of any histologic block, we plan to adapt an agar–gelatin mix protocol,
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Figure 5. Improved cellular homogeneity of YURIF, YUKSI, and YUVON cell lines across deep sections using Cell and Tissue Display (CTD) methodology. (A) Representative photomicrographs showing melanoma cell lines of varied cellularity: A low-cellularity cell line, YURIF; moderate-cellularity cell line, YUKSI; and high-cellularity cell line, YUVON. (B) Relatively uniform cell number of YURIF, YUKSI, and YUVON cells on an early cut (cut no. 10), on an intermediate cut (cut no. 50), and on a deep cut (cut no. 100). (C) Relatively uniform cell distribution of YURIF YUKSI, and YUVON cells on an early cut (cut no. 10), on an intermediate cut (cut no. 50), and on a deep cut (cut no. 100). Final magnification, part (A), 400 ×; black scale bar denotes 60 µm. Abbreviation: NND, nearest neighbor distance.
originally developed by Jones and Calabresi,20 to the next iteration of the CTD methodology. In addition to CTD, there are two alternative nontraditional methods for creating histologic blocks from paucicellular specimens. The first is the Cellient™ cell block, the method used to create the cytology blocks for comparison in this study.21 The second method is the rapid formaldehyde fixed-paraffin embedded (FFPE) cell block.22 In the present report, we observed a qualitative improvement in cellularity in the slides procured with the CTD method compared with the Cellient-prepared cytology blocks. The rapid FFPE method entails wrapping cellular pellets in gauze to improve cellularity, prior to histologic block-making, and was not evaluated in the current study. The potential for CTD to improve scientific research is also significant. Low-cost assemblies of highly homogeneous arrays of cell lines can be used to rapidly screen for expression of markers of interest without the need for retrieval of frozen stocks or the growth of difficult-to-culture lines. The improved stability of cell lines preserved in this manner may also facilitate sharing of cultured tissue between institutions with fewer concerns over sample viability. In addition, the ability to take multiple cuts
from a highly homogeneous block will allow for rapid parallel staining for a large number of markers, without the need to simultaneously process large numbers of separately grown cultures. Herein, we report the development of a novel protocol for histologic block-making, CTD, which allows for more nuanced and controlled manipulation of cell lines, as well as the multiplexing of cell lines with tissue within the same histologic block. Using CTD allows for better control of the number of cells visible on a given slide, which may allow the construction of histologic blocks from markedly paucicellular specimens, such as primary cells. We demonstrated the utility of CTD by rapidly generating histologic blocks from normal murine tissue, as well as from multiplexed melanoma cell lines. The versatility of this technique lends itself to adaptation to a wide variety of research and clinical needs, which could help improve the utility of histology in mainstream investigative research. Acknowledgments The authors thank Drs. Ruth Halaban and Antonietta Bacchiocchi for providing the human melanoma cell lines used to validate this technique. We also thank Mrs. Nancy Troiano, of
Cell and Tissue Display: A New Microscopy Tool Cell and Tissue Display: A New Microscopy Tool the Orthopaedic Histology and Histomorphometry Laboratory, for preparing the tissue slides from the Cell and Tissue Display (CTD) blocks. The authors also thank the Yale Dermatopathology Laboratory for performing immunostaining studies.
Author Contributions NR designed the study; NT, GM, and NR constructed the Cell and Tissue Display (CTD) blocks; NT and NR performed image processing and analysis; MWB provided project oversight and critically examined intellectual content; NT and NR wrote the manuscript. All authors have read and approved the final manuscript.
Competing Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Department of Dermatology at the Yale School of Medicine.
Literature Cited 1. Volpi EV, Bridger JM. FISH glossary: an overview of the fluorescence in situ hybridization technique. Biotechniques. 2008;45:385–86, 388, 390 passim. 2. Bhattacharyya D, Hammond AT, Glick BS. High-quality immunofluorescence of cultured cells. Methods Mol Biol. 2010;619:403–10. 3. Battafora H. The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest. 1986;55:244–48. 4. Taylor MS, Lacava J, Mita P, Molloy KR, Huang CR, Li D, Adney EM, Jiang H, Burns KH, Chait BT, Rout MP, Boeke JD, Dai L. Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell. 2013;155:1034–48. 5. Battafora H, Mehta P. The checkerboard tissue block. An improved multitissue control block. Lab Invest. 1990;63:722–24. 6. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schrami P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4:844–47. 7. Hoos A, Cordon-Cardo C. Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations. Lab Invest. 2001;81:1331–38. 8. Ferrer B, Bermudo R, Thomson T, Nayach I, Soler M, Sanchez M, Castillo M, Calvo J, Campo E, Fernandez PL. Paraffin-embedded cell line microarray (PECLIMA): development and validation of a high-throughput method for antigen profiling of cell lines. Pathobiology. 2005;72:225–32.
411 9. Waterworth A, Hanby A, Speirs V. A novel cell array technique for high-throughput, cell-based analysis. In Vitro Cell Dev Biol Anim. 2005;41:185–87. 10. Andersson AC, Stromberg S, Backvall H, Kampf C, Uhlen M, Wester K, Ponten F. Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics. J Histochem Cytochem. 2006;54:1413–23. 11. Choi SJ, Choi YI, Kim L, Park IS, Han JY, Kim JM, Chu YC. Preparation of compact agarose cell blocks from the residues of liquid-based cytology samples. Korean J Pathol. 2014;48:351–60. 12. Moskaluk CA, Stoler MH. Agarose mold embedding of cultured cells for tissue microarrays. Diagn Mol Pathol. 2002;11:234–38. 13. Halaban R, Zhang W, Bacchiocchi A, Cheng E, Parisi F, Ariyan S, Krauthammer M, McCusker JP, Kluger Y, Sznol M. PLX4032, a selective BRAF(V600E) kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAF melanoma cells. Pigment Cell Melanoma Res. 2010;23:190–200. 14. Dolled-Filhart M, Mccabe A, Giltnane J, Cregger M, Camp RL, Rimm DL. Quantitative in situ analysis of beta-catenin expression in breast cancer shows decreased expression is associated with poor outcome. Cancer Res. 2006;66:5487–94. 15. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–75. 16. Sfanos KS, Aloia AL, Hicks JL, Esopi DM, Steranka JP, Shao W, Sanchez-Martinez S, Yegnasubramanian S, Burns KH, Rein A, De Marzo AM. Identification of replication competent murine gammaretroviruses in commonly used prostate cancer cell lines. PLoS ONE. 2011;6:e20874. 17. Barker K. At the bench: a laboratory navigator. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2005. 18. Thiede C, Prange-Krex G, Freiberg-Richter J, Bornhauser M, Ehninger G. Buccal swabs but not mouthwash samples can be used to obtain pretransplant DNA fingerprints from recipients of allogeneic bone marrow transplants. Bone Marrow Transplant. 2000;25:575–77. 19. Engel KB, Moore HM. Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med. 2011;135:537–43. 20. Jones MV, Calabresi PA. Agar-gelatin for embedding tissues prior to paraffin processing. Biotechniques. 2007;42:569–70. 21. Wagner DG, Russell DK, Benson JM, Schneider AE, Hoda RS, Bonfiglio TA. Cellient automated cell block versus traditional cell block preparation: a comparison of morphologic features and immunohistochemical staining. Diagn Cytopathol. 2011;39:730–36. 22. Zhao J, Lin DL, Zhai LH, Wang JG. Evaluation of ultrasound-processed rapid cell blocks in the cytopathologic diagnosis of cavity fluids. Acta Cytol. 2014;58:182–91.
JHCXXX10.1369/0022155416651065Cell and Tissue Display: A New Microscopy ToolTheodosakis et al.
Article Journal of Histochemistry & Cytochemistry 2016, Vol. 64(7) 403–411 © 2016 The Histochemical Society Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1369/0022155416651065 jhc.sagepub.com
Cell and Tissue Display: An Alternative Multipurpose Tool for Microscopy Nicholas Theodosakis, Goran Micevic, Marcus W. Bosenberg, and Nemanja Rodić
Department of Pathology (NT, GM, MWB) and Division of Dermatopathology, Department of Dermatology (NT, GM, MWB, NR), Yale University, New Haven, Connecticut
Summary We developed a method, termed Cell and Tissue Display (CTD), for embedding 16 or more different tissue samples in multicompartment agarose blocks. The CTD-generated blocks allow uniform multiplexing of cell lines and small tissue fragments within a single histologic block. The distribution of individual cells within the CTD blocks is improved, likely because the individual agarose compartments are small and uniform. The composition of each CTD block can be customized based on intended use. Some potential uses of CTD histologic blocks include improved sectioning of small tissue fragments, such as needle biopsy specimens or punch biopsies; multiplexing of tissue fragments within a single block; and the generation of control slides for laboratory proficiency testing. (J Histochem Cytochem 64:403–411, 2016) Keywords Cell and Tissue Display, histologic block, histology
Introduction A complex need for detailed histologic and molecular analysis in clinical and research biomedicine has resulted in the development of a wide range of assays, such as in situ hybridization, immunofluorescence, and immunohistochemistry.1–3 Reliable preservation of both normal and tumor tissue continues to be integral to the proper use of these assays. One economical and rapid method in the clinical setting is to simply place multiple tissue specimens into a single histologic block. In research investigations, where hypocellular specimens are often a major limitation, investigators frequently turn to adhering cells to slides by culturing cells in vitro or by manually spreading cells on slides.4 Histologic block-making has undergone iterative improvement over the last several decades. The first hallmark improvement was devised in 1986, when Dr. Hector Battifora developed a “sausage” histologic block method, which entailed the arrangement of multiple approximately 1 mm thick tissue fragments obtained from different specimens in a segment of
small intestine.3 This method marked a significant technological advancement, allowing for multiple tissue specimens to be easily evaluated under identical processing conditions. However, the individual tissue fragments could not be identified with certainty. In 1990, Dr. Battifora modified his original method and termed his new technique the “checkerboard” tissue block. This iteration allowed even distribution and identification of multiple tissue specimens in one block.5 The next major technological advancement was the development of tissue microarray (TMA) technology by Kononen et al.6 TMA allowed for the identical configuration of hundreds of tissue cores within a block. However, technical limitations persisted despite near-universal application of TMA in research settings. Most notably, tissue loss was detected in 10% to 30% of assembled Received for publication October 22, 2015; accepted April 18, 2016. Corresponding Author: Nemanja Rodić, Yale Dermatopathology Laboratory, 15 York Street, LMP5031, P.O. Box 208059, New Haven, CT 06520-8059, USA. E-mail: [email protected]
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404 cores, resulting either from loss during cutting or from the transfer of array sections from archival blocks.7 The most common approach for correcting uneven cell display of tissue fragments on slides was to create multiple cores of an identical case. Similarly, the most common approach used to correct uneven cell displays of cell lines on slides was to massively increase the input, often by increasing the number of cells submitted for the construction of the histologic block. While this may be readily achieved for untreated established cancer cell lines, cells grown in culture are often treated with reagents that cause apoptosis or inhibit cancer cell growth. In those instances, it becomes difficult to procure sufficient numbers of lesional cells to generate blocks. Other researchers have previously pioneered the incorporation of cell lines in TMAs8,9 and agarose blocks10,11 or molds12 to increase yield from paucicellular specimens. Herein, we expand on this knowledge and describe a novel method, Cell and Tissue Display (CTD), which achieves both improved cellular homogeneity of the x–y planes of a z-axis and also permits rapid multiplexing and uniform arrangement of both tissues and cell lines in one block.
Materials and Methods Cell Culture Cell lines were provided as a gift by Dr. Ruth Halaban (Yale University, New Haven, CT).13 Surgical melanoma resections were used to generate the lines. Specimens were collected with patients’ informed consent in accordance with the Health Insurance Portability and Accountability Act (HIPAA) under a Human Investigations Committee protocol. All melanoma cell lines (YUVON, YUGASP, YUKOLI, YUHEF, YUROB, YUKSI, YUSIK, YUTIKA, YURIF, WM1346, and YUGEN8) were grown routinely in Opti-MEM® (Invitrogen, Carlsbad, CA) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum, and maintained in a 37C incubator with 5% CO2.
Human Normal Tissue Procurement Following review, written permission was obtained from the director of autopsy services at Johns Hopkins Hospital to procure fresh normal skin tissue from routine adult autopsies. Apart from designating the tissue of origin for each specimen, no additional identifiers were recorded.
Animal Tissue Preparation All mice were bred on a C57BL/6 inbred genetic background. All experiments involving animals were
Theodosakis et al. reviewed and approved by the Yale Institutional Animal Care and Use Committee. The mice were euthanized according to the Yale University animal protocol. Tissue was harvested and freshly embedded in agarose mold. CTD agarose blocks were fixed in 1% neutral buffer formalin solution for ~1 hr prior to submitting the blocks for routine processing at the Orthopaedic Histology and Histomorphometry Laboratory at the Yale School of Medicine.
Histologic Block Construction Traditional histologic blocks used as controls were prepared according to previously published protocols.14-16 Cytologic blocks were prepared routinely by the Cytology Service at the Yale School of Medicine using standard Cellient™ automated cell block technology (Hologic, Inc., Bedford, MA). The CTD histologic block procurement method was performed as follows. First, 3 g of standard melting point agarose (UltraPure Agarose, Invitrogen, Inc.) powder was dissolved in 100 ml of 1× phosphate-buffered saline (PBS) solution by heating in a standard microwave for 60 to 90 sec. Molten agarose solution was poured into an inverted pipette container lid from a BioDOT Universal Fit pipette tips container (DOT Scientific, Inc., Burton, MI). Next, either a MicroAmp Optical 96-well reaction plate or a 384-well reaction plate (Life Technologies, Inc., Carlsbad, CA) was placed onto the molten agarose solution. The mixing step, in which cells/tissue are mixed with molten agarose, is critical to perform prior to deposition into the agarose mold to prevent any shrinkage artifact. The preparation was then left at room temperature for 30 min to allow the agarose to solidify. Afterward, the container lid was removed, and the agarose mold was carefully extricated from the plastic lid. The agarose mold was then cut and trimmed to fit into a closed anatomic pathology cassette. Excess agarose mold may be placed in 1 × PBS solution and stored in a refrigerator at 4C for at least 1 month. Cells were routinely retrieved from cell culture plates,17 then fixed in 1% buffered formalin solution, optimally at a concentration of 1 × 106 cells/ml. Next, 50 ml aliquots were removed and placed into new microcentrifuge tubes. The cells were allowed to settle out of suspension on the benchtop for 30 to 60 min. After the cells have settled, supernatant solvent was carefully removed with a pipette. The remaining cells were resuspended in approximately 50 µl of 1% molten agarose solution and injected into the agarose spots or wells created by the bottoms of the 96- or 348-well reaction plates. The agarose mold was then quickly placed at the corner of an inverted Corning Costar® cell culture
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Cell and Tissue Display: A New Microscopy Tool Cell and Tissue Display: A New Microscopy Tool plate lid (Sigma-Aldrich, Inc., St. Louis, MO) and spun for 1 to 3 min at 1500 rpm using a Beckman microplate carrier 270-453790-B and G.H. 3.7 Rotor/Centrifuge (Beckman, Inc., Brea, CA) to achieve better cellular homogeneity. For tissue fragment processing, each fragment was placed in a spot or well and covered with molten agarose solution. Cells were enumerated using Countess® Cell Counting Chamber Slides (Life Technologies, Inc., Carlsbad, CA). The agarose blocks were first allowed to rest on the benchtop for 30 min. They were then placed in 1% buffered formalin solution and taken to the histology laboratory for paraffin embedding. Histoprocessing was performed routinely using a standard vacuum infiltration processor (Shandon Excelsior ES; Thermo Fisher Scientific, Inc., Waltham, MA) at the Orthopaedic Histology and Histomorphometry Laboratory, Yale School of Medicine. We used Paraplast Plus® embedding media (supplier no. 39502004; VWR International, Inc., Radnor, PA) for embedding. The paraffin-embedded CTD histologic blocks were stored routinely at room temperature. At the time of publication of this report (1 year following the original CTD histologic block constructions), we examined the CTD histologic blocks for gross evidence of shrinkage. We detected no shrinkage between specimen and agarose matrix, nor between agarose matrix and surrounding paraffin. Deeper step sections (cut nos. 30, 40, 50, 60, 70, 80, 90, and 100) into CTD histologic blocks were cut routinely, and both unstained sections and hematoxylin and eosin (H&E) stained slides were produced. By cut no. 70, we had cut through two spots or wells so that 10 out of 12 spots/wells were visualized. By cut no. 100, we cut through an additional one spot/well, so that nine out of 12 were visualized. As CTD entails working with molten agarose, experimenters should use laboratory coats, gloves, silicone insulation sandwiched gloves, and safety goggles. Rarely, molten agarose solution can become superheated and violently boil over immediately after mechanical agitation, such as immediately after lifting the beaker from the microwave.
was obtained from Life Technologies, Inc. (Grand Island, NY). Monoclonal mouse anti-human primary antibodies against microphthalmia-associated transcription factor (MITF; product no. M362129-2) at a dilution of 1:100, A1/A3 (product no. M351529-2) at a dilution of 1:50, polyclonal rabbit anti-human primary antibodies against S-100 (product no. Z031129-2) at a dilution of 1:400, and cytokeratin (CK116; product no. Z062201-2) at a dilution of 1:500 were used according to the manufacturer’s instructions (Dako North America, Inc.). Secondary anti-mouse antibodies conjugated to horseradish peroxidase (HRP; catalog no. M32207) at a 1:2000 dilution (Thermo Fisher Scientific, Inc.), anti-rabbit antibodies conjugated to fluorescein isothiocyanate (FITC; catalog no. F9887) at a dilution of 1:200 (Sigma-Aldrich Inc.), and anti-rabbit antibodies conjugated to Cy3 (catalog no. C2306) at a dilution of 1:200 (Sigma-Aldrich, Inc.) were used according to the manufacturer’s instructions. All antibodies incubations were performed overnight at 4C in the humid chamber.
Antigen Detection
We have developed an alternative approach, termed CTD, to embed cells and/or tissue fragments in small potential spaces, herein termed spots or wells. To begin to create the agarose mold, we started by pouring molten agarose into a plastic receptacle. We then placed empty 96- or 384-well reaction plates over the molten agar to create spots/wells within the agarose (Fig. 1F). Following solidification of the agarose, cells or tissue fragments were placed in the spots/wells (Fig. 1G). Agarose molds were cut to fit standard histology cassettes (Fig. 1H). The specimens were then
Both immunohistochemistry and immunofluorescence were performed by the Yale Dermatopathology Laboratory. Briefly, antigens were first unmasked in 10 mM sodium citrate buffer (pH 6.0) antigen-retrieval solution using PT Link Module Plus (Dako North America, Inc., Carpinteria, CA). Tissues slides were then processed routinely on Dako Autostainer Plus (Dako North America, Inc.). DAPI (4′,6-diamidino2-phenylindole, dihydrochloride; product no. D1306)
Image Analysis Representative digital photomicrographs (400× final magnification) of tissue spots/wells were captured using laboratory microscopes (BX41 microscope with a DP26 camera, Olympus America, Inc., Melville, NY) equipped with imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI). We then used ImageJ analysis software (version 1.49) to measure cellular homogeneity within each photomicrograph. Shapes within each photomicrograph of >100 square pixels with a roundness coefficient between 0.2 and 1 were counted as cells. The contrast threshold was adjusted manually to account for background on each image. Once the cells were defined, a nearest neighbor distance (NND) calculator plugin for ImageJ was used to calculate the NND value for each particle in the field, according to described specifications.16
Results
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Figure 1. Key features of Cell and Tissue Display (CTD) method. (A–F) Pictorial representation of agarose mold preparation from molten agarose (first two diagrams). (G, H) Cells and/or tissue fragments can be deposited into spots/wells (with or without subsequent centrifugation) and submitted for routine histologic block-making.
delivered to the histopathology laboratory for routine embedding and slide preparation. We compared the performance characteristics of slides created from CTD cell line blocks to three commonly used methods in a series of qualitative (Figs. 2 and 3) and quantitative (Fig. 4) tests.14-16 Two of these
three methods are traditional histologic techniques used to prepare histologic blocks in the research setting at Johns Hopkins Hospital and the Yale School of Medicine. The third cytologic block method is a standard technique to procure cytologic specimens for routine clinical evaluation.
Cell and Tissue Display: A New Microscopy Tool Cell and Tissue Display: A New Microscopy Tool
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Figure 2. Additional qualitative features of histologic sections made by Cell and Tissue Display (CTD) method. (A–C) Representative photographs from histologic blocks made with traditional methods; provided to illustrate comparable inhomogeneity of visible cellular aggregates and clusters in those preparations. (D–I) Representative images showing hematoxylin and eosin (H&E) stained sections from histologic blocks made by CTD methodology. (D) Image of CTD slide multiplexed with 16 murine tissue fragments. (E) Photographs of CTD slide multiplexed with 11 human melanoma cell lines and one normal human skin tissue fragment. Representative photomicrographs of CTD spots/wells containing (F) murine colon; (G) normal murine brain; (H) low-cellularity melanoma cell line, YURIF; and (I) high-cellularity melanoma cell line, YUGASP. Final magnification, parts (F–I), 200×; white scale bar in (A, D) denotes 5 mm; black scale bar in (F) denotes 250 µm.
Both the manual inspections and the microscopic evaluations of the CTD sections showed that they can be multiplexed and tiled in consecutive uniform rows and columns, similar to the slides made by TMA technology. Either cells or tissues can be arranged in spots or wells (Fig. 2D–I), a feature which represents a technological improvement from the other three common methods of histologic block-making (Fig. 2A–C). In addition, mixed cultured cell and tissue fragments can be multiplexed within the same CTD block (Fig. 2E, 2G–I). Next, we generated another CTD block that contained normal murine tissues procured from routine animal necropsy (Fig. 2D, F). Within a single histologic block, we arranged 16 unique tissue fragments and achieved full cross-sections of all 16 tissue fragments on a single slide. Upon microscopic evaluation of the slides made with the CTD method, we noted a marked qualitative improvement in cell homogeneity (Fig. 3A, lower panel) relative to the other three methods (Fig. 3A, upper panel). Importantly, cells procured by CTD maintained appropriate epitope antigenicity by immunohistochemistry and immunofluorescence (Fig. 3B). Upon further optimizing the protocol to include an additional centrifugation step
prior to agarose-based mold solidification, we observed further enhancement in microscopic homogeneity. To quantify this effect, we applied ImageJ software to representative photomicrographs and evaluated a correction value, NND, which represents the intercellular distances between individual cells. We measured hundreds of NND values within a given field of view. The scores were then reported as relative coefficients of variation (CVs) for NND values (Fig. 4). Finally, we determined the cell count and distribution in deeper sections to measure homogeneity through various levels of the z-axis. We focused our analysis of cellularity and distribution on three representative cell lines: a low-cellularity cell line, YURIF; moderate-cellularity cell line, YUKSI; and high-cellularity cell line, YUVON (Fig. 5A). By applying ImageJ analysis on representative photomicrographs, we determined that the cellularity of all three cell lines remained relatively uniform (Fig. 5B). On an early cut (cut no. 10), we detected 33 YURIF cells; on an intermediate cut (cut no. 50), we detected 46 YURIF cells; and on a deep cut (cut no. 100), we detected 34 YURIF cells. The number of YUKSI cells was 154, 99, and 113 on cuts 10, 50, and 100, respectively. The number of
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Figure 3. Homogeneity and epitope antigenicity are preserved by the Cell and Tissue Display (CTD) method. (A) Representative photomicrographs showing YUGEN8 cells procured via traditional methods (upper panels), compared with cells procured via CTD (lower panels, low to moderate to high cellular density for comparison). Final magnification, part (A), 200×; black scale bar in (A) denotes 250 µm. (B) Representative photomicrographs showing appropriate epitope detection following CTD histologic block-making with immunohistochemistry (upper panel) and immunofluorescence (lower panel). Final magnification, part (B), 400×; black and white scale bars denote 60 µm. Abbreviations: H&E, hematoxylin and eosin; DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride.
Cell and Tissue Display: A New Microscopy Tool Cell and Tissue Display: A New Microscopy Tool
Figure 4. Improved YUGEN8 cellular homogeneity in x- and y-planes using Cell and Tissue Display (CTD) methodology. The y-axis denoted coefficient of variation (CV) for value nearest neighbor distance (NND), which represents the intercellular distances among individual cells within a given field of view. Variegated and comparably higher CV in histologic blocks procured using traditional methods (traditional methods 1 and 2, and the cytologic block method) compared with several iterations of CTD histologic block preparations.
YUVON cells was 530, 605, and 541 on cuts 10, 50, and 100, respectively. To evaluate the distribution of cells, we determined number of cells and their corresponding NNDs. Both values remained largely unchanged among an early cut (cut no. 10) and later serial cut (cuts no. 50 and 100; Fig. 5C). YURIF cells retained similar distribution (NND ± CV) on an early cut (149 ± 49), on an intermediate cut (157 ± 43), and on a deep cut (174 ± 52) as well. NND and CV values for YUKSI and YUVON cell lines were 91 ± 31 (cut no. 10), 107 ± 42 (cut no. 50), 103 ± 38 (cut no. 100), and 49 ± 24 (cut no. 10), 43 ± 21 (cut no. 50), 52 ± 22 (cut no. 100), respectively. Taken together, our findings support the hypothesis that microscopic homogeneity in the z-plane of sectioning is reliably achieved with CTD. This feature of our method supports its use in the clinical setting for making histologic blocks using markedly hypocellular specimens, such as buccal swabs.18
Discussion The potential applications of CTD for rapidly generating reliable testing controls in the clinical setting are numerous. The need for reliable positive and negative controls in laboratory medicine and clinical pathology necessitates dependable reproducibility from test to test. The marked homogeneity of tissue embedded using CTD from section to section, as well as long-term preservation of antigenicity mark it as ideal for this purpose. We
409 also believe that coupling of CTD to cell-sorting tools such as flow cytometry could be used to construct control blocks containing cell lines both enriched and homogeneous for markers of interest, further improving both reproducibility and utility for quality control. Furthermore, the ability to mix purified cell lines expressing different markers would provide a potential test modality to verify the specificity of new antibodies or staining techniques for clinical use in a controlled manner. Analytic sensitivity may be measured in an analogous fashion by mixing purified cells with variable expressivity of important markers to determine detection thresholds. The biological homogeneity offered by cell lines may also allow better evaluation of the effects of different pre-analytical variables, with controlled manipulation of characteristics such as fixative and length of fixation, yielding improved characterization of false-detection rates. While validation work would have to be done, given that cell lines often express proteins in non-physiologic ranges, comparison with marker-expressing tissue samples may be of use in certain settings. CTD has the additional advantage of allowing multiple tissue fragments to be tiled directly from the grossing station (in clinical investigations) or the benchtop (in research applications), whereas histologic blocks must be retrieved to create TMAs. Alternatively, if there is a collection of fixed tissue fragments, they can simply be tiled into CTD spots/wells, and the agarose block can be submitted for histopathologic processing. We termed this protocol a prospective, as opposed to retrospective, construction. CTD can also easily be incorporated into a TMA construction by coring the foci of interest. Using the principles presented herein, any clinical laboratory can construct in-house CTDs that are tailored to their own practice. CTDs can be constructed centrally, then distributed to the practicing clinical laboratories to help improve quality control. Alternatively, the CTD method may prove useful as an alternative platform for initiatives toward synchronization of immunohistochemical staining protocols.19 With regard to guaranteeing quality from the CTD protocol, we have noted only a handful of minor issues. First, we rarely observed small (approximately 100– 200 µm2 areas, best seen in Fig. 3, lower panel, center of middle image) cell-free regions, which may be due to artifactual introduction of air bubbles during molten agarose pipetting. This feature indicates that while CTD signifies an improvement in histologic block-making, it is not a perfect method. Second, we rarely observed tissue folding during the microtome-cutting step of the CTD-made blocks. While tissue folding can occur during the routine processing of any histologic block, we plan to adapt an agar–gelatin mix protocol,
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Figure 5. Improved cellular homogeneity of YURIF, YUKSI, and YUVON cell lines across deep sections using Cell and Tissue Display (CTD) methodology. (A) Representative photomicrographs showing melanoma cell lines of varied cellularity: A low-cellularity cell line, YURIF; moderate-cellularity cell line, YUKSI; and high-cellularity cell line, YUVON. (B) Relatively uniform cell number of YURIF, YUKSI, and YUVON cells on an early cut (cut no. 10), on an intermediate cut (cut no. 50), and on a deep cut (cut no. 100). (C) Relatively uniform cell distribution of YURIF YUKSI, and YUVON cells on an early cut (cut no. 10), on an intermediate cut (cut no. 50), and on a deep cut (cut no. 100). Final magnification, part (A), 400 ×; black scale bar denotes 60 µm. Abbreviation: NND, nearest neighbor distance.
originally developed by Jones and Calabresi,20 to the next iteration of the CTD methodology. In addition to CTD, there are two alternative nontraditional methods for creating histologic blocks from paucicellular specimens. The first is the Cellient™ cell block, the method used to create the cytology blocks for comparison in this study.21 The second method is the rapid formaldehyde fixed-paraffin embedded (FFPE) cell block.22 In the present report, we observed a qualitative improvement in cellularity in the slides procured with the CTD method compared with the Cellient-prepared cytology blocks. The rapid FFPE method entails wrapping cellular pellets in gauze to improve cellularity, prior to histologic block-making, and was not evaluated in the current study. The potential for CTD to improve scientific research is also significant. Low-cost assemblies of highly homogeneous arrays of cell lines can be used to rapidly screen for expression of markers of interest without the need for retrieval of frozen stocks or the growth of difficult-to-culture lines. The improved stability of cell lines preserved in this manner may also facilitate sharing of cultured tissue between institutions with fewer concerns over sample viability. In addition, the ability to take multiple cuts
from a highly homogeneous block will allow for rapid parallel staining for a large number of markers, without the need to simultaneously process large numbers of separately grown cultures. Herein, we report the development of a novel protocol for histologic block-making, CTD, which allows for more nuanced and controlled manipulation of cell lines, as well as the multiplexing of cell lines with tissue within the same histologic block. Using CTD allows for better control of the number of cells visible on a given slide, which may allow the construction of histologic blocks from markedly paucicellular specimens, such as primary cells. We demonstrated the utility of CTD by rapidly generating histologic blocks from normal murine tissue, as well as from multiplexed melanoma cell lines. The versatility of this technique lends itself to adaptation to a wide variety of research and clinical needs, which could help improve the utility of histology in mainstream investigative research. Acknowledgments The authors thank Drs. Ruth Halaban and Antonietta Bacchiocchi for providing the human melanoma cell lines used to validate this technique. We also thank Mrs. Nancy Troiano, of
Cell and Tissue Display: A New Microscopy Tool Cell and Tissue Display: A New Microscopy Tool the Orthopaedic Histology and Histomorphometry Laboratory, for preparing the tissue slides from the Cell and Tissue Display (CTD) blocks. The authors also thank the Yale Dermatopathology Laboratory for performing immunostaining studies.
Author Contributions NR designed the study; NT, GM, and NR constructed the Cell and Tissue Display (CTD) blocks; NT and NR performed image processing and analysis; MWB provided project oversight and critically examined intellectual content; NT and NR wrote the manuscript. All authors have read and approved the final manuscript.
Competing Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Department of Dermatology at the Yale School of Medicine.
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