DOI:10.1111/cyt.12138
An improved high-output cell microarray technology Q. Hu, Y. Shi, X. Li, Y. Hou, D. Jiang, J. Huang, J. Su, H. Zeng and Y. Tan Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China Accepted for publication 5 January 2014
Q. Hu, Y. Shi, X. Li, Y. Hou, D. Jiang, J. Huang, J. Su, H. Zeng and Y. Tan An improved high-output cell microarray technology Aims: Cell microarray (CMA) is a high-throughput scientific research tool, which has greatly accelerated many analyses based at the cellular level. However, there are few described methods for constructing CMAs. Here, we introduce a new, simple, high-output CMA method that is applicable to a broad range of cellular samples. Methods: In this method, a recipient block (length, 3.6 cm; width, 2.7 cm; depth, 2 cm) with 40 dot markers was moulded using a transparent plastic box. Adenocarcinoma cells were collected from malignant pleural effusions, cell cylinders were moulded with plastic piping and the cylinders were manually arrayed one by one into the corresponding location of the 60 °C pre-softened recipient block using the guide holes drilled with a steel needle. We constructed a 40-cylinder CMA to prove this method. The expression of cytokeratin 7 (CK7) in the CMA was examined to confirm antigen preservation and epidermal growth factor receptor (EGFR) gene mutation was screened for in five samples. Results: The CMA prepared by this method had well-defined array configurations, good cellular morphology and well-preserved proteins and DNA. A total of 1000 sections could be easily gained from this CMA block. Conclusions: This simple and low-cost method provides a novel way of preparing a high-output CMA. Keywords: cell sediment, cell cylinder, cell microarray, high output, pleural effusion
Introduction Tissue microarray (TMA) is a high-throughput research tool, which has greatly facilitated and accelerated tissue analyses by in situ technologies.1 In 1986, Battifora2 devised an ingenious idea for combining multiple tissue samples into a single ‘sausage’ composite held together with a wrapper of intestinal casing. Several years after this pioneering and prototypical work, Kononen et al.3 developed a novel high-throughput TMA technique in 1998. As a highthroughput research method, the cell microarray (CMA), like TMA, is one of the most important and powerful biochip technologies that allows the
Correspondence: Dr Y. Hou, Department of Pathology, Zhongshan Hospital, Fudan University, Fenglin Road #180, Shanghai 200032, China Tel.: +86-21-64041990-2732; Fax: +86-21-64038472; E-mail: [email protected] Qin Hu and Yuan Shi contributed equally to this project.
44
analysis of a large number of samples simultaneously. Recently, Kim et al.4 constructed a CMA containing a panel of 40 pancreatic cancer cell lines to rapidly test the expression of molecules of interest in these cancer cell lines. Cardano et al.5 constructed a CMA containing six cell lines to analyse the expression of SEL1L in these cell lines by immunohistochemistry and immunofluorescence techniques. Their results showed the utility of CMAs as a resource for rapid screening of molecules of interest. They suggested that CMAs could become a universal standard platform in cancer research. However, methods for the construction of CMAs are not widely described in the literature. A CMA with eight cylinders, constructed by punching cores from cell blocks with a manual arrayer (Beecher Instrument, Silver Spring, MD, USA), was introduced by Moskaluk and Stoler6 in 2002, the cell blocks being embedded by three different procedures: agarose matrix method, cell pellet method and agarose mould method. Wen et al.7 later reported a CMA with 25 agar-mixed cell cylinders built by punch extractor (Pin-Extractor, Ho Hua Electricity, Taiwan) © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
High output cellular microarray
in 2007. Both of the above methods were similar to traditional TMA; the number of sections was therefore restricted by the thickness of the paraffinembedded cell blocks with only between 100 and 300 sections available from one CMA constructed by punching cylinders from cell blocks. Recently, we have described an alternative method using various tools, including several inhouse instruments, to make a high-output TMA.8 We have now developed a high-output CMA based on these simple tools with some improvement. To our knowledge, this is the first report on making 1000 sections from one CMA block. We describe this new simple technique which is fundamentally different from previous methods of CMA construction. Methods Selection of appropriate cell sediment with adenocarcinoma in pleural effusion Forty cases of adenocarcinoma in pleural effusion samples were collected from the Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China from August 2010 to March 2013. These cases were confirmed in smears by two pathologists (Qin Hu and Yuan Shi). Prior written informed consent was signed by all patients and the study protocol was approved by the ethics committee board at Zhongshan Hospital, Fudan University. The specimens were centrifuged for 5 minutes at 581 g and the supernatant was discarded. The sediment was separated into two parts: the first part was used to make a cell block,9 and the second part was prepared for the ‘cell cylinder’. Preparation of the ‘cell cylinder’ For this procedure, cell sediment was collected and packaged in a piece of intestinal casing, formalin fixed at this stage for about 2 hours, and then dehydrated and waxed by automatic tissue dehydration (Leica ASP300S; Leica Microsystems Nussloch GmbH, Nussloch, Germany). The waxed cell sediment was scraped from the intestinal casing into a 1.5-ml Eppendorf tube and melted in a 60 °C oven. The volume of total wax was adjusted to 0.5 ml in the Eppendorf tube. A 1-ml injector without a needle was placed on a plastic tube with an inner diameter of 3 mm and the injector was used to withdraw the melted wax from the Eppendorf tube in which the © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
(a)
(b) (c)
(d)
Figure 1. The cell cylinder preparation procedure. (a, b) The injector with plastic tube was used to draw the melted wax containing dispersed cells into an Eppendorf tube; (c) the cell cylinder was moulded; (d) the cylinder was pulled out.
cell sediment was contained (Figure 1a,b). The injector was placed in a refrigerator at 20 °C for 10 minutes until the cell cylinder was moulded (Figure 1c); thereafter, the 3-mm-diameter cell cylinder was taken out (Figure 1d), trimmed to 10 mm in length and temporarily stored in a 96-well culture plate. Preparation of the ‘recipient’ block and implantation of the cell sediment cylinders For this procedure, a recipient block (length, 3.6 cm; width, 2.7 cm; depth, 2 cm) was first made using a transparent plastic box as a mould. The detailed methods and steps were performed according to the protocols reported previously.8 In short, a plastic board etched with a 40-dot marker was placed in a topless and bottomless transparent plastic box; melting paraffin was poured into the box to cover the whole area and then cooled at room temperature until the plastic board could be separated from the transparent box. The recipient block, together with the transparent box, was incubated at 60 °C for 5 minutes prior to planting the cell cylinders to maintain the paraffin in a soft, but not melted state. A hole was made at the starting spot using a steel needle; a starting cylinder was inserted into the drilled hole. Donor cell cylinders were then manually planted into the recipient block one by one, according to the corresponding location. The recipient block held the cell cylinders firmly without slanting as the
45
46
Q. Hu et al.
paraffin was pushed to the former hole when drilling the next hole because of the restriction of the transparent box. The planting surface was aggregated at 55 °C in an incubator for 2 hours. Then, the recipient block with the transparent box was placed into a 4 °C refrigerator for 10 minutes until the paraffin could be easily separated from the box. After this, the CMA block was taken out and sectioned on a routine microtome. Slides at an interval of 100 were chosen for haematoxylin and eosin (H&E) staining. Immunohistochemistry For immunohistochemistry, all incubations were at room temperature and washes were performed with Tris-buffered saline and Tween 20 (TBST). The CMA was sectioned at 4 lm onto slides which were dewaxed and rehydrated. Antigen retrieval was performed in a pressure cooker at 110 °C for 5 minutes in retrieval buffer (S2367; Dako Denmark A/s, Glostrup, Denmark). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (S2023; DAKO). Sections were incubated with cytokeratin 7 (CK7) antibody (DAKO) for 30 minutes, incubated with EnVision/horseradish peroxidase (HRP) complex and then visualized with diaminobenzidine (K3468; DAKO) for 10 minutes. Analysis of epidermal growth factor receptor (EGFR) gene sequences First, five consecutive sections were prepared and the same cylinder on the consecutive sections was collected for genetic testing. The sequence analysis of EGFR (exons 18, 19, 20 and 21) was performed according to the protocols reported previously.10 In brief, genomic DNA was isolated from cellular samples using a standard phenol/chloroform organic extraction protocol. Sequencing reactions were conducted in both forward and reverse directions using primers for exons 18, 19, 20 and 21 of the EGFR gene: 50 -gagg tgacccttgtctctgtgt-30 for forward and 50 -cccaaacactcag tgaaacaaa-30 for reverse, 50 -tgccagttaacgtcttccttct-30 for forward and 50 -tgaacatttaggatgtggagat-30 for reverse, 50 -acttcacagccctgcgtaaac-30 for forward and 50 -atgggacaggcactgatttgt-30 for reverse, 50 -gagcttcttcc catgatgatct-30 for forward and 50 -gaaaatgctggc tgacctaaag-30 for reverse, respectively. The results were compared with the sequences of the human EGFR gene in the National Center for Biotechnology Information.
Results Sectioning and staining A CMA with 40 cylinders was constructed to demonstrate this method; the front and lateral views of the CMA are shown in Figure 2(a,b). Up to 1000 consecutive 3–5-lm sections could be gained by a trained histotechnologist because of the thickness of the CMA. The sections adhered to the special slides and were stained with H&E (Figure 2c,e). No spots were observed to have been missed after 200, 400, 800 and 1000 sections: 100% remained at at section number 1000. The morphology of all the spots was consistent with the original slides. Cellularity evaluation In this study, we examined smears of the 40 cases of adenocarcinoma in pleural fluid, and assigned each to a null, low, moderate or high cellularity category. The cellularity was defined as follows: null – red blood cells, mesothelial cells or inflammatory cells present only; low cellularity – 10 or fewer cell clusters; moderate cellularity – 11–30 clusters; high cellularity – more than 30 clusters. A cell cluster was defined as five or more cells.11 The tumour cellularity results in smears were as follows: null, 0 cases; low cellularity, one case; moderate cellularity, four cases; high cellularity, 35 cases. Correspondingly, we observed the tumour cellularity in each cell cylinder in the CMA section. The tumour cell cluster results were as follows: null, 0 cases; low cellularity, one case; moderate cellularity, eight cases; high cellularity, 31 cases. The relationship between the tumour cellularity on the smear slides and the cell cylinders in the CMA is shown in Table 1. Immunohistochemistry on the CMA sections Among the 40 tumours, 37 were positive for CK7 (Figure 2d,f). All immunohistochemistry results for the CMA-derived sections were consistent with those of their corresponding original cell block slides. Analysis of EGFR sequences The sequences of the analysed genes obtained from CMA were consistent with those obtained from their corresponding original cell block slides. Figure 3 © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
High output cellular microarray
(a)
(c)
(e)
(b)
(d)
(f)
Figure 2. Images of the prepared cell microarray (CMA). (a) Front view of a CMA (length, 3.6 cm; width, 2.7 cm). (b) Lateral view of a CMA showing that the cell cylinder depth is vaguely visible. Histological results of CMA samples. (c) Haematoxylin and eosin (H&E) staining of CMA samples, showing a total of 40 spots in the section. (d) Immunohistochemistry of CMA sections using cytokeratin 7 (CK7) antibody. (e) H&E staining of a spot at higher magnification. (f) Immunohistochemistry of CK7 staining of a spot at higher magnification. Table 1. Correlation of cellularity of smears and cell cylinders in cell microarays Cell cylinder of cell microarray Smear
Low
Moderate
High
Total
Low Moderate High Total
1 0 0 1
0 1 7 8
0 3 28 31
1 4 35 40
shows the point mutation of CTG to CGG in exon 21 of the EGFR gene. Discussion Development of CMA and shortcomings of traditional methods As a high-throughput research tool, CMA, also known as a chip technique, is similar to TMA. However, there are few methods described for the construction of CMAs. Traditional TMA technology involved the extraction of a tissue cylinder from a tissue block by a punching method,3,12 and previous reports on the construction of CMAs were based on this TMA methodology.6,7 However, there were shortcomings in these methods of construction of CMAs. © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
Figure 3. Sequence of epidermal growth factor receptor (EGFR) gene at codon 858, showing the point mutation of CTG to CGG in exon 21.
First, the thickness of the cell block was usually limited to less than 2 mm after being sectioned for H&E staining; if arrays were not well constructed or some cylinders were shorter than others, some cylinders would be ‘sectioned out’ before others. Therefore, the yield from one traditional CMA block may be limited. Second, the cell blocks can be made with or without agarose. If the cell block was made without agarose, it was difficult to construct a CMA as the cell
47
48
Q. Hu et al.
cylinders dispersed when aggregated.1,3,12 However, the cellularity in cylinders would be lost during the process of making an agarose-mixed cell block. Moskaluk and Stoler6 investigated the difference in cell density between three methods: agarose matrix method, cell pellet method and agarose mould method. The study showed that the agarose matrix method yielded the lowest cell density. The cell pellet method yielded the highest average cell number, as the cells were not suspended in agarose, and hence were not diluted. Invention of a new high-output CMA technique This new method could improve the output of CMA in terms of section yield. As demonstrated, by using appropriate simple tools, it was possible to construct a CMA with long cylinders and to produce up to 1000 sections. By withdrawing the melted wax containing the cell clusters into a plastic tube, long cylinders of the same length are moulded, which can then be implanted into the drilled holes in the recipient block in a vertical orientation, thus producing a CMA of greater depth than can be achieved with cell blocks. We found that the cell cylinders were easily dispersed when traditional TMA aggregation methods or pouring wax methods7 were used in the aggregation stage, and that temperature and time control during the CMA aggregation process were very important. Cylinders dispersed and touched each other at high temperature, and could not homogenize with wax in between at low temperature. To prevent this, we recommend that the recipient block with the transparent box is placed in an incubator at 55 °C for 2 hours. Cylinders are thus maintained in good shape, location and merge well with the recipient paraffin (Figure 2a,b). After aggregation and separation from the transparent box, all 40 cylinders and starting spot were well aligned (Figure 2a,b), which enabled the production of approximately 1000 sections from each cylinder to be used for H&E (Figure 2c,e) and immunohistochemistry (Figure 2d,f) staining. Furthermore, as our results show, the cellularity of the cell spots was maintained throughout and was comparable with the original slide, and immunohistochemistry results were consistent with the corresponding cell block. The DNA sequencing results (Figure 3) showed that the CMA cell maintained its DNA after the cylinders of cell sediment were remoulded and planted in the recipient block.
Advantages and limitations of our high-output CMA technique The advantages of our current approach are summarized as follows: (i) the procedure and facility are simple and no special, expensive instrumentation is required; with only a little practice, an operator can successfully plant cell cylinders into a recipient paraffin block; (ii) we have found an appropriate temperature and time range in which the CMA can be aggregated well without melting of wax, movement of cylinders and dispersion of cells; and (iii) this method has a very high output in terms of sections, which is the greatest improvement over previous methods. There are, however, limitations to this method of CMA construction. For example, where the number of cell clusters in the slide prepared from a fluid is low, i.e. less than 10, there is likely to be insufficient cells for inclusion in a CMA, as the cell clusters will be further diluted in the paraffin and will therefore not be represented in each CMA section. In addition, if the sediment contains a large amount of red blood cells, inflammatory cells or mesothelial cells and only a small proportion of cancer cells, it would be unsuitable for making a high-output CMA, as the small proportion of cancer cells would also be diluted by paraffin and the other cell types. Conclusion As compared with CMA methods based on the punching of cylindrical cores, the CMA preparation technique described in this study is relatively simple, economic and highly efficient. Construction of a CMA is inexpensive, large numbers of sections can be produced and the CMA performs well with respect to the preservation of cell morphology, proteins and DNA. This method would be feasible for use by general research groups employing microarray technology. Acknowledgments This study was funded by: (i) The National Natural Science Foundation of Youth Science Foundation, grant number 81101809; (ii) Shanghai Science and Technology grant number 11140902502; and (iii) Shanghai Science and Technology grant number 13411950802. © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
High output cellular microarray
References 1. Avninder S, Ylaya K, Hewitt SM. Tissue microarray: a simple technology that has revolutionized research in pathology. J Postgrad Med 2008;54:158–62. 2. Battifora H. The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest 1986;55:244–8. 3. Kononen J, Bubendorf L, Kallioniemi A et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4:844–7. 4. Kim MS, Kuppireddy SV, Sakamuri S et al. Rapid characterization of candidate biomarkers for pancreatic cancer using cell microarrays (CMAs). J Proteome Res 2012;11:5556–63. 5. Cardano M, Diaferia GR, Falavigna M et al. Cell and tissue microarray technologies for protein and nucleic acid expression profiling. J Histochem Cytochem 2013;61:116–24. 6. Moskaluk CA, Stoler MH. Agarose mold embedding of cultured cells for tissue microarrays. Diagn Mol Pathol 2002;11:234–8.
© 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
7. Wen CH, Su YC, Wang SL, Yang SF, Chai CY. Application of the microarray technique to cell blocks. Acta Cytol 2007;51:42–6. 8. Shi Y, He D, Hou Y et al. An alternative high output tissue microarray technique. Diagn Pathol 2013;8:9. 9. Jalal R, Aftab K, Hasan SH, Pervez S. Diagnostic value of clot examination for malignant cells in serous effusions. Cytopathology 2009;20:231–4. 10. Wu X, Zhang J, Zhen R et al. Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models. J Transl Med 2012;10:180. 11. Layfield LJ, Mooney EE, Glasgow B, Hirschowitz S, Coogan A. What constitutes an adequate smear in fineneedle aspiration cytology of the breast? Cancer 1997;81:16–21. 12. Anagnostou VK, Lowery FJ, Syrigos KN, Cagle PT, Rimm DL. Quantitative evaluation of protein expression as a function of tissue microarray core diameter: is a large (1.5 mm) core better than a small (0.6 mm) core? Arch Pathol Lab Med 2010;134:613–9.
49
Copyright of Cytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
An improved high-output cell microarray technology Q. Hu, Y. Shi, X. Li, Y. Hou, D. Jiang, J. Huang, J. Su, H. Zeng and Y. Tan Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China Accepted for publication 5 January 2014
Q. Hu, Y. Shi, X. Li, Y. Hou, D. Jiang, J. Huang, J. Su, H. Zeng and Y. Tan An improved high-output cell microarray technology Aims: Cell microarray (CMA) is a high-throughput scientific research tool, which has greatly accelerated many analyses based at the cellular level. However, there are few described methods for constructing CMAs. Here, we introduce a new, simple, high-output CMA method that is applicable to a broad range of cellular samples. Methods: In this method, a recipient block (length, 3.6 cm; width, 2.7 cm; depth, 2 cm) with 40 dot markers was moulded using a transparent plastic box. Adenocarcinoma cells were collected from malignant pleural effusions, cell cylinders were moulded with plastic piping and the cylinders were manually arrayed one by one into the corresponding location of the 60 °C pre-softened recipient block using the guide holes drilled with a steel needle. We constructed a 40-cylinder CMA to prove this method. The expression of cytokeratin 7 (CK7) in the CMA was examined to confirm antigen preservation and epidermal growth factor receptor (EGFR) gene mutation was screened for in five samples. Results: The CMA prepared by this method had well-defined array configurations, good cellular morphology and well-preserved proteins and DNA. A total of 1000 sections could be easily gained from this CMA block. Conclusions: This simple and low-cost method provides a novel way of preparing a high-output CMA. Keywords: cell sediment, cell cylinder, cell microarray, high output, pleural effusion
Introduction Tissue microarray (TMA) is a high-throughput research tool, which has greatly facilitated and accelerated tissue analyses by in situ technologies.1 In 1986, Battifora2 devised an ingenious idea for combining multiple tissue samples into a single ‘sausage’ composite held together with a wrapper of intestinal casing. Several years after this pioneering and prototypical work, Kononen et al.3 developed a novel high-throughput TMA technique in 1998. As a highthroughput research method, the cell microarray (CMA), like TMA, is one of the most important and powerful biochip technologies that allows the
Correspondence: Dr Y. Hou, Department of Pathology, Zhongshan Hospital, Fudan University, Fenglin Road #180, Shanghai 200032, China Tel.: +86-21-64041990-2732; Fax: +86-21-64038472; E-mail: [email protected] Qin Hu and Yuan Shi contributed equally to this project.
44
analysis of a large number of samples simultaneously. Recently, Kim et al.4 constructed a CMA containing a panel of 40 pancreatic cancer cell lines to rapidly test the expression of molecules of interest in these cancer cell lines. Cardano et al.5 constructed a CMA containing six cell lines to analyse the expression of SEL1L in these cell lines by immunohistochemistry and immunofluorescence techniques. Their results showed the utility of CMAs as a resource for rapid screening of molecules of interest. They suggested that CMAs could become a universal standard platform in cancer research. However, methods for the construction of CMAs are not widely described in the literature. A CMA with eight cylinders, constructed by punching cores from cell blocks with a manual arrayer (Beecher Instrument, Silver Spring, MD, USA), was introduced by Moskaluk and Stoler6 in 2002, the cell blocks being embedded by three different procedures: agarose matrix method, cell pellet method and agarose mould method. Wen et al.7 later reported a CMA with 25 agar-mixed cell cylinders built by punch extractor (Pin-Extractor, Ho Hua Electricity, Taiwan) © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
High output cellular microarray
in 2007. Both of the above methods were similar to traditional TMA; the number of sections was therefore restricted by the thickness of the paraffinembedded cell blocks with only between 100 and 300 sections available from one CMA constructed by punching cylinders from cell blocks. Recently, we have described an alternative method using various tools, including several inhouse instruments, to make a high-output TMA.8 We have now developed a high-output CMA based on these simple tools with some improvement. To our knowledge, this is the first report on making 1000 sections from one CMA block. We describe this new simple technique which is fundamentally different from previous methods of CMA construction. Methods Selection of appropriate cell sediment with adenocarcinoma in pleural effusion Forty cases of adenocarcinoma in pleural effusion samples were collected from the Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China from August 2010 to March 2013. These cases were confirmed in smears by two pathologists (Qin Hu and Yuan Shi). Prior written informed consent was signed by all patients and the study protocol was approved by the ethics committee board at Zhongshan Hospital, Fudan University. The specimens were centrifuged for 5 minutes at 581 g and the supernatant was discarded. The sediment was separated into two parts: the first part was used to make a cell block,9 and the second part was prepared for the ‘cell cylinder’. Preparation of the ‘cell cylinder’ For this procedure, cell sediment was collected and packaged in a piece of intestinal casing, formalin fixed at this stage for about 2 hours, and then dehydrated and waxed by automatic tissue dehydration (Leica ASP300S; Leica Microsystems Nussloch GmbH, Nussloch, Germany). The waxed cell sediment was scraped from the intestinal casing into a 1.5-ml Eppendorf tube and melted in a 60 °C oven. The volume of total wax was adjusted to 0.5 ml in the Eppendorf tube. A 1-ml injector without a needle was placed on a plastic tube with an inner diameter of 3 mm and the injector was used to withdraw the melted wax from the Eppendorf tube in which the © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
(a)
(b) (c)
(d)
Figure 1. The cell cylinder preparation procedure. (a, b) The injector with plastic tube was used to draw the melted wax containing dispersed cells into an Eppendorf tube; (c) the cell cylinder was moulded; (d) the cylinder was pulled out.
cell sediment was contained (Figure 1a,b). The injector was placed in a refrigerator at 20 °C for 10 minutes until the cell cylinder was moulded (Figure 1c); thereafter, the 3-mm-diameter cell cylinder was taken out (Figure 1d), trimmed to 10 mm in length and temporarily stored in a 96-well culture plate. Preparation of the ‘recipient’ block and implantation of the cell sediment cylinders For this procedure, a recipient block (length, 3.6 cm; width, 2.7 cm; depth, 2 cm) was first made using a transparent plastic box as a mould. The detailed methods and steps were performed according to the protocols reported previously.8 In short, a plastic board etched with a 40-dot marker was placed in a topless and bottomless transparent plastic box; melting paraffin was poured into the box to cover the whole area and then cooled at room temperature until the plastic board could be separated from the transparent box. The recipient block, together with the transparent box, was incubated at 60 °C for 5 minutes prior to planting the cell cylinders to maintain the paraffin in a soft, but not melted state. A hole was made at the starting spot using a steel needle; a starting cylinder was inserted into the drilled hole. Donor cell cylinders were then manually planted into the recipient block one by one, according to the corresponding location. The recipient block held the cell cylinders firmly without slanting as the
45
46
Q. Hu et al.
paraffin was pushed to the former hole when drilling the next hole because of the restriction of the transparent box. The planting surface was aggregated at 55 °C in an incubator for 2 hours. Then, the recipient block with the transparent box was placed into a 4 °C refrigerator for 10 minutes until the paraffin could be easily separated from the box. After this, the CMA block was taken out and sectioned on a routine microtome. Slides at an interval of 100 were chosen for haematoxylin and eosin (H&E) staining. Immunohistochemistry For immunohistochemistry, all incubations were at room temperature and washes were performed with Tris-buffered saline and Tween 20 (TBST). The CMA was sectioned at 4 lm onto slides which were dewaxed and rehydrated. Antigen retrieval was performed in a pressure cooker at 110 °C for 5 minutes in retrieval buffer (S2367; Dako Denmark A/s, Glostrup, Denmark). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (S2023; DAKO). Sections were incubated with cytokeratin 7 (CK7) antibody (DAKO) for 30 minutes, incubated with EnVision/horseradish peroxidase (HRP) complex and then visualized with diaminobenzidine (K3468; DAKO) for 10 minutes. Analysis of epidermal growth factor receptor (EGFR) gene sequences First, five consecutive sections were prepared and the same cylinder on the consecutive sections was collected for genetic testing. The sequence analysis of EGFR (exons 18, 19, 20 and 21) was performed according to the protocols reported previously.10 In brief, genomic DNA was isolated from cellular samples using a standard phenol/chloroform organic extraction protocol. Sequencing reactions were conducted in both forward and reverse directions using primers for exons 18, 19, 20 and 21 of the EGFR gene: 50 -gagg tgacccttgtctctgtgt-30 for forward and 50 -cccaaacactcag tgaaacaaa-30 for reverse, 50 -tgccagttaacgtcttccttct-30 for forward and 50 -tgaacatttaggatgtggagat-30 for reverse, 50 -acttcacagccctgcgtaaac-30 for forward and 50 -atgggacaggcactgatttgt-30 for reverse, 50 -gagcttcttcc catgatgatct-30 for forward and 50 -gaaaatgctggc tgacctaaag-30 for reverse, respectively. The results were compared with the sequences of the human EGFR gene in the National Center for Biotechnology Information.
Results Sectioning and staining A CMA with 40 cylinders was constructed to demonstrate this method; the front and lateral views of the CMA are shown in Figure 2(a,b). Up to 1000 consecutive 3–5-lm sections could be gained by a trained histotechnologist because of the thickness of the CMA. The sections adhered to the special slides and were stained with H&E (Figure 2c,e). No spots were observed to have been missed after 200, 400, 800 and 1000 sections: 100% remained at at section number 1000. The morphology of all the spots was consistent with the original slides. Cellularity evaluation In this study, we examined smears of the 40 cases of adenocarcinoma in pleural fluid, and assigned each to a null, low, moderate or high cellularity category. The cellularity was defined as follows: null – red blood cells, mesothelial cells or inflammatory cells present only; low cellularity – 10 or fewer cell clusters; moderate cellularity – 11–30 clusters; high cellularity – more than 30 clusters. A cell cluster was defined as five or more cells.11 The tumour cellularity results in smears were as follows: null, 0 cases; low cellularity, one case; moderate cellularity, four cases; high cellularity, 35 cases. Correspondingly, we observed the tumour cellularity in each cell cylinder in the CMA section. The tumour cell cluster results were as follows: null, 0 cases; low cellularity, one case; moderate cellularity, eight cases; high cellularity, 31 cases. The relationship between the tumour cellularity on the smear slides and the cell cylinders in the CMA is shown in Table 1. Immunohistochemistry on the CMA sections Among the 40 tumours, 37 were positive for CK7 (Figure 2d,f). All immunohistochemistry results for the CMA-derived sections were consistent with those of their corresponding original cell block slides. Analysis of EGFR sequences The sequences of the analysed genes obtained from CMA were consistent with those obtained from their corresponding original cell block slides. Figure 3 © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
High output cellular microarray
(a)
(c)
(e)
(b)
(d)
(f)
Figure 2. Images of the prepared cell microarray (CMA). (a) Front view of a CMA (length, 3.6 cm; width, 2.7 cm). (b) Lateral view of a CMA showing that the cell cylinder depth is vaguely visible. Histological results of CMA samples. (c) Haematoxylin and eosin (H&E) staining of CMA samples, showing a total of 40 spots in the section. (d) Immunohistochemistry of CMA sections using cytokeratin 7 (CK7) antibody. (e) H&E staining of a spot at higher magnification. (f) Immunohistochemistry of CK7 staining of a spot at higher magnification. Table 1. Correlation of cellularity of smears and cell cylinders in cell microarays Cell cylinder of cell microarray Smear
Low
Moderate
High
Total
Low Moderate High Total
1 0 0 1
0 1 7 8
0 3 28 31
1 4 35 40
shows the point mutation of CTG to CGG in exon 21 of the EGFR gene. Discussion Development of CMA and shortcomings of traditional methods As a high-throughput research tool, CMA, also known as a chip technique, is similar to TMA. However, there are few methods described for the construction of CMAs. Traditional TMA technology involved the extraction of a tissue cylinder from a tissue block by a punching method,3,12 and previous reports on the construction of CMAs were based on this TMA methodology.6,7 However, there were shortcomings in these methods of construction of CMAs. © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
Figure 3. Sequence of epidermal growth factor receptor (EGFR) gene at codon 858, showing the point mutation of CTG to CGG in exon 21.
First, the thickness of the cell block was usually limited to less than 2 mm after being sectioned for H&E staining; if arrays were not well constructed or some cylinders were shorter than others, some cylinders would be ‘sectioned out’ before others. Therefore, the yield from one traditional CMA block may be limited. Second, the cell blocks can be made with or without agarose. If the cell block was made without agarose, it was difficult to construct a CMA as the cell
47
48
Q. Hu et al.
cylinders dispersed when aggregated.1,3,12 However, the cellularity in cylinders would be lost during the process of making an agarose-mixed cell block. Moskaluk and Stoler6 investigated the difference in cell density between three methods: agarose matrix method, cell pellet method and agarose mould method. The study showed that the agarose matrix method yielded the lowest cell density. The cell pellet method yielded the highest average cell number, as the cells were not suspended in agarose, and hence were not diluted. Invention of a new high-output CMA technique This new method could improve the output of CMA in terms of section yield. As demonstrated, by using appropriate simple tools, it was possible to construct a CMA with long cylinders and to produce up to 1000 sections. By withdrawing the melted wax containing the cell clusters into a plastic tube, long cylinders of the same length are moulded, which can then be implanted into the drilled holes in the recipient block in a vertical orientation, thus producing a CMA of greater depth than can be achieved with cell blocks. We found that the cell cylinders were easily dispersed when traditional TMA aggregation methods or pouring wax methods7 were used in the aggregation stage, and that temperature and time control during the CMA aggregation process were very important. Cylinders dispersed and touched each other at high temperature, and could not homogenize with wax in between at low temperature. To prevent this, we recommend that the recipient block with the transparent box is placed in an incubator at 55 °C for 2 hours. Cylinders are thus maintained in good shape, location and merge well with the recipient paraffin (Figure 2a,b). After aggregation and separation from the transparent box, all 40 cylinders and starting spot were well aligned (Figure 2a,b), which enabled the production of approximately 1000 sections from each cylinder to be used for H&E (Figure 2c,e) and immunohistochemistry (Figure 2d,f) staining. Furthermore, as our results show, the cellularity of the cell spots was maintained throughout and was comparable with the original slide, and immunohistochemistry results were consistent with the corresponding cell block. The DNA sequencing results (Figure 3) showed that the CMA cell maintained its DNA after the cylinders of cell sediment were remoulded and planted in the recipient block.
Advantages and limitations of our high-output CMA technique The advantages of our current approach are summarized as follows: (i) the procedure and facility are simple and no special, expensive instrumentation is required; with only a little practice, an operator can successfully plant cell cylinders into a recipient paraffin block; (ii) we have found an appropriate temperature and time range in which the CMA can be aggregated well without melting of wax, movement of cylinders and dispersion of cells; and (iii) this method has a very high output in terms of sections, which is the greatest improvement over previous methods. There are, however, limitations to this method of CMA construction. For example, where the number of cell clusters in the slide prepared from a fluid is low, i.e. less than 10, there is likely to be insufficient cells for inclusion in a CMA, as the cell clusters will be further diluted in the paraffin and will therefore not be represented in each CMA section. In addition, if the sediment contains a large amount of red blood cells, inflammatory cells or mesothelial cells and only a small proportion of cancer cells, it would be unsuitable for making a high-output CMA, as the small proportion of cancer cells would also be diluted by paraffin and the other cell types. Conclusion As compared with CMA methods based on the punching of cylindrical cores, the CMA preparation technique described in this study is relatively simple, economic and highly efficient. Construction of a CMA is inexpensive, large numbers of sections can be produced and the CMA performs well with respect to the preservation of cell morphology, proteins and DNA. This method would be feasible for use by general research groups employing microarray technology. Acknowledgments This study was funded by: (i) The National Natural Science Foundation of Youth Science Foundation, grant number 81101809; (ii) Shanghai Science and Technology grant number 11140902502; and (iii) Shanghai Science and Technology grant number 13411950802. © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
High output cellular microarray
References 1. Avninder S, Ylaya K, Hewitt SM. Tissue microarray: a simple technology that has revolutionized research in pathology. J Postgrad Med 2008;54:158–62. 2. Battifora H. The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest 1986;55:244–8. 3. Kononen J, Bubendorf L, Kallioniemi A et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4:844–7. 4. Kim MS, Kuppireddy SV, Sakamuri S et al. Rapid characterization of candidate biomarkers for pancreatic cancer using cell microarrays (CMAs). J Proteome Res 2012;11:5556–63. 5. Cardano M, Diaferia GR, Falavigna M et al. Cell and tissue microarray technologies for protein and nucleic acid expression profiling. J Histochem Cytochem 2013;61:116–24. 6. Moskaluk CA, Stoler MH. Agarose mold embedding of cultured cells for tissue microarrays. Diagn Mol Pathol 2002;11:234–8.
© 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 44–49
7. Wen CH, Su YC, Wang SL, Yang SF, Chai CY. Application of the microarray technique to cell blocks. Acta Cytol 2007;51:42–6. 8. Shi Y, He D, Hou Y et al. An alternative high output tissue microarray technique. Diagn Pathol 2013;8:9. 9. Jalal R, Aftab K, Hasan SH, Pervez S. Diagnostic value of clot examination for malignant cells in serous effusions. Cytopathology 2009;20:231–4. 10. Wu X, Zhang J, Zhen R et al. Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models. J Transl Med 2012;10:180. 11. Layfield LJ, Mooney EE, Glasgow B, Hirschowitz S, Coogan A. What constitutes an adequate smear in fineneedle aspiration cytology of the breast? Cancer 1997;81:16–21. 12. Anagnostou VK, Lowery FJ, Syrigos KN, Cagle PT, Rimm DL. Quantitative evaluation of protein expression as a function of tissue microarray core diameter: is a large (1.5 mm) core better than a small (0.6 mm) core? Arch Pathol Lab Med 2010;134:613–9.
49
Copyright of Cytopathology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
