Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides Flavio de Oliveira Lima1,2, Joaquim Soares de Almeida2, Henrique de Oliveira Costa2, Nathanael F Pinheiro, Jr2, Celina Tizuko Oshima2, Marcello F de Franco1,2 1
Department of Pathology, Botucatu Medical School, UNESP, Sa˜o Paulo, Brazil, 2Department of Pathology, Escola Paulista de Medicina, Federal University of Sa˜o Paulo – UNIFESP, Sa˜o Paulo, Brazil
The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1.2 mm apart. Section of 4 mm were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer’s recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (.80%), almost complete (75–79%), or partial (50–74%). The commercial adhesive tape group presented lesser total loss (1.1 versus 6.4%), almost complete loss (2.2 versus 3.5%), and partial loss (2.1 versus 3.8%) than the silanized slide group (ANOVA, P,0.05). The sum of total and almost complete losses in the silanized slide group was 9.9%, greater than the losses in slides using commercial adhesive tapes (3.3%) and less than reported and considered acceptable in the literature (10–30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research. Keywords: Immunohistochemistry, Sample losses, Tissue microarray
Introduction In 1986, Batiffora proposed the first tissue microarray (TMA) method,1 which was quickly adopted and improved.2,3 It was in 1998 that Kononen4 developed the basis of the technique currently used, with the use of a fixed-base mechanical device (Beecher Instruments, Silver Spring, MD, USA), which amplified the possibilities of the method. Next, other authors suggested alterations in the technique5 and in slide analysis.6 Currently, a number of publications exist that sought to diminish the cost of research involving TMA, principally through the adoption of alternative methods of block construction.7–11
Correspondence to: Flavio de Oliveira Lima, Department of Pathology, Botucatu Medical School, UNESP, Rubia˜o Junior District, Botucatu, Sa˜o Paulo, CEP 18618-000, Brazil. Email:
[email protected]
ß National Society for Histotechnology 2011 DOI 10.1179/2046023611Y.0000000001
One of the problems related to the TMA technique is the detachment of the samples after sectioning the block. Researchers who initially worked with the technique, using a fixed-base device, used adhesive tape to fix the samples in place and reported low cylinder losses.4,5,12,13 In contrast, several other researchers have reported good results without its use, emphasizing cost reduction.14–16 Other works have highlighted the poorer performance of the method using adhesive tape in fluorescence in situ hybridization and chromogenic in situ hybridization, or for samples in immunohistochemical reaction.17,18 The objective of this work was to compare the percentage of TMA tissue block sample loss prepared for use in a fixed-base device, on commercial slides (Instrumedics Inc., Hackensak, NJ, USA) using adhesive tape or silanized slides.
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Materials and Methods Materials Eight blocks of varying tissues (heart, spleen, lung, gut, central nerve system, breast fat, kidney, and liver) from autopsies, fixed in formalin and paraffin embedded, and two blocks from cell-blocks collected by fine-needle aspiration were randomly selected from the archives of Department of Pathology, Sao Paulo Federal University (UNIFESP). One block of liver was used as a cylinder marker.
TMA construction The construction of the TMA block followed the recommendations of Kononen,4 using a fixed-base device (Beecher Instruments) and 1 mm diameter needles. The block was elaborated with 108 mirrored cylinders (in duplicate), in a total of 216 samples, placed at 1.2 mm spacing between the cylinders centers. Sections of 4 mm were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides. For silanization, the glass slides were immersed in neutral detergent for 2 hours and then washed in running water to remove any excess detergent. Next, they were sequentially immersed in three baths of 100% alcohol, followed by immersion in 4% silane in acetone (v/v). After a further three baths in 100% alcohol, the slides were dried in a heater at 58uC. The other 30 slides were adhered to commercial glass slides using adhesive tape (Instrumedics Inc.) and submitted to solvents and UV light baths according to the manufacture’s recommendations. One pair of glass slides from each group was stained with hematoxylin–eosin, slides 1 and 15 in the sectioning sequence. The remaining slides were submitted to the immunohistochemical technique described below.
Immunohistochemistry After being maintained in a heater at 57uC overnight, the slides were immersed in three baths of xylene, the first for 30 minutes and the second and third for 5 minutes each, followed by three more baths of 5 minutes in 100% ethylic alcohol and washing in running water for 5 minutes. Antigenic recovery was realized in citrate buffer, pH 6.0, in a microwave oven for 30 minutes at 750 W. After cooling, the slides were washed in running water for a further 5 minutes. Endogenous peroxidase blocking was realized by four 5-minute baths in 3% hydrogen peroxide (v/v). Next, the slides were again washed in running water for 5 minutes, immersed in phosphatebuffered saline (PBS) buffer, pH 7.4, and incubated 70
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with anti-vimentin primary antibody (Clone V9, Code M 0725; DakoCytomation, Carpinteria, CA, USA) at 1:100 dilution in 1% bovine serum albumin, overnight. After washing in PBS buffer, the slides were incubated in the secondary antibody of LSABz System HRP kit (DakoCytomation) for 30 minutes, and washed in PBS. Colors were developed with 3,39diaminobenzidine (DAB; Sigma Chemical Co., St Louis, MO, USA). The slides were again washed in running water, counter-stained with hematoxylin for 3 minutes, washed in running water, dehydrated in ethylic alcohol, diaphanized in xylene, and mounted with a coverslip and resin.
Evaluation of tissue loss in the cores Silanized glass slides using commercial adhesive tapes and silanized glass slides only were analyzed separately by two pathologists to evaluate loss by sample detachment. For standardization, total loss was considered as the absence of more than 80% of the core area (A); almost complete loss, the absence between 75 and 79% of the core area (B); losses between 74 and 50% were considered partial (C); and when above 50% of core area was present in the slide, it was not considered a loss (D) (Fig. 1).
Statistical analysis The data were tabled and submitted to the t test and to ANOVA, followed by the post hoc Bonferroni test, when using proportions and not actual numbers.
Results The silanized glass slides using commercial adhesive tapes showed a total loss of 1.1% and an almost complete loss of 2.2%. The use of silanized only glass slides showed a total loss of 6.4% and an almost complete loss of 3.5%. The commercial adhesive tape group presented smaller losses compared to the silanized only glass slide group (Fig. 2). There were not any differences in the quality of stain (hematoxylin–eosin or immunohistochemistry) between the two tested groups. T test analysis showed the superiority of the method using commercial adhesive tape (t test, P,0.0001). The rate of 9.9% for the sum of total and almost complete losses for silanized only glass slides (Fig. 3) was greater than the combined losses of the glass slides prepared using commercial adhesive tape (3.3%). Visual analysis of the slides revealed that, despite the statistical differences found, the two methods were equivalent and both were adequate for research use (Fig. 4).
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Figure 1 Evaluation of cores: total loss was considered as the absence of more than 80% of the core area (A), almost complete loss, the absence between 75 and 79% of the core area (B); losses between 74 and 50% were considered partial (C); and when above 50% of core area are present in the slide, it was not considered a loss (D).
Discussion The advent of the TMA technique provided research of immunohistochemical markers in pathology with a robust tool that permits the evaluation of dozens to hundreds of tissue samples from numerous patients in a single block, optimizing costs by saving time and reagents, as well as providing uniformity among the results, with the realization of reactions in a single assay.4
Figure 2 Comparison of sample losses between silanized glass slides using commercial adhesive tape and silanized only slides. There were less partial (ANOVA, p,0.05), almost complete (p,0.01), or complete (p,0.001) sample losses in silanized glass slides using commercial adhesive tapes as compared to silanized glass slides only.
The use of a fixed-base device, like the Beecher model, became the gold standard for TMA block preparation in many research services around the world. However, one limiting factor is the high cost of implantation and maintenance. Aimed at reducing procedural costs, several authors have described alternative TMA block construction techniques.7,9,10,18
Figure 3 Comparison of complete or almost complete sample losses in silanized glass slides using commercial adhesive tape and silanized slides only. Silanized glass slides with commercial adhesive tapes lose less samples as compared to silanized slides (3.3 vs 9.9% respectively; t test, p,0.0001).
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Figure 4 Magnified view of silanized glass slides using commercial adhesive tape (A) and silanized slides only (B).
One of the major problems of histological sections derived from TMA blocks is the loss of samples due to detachment during processing. Standardization of the use of adhesive tapes, together with special slides, solvent solutions, and ultraviolet lights was realized in order to deal with this problem.4,5,12,13,19 This procedure increased the technical difficulty of sectioning the TMA blocks, demanding complementary technical training, with accompanying cost increases for maintenance and supplies; as well as reports regarding technical preparation for the realization of fluorescence in situ hybridization, chromogenic in situ hybridization, and even immunohistochemical tests.17,18 On the other hand, good results have been reported when using conventional silanized slides, which do not require the use of commercial adhesive tapes.14,16 Our group addressed a previous study comparing sample loss between adhesive tapes, a modified silanized technique and silanized only slides, but with the use of 2.0 mm needle, anti-cytoqueratin 18 antibody and fewer samples from liver tissue.20 In this study, comparisons were made using the TMA technique regarding material submitted to immunohistochemical tests, on histological sections using commercial adhesive tape and silane. Analysis revealed the superiority of commercial adhesive tape technique. However, despite the fact that the combined total and almost complete losses for silanized only glass slides (9.9%) were greater than the losses for glass slides using commercial adhesive tapes (3.3%), this value is lower than that reported in the literature (10–30%) and is considered acceptable from a methodological point of view.14,16 Considering that the use of duplicate mirrored samples prevents and diminishes 72
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relevant data loss, an even smaller loss should have occurred in the duplicated cylinders. Another question that should be considered is the fact that the use of commercial adhesive tapes, with their solvents, UV lights, and own specific resin, requires specific training for the technicians involved, aside from the training required to construct the TMA blocks. During this study, there were sample losses when using commercial adhesive tapes due to artifactual problems resulting from a lack of experience of the histological technicians, a fact that obliged us to repeat the assays, with the consequent expenditure of extra time and materials. TMA block sectioning with the use of silanized only glass slides did not present these problems, since the technical team already had experience and refined expertise in this technique, which is similar to the routine of histological technicians. Finally, Milanes-Yearsley14 reported difficulties in staining and background in immunohistochemical reactions when using commercial adhesive tapes, a fact not observed in this study.
Conclusion The present study demonstrated that the use of commercial adhesive tapes resulted in a lower rate of sample losses compared to conventional silanized only glass slides. On the other hand, conventionally prepared glass slides, not using commercial adhesive tape, revealed highly satisfactory results and demanded less specific training. This results in the economy of financial resources without significant losses in the quality of the scientific information.
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Acknowledgement The authors would like to thank Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo for ‘Apoptose em Tumores’ project no. 2004/09932-4.
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