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We are a group of people who like frozen tissue arrays, work with frozen tissue arrays and want to make the best use of frozen tissue arrays. We would like to share our experience in frozen tissue arrays with you. Hope you may also share your experience with us.

About Frozen Array

Paraffin tissue arrays are produced by a method of re-locating tissue from conventional histologic paraffin blocks such that tissue from multiple patients or blocks can be seen on the same slide. This is done by using a needle to biopsy a standard histologic sections and placing the core into an array on a recipient paraffin block. This technique, originally described by in 1987 by Wan, Fortuna and Furmanski in Journal of Immunological Methods. They published a modification of Battifora's "sausage" block technique whereby tissue cores were placed in specific spatially fixed positions in a block. The technique was popularized by Kononen and colleagues in the laboratory of Ollie Kallioneimi after a publication in Nature Medicine in 1998. (1)

Tissue microarrays (TMAs) are widely used to analyze gene expression in multiple pathological samples on a single slide. However, one difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. Stronger fixatives can partially degrade proteins and nucleotides. (Schoenberg Fejzo and Slamon 2001) Tissue microarrays provide a high-throughput tool for the study of gene dosage and protein expression patterns in a large number of individual tissues for rapid and comprehensive molecular profiling of cancer and other diseases, without exhausting limited tissue resources.

Tissues are not fixed before embedding, and sections from the array are evaluated without fixation or post fixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. Frozen tissues are recommended for optimal recovery of DNA, RNA, and protein.

With the improvment of microarray technology a new kind of TMAs was developed by using frozen tissues embedded in tissue embedding compound. Frozen tissue arrays are produced by using a needle to biopsy frozen tissues embedded in O.C.T. compound and placing the core into an array on a recipient O.C.T. compound block.

This technology helps expedite discovery of the novel targets important in cancer treatment by providing a tool for high-throughput screening of multiple tumor tissues using immunohistochemical, in situ hybridization, and fluorescent in situ hybridization (FISH) analyses.

Tissue is not fixed prior to embedding, and sections from the array are evaluated without fixation or post-fixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. Unlike paraffin tissue arrays which can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, the disclosed methods allow optimal evaluation by each technique and uniform fixation across the array panel. The disclosed arrays work well for DNA, RNA, and protein analyses, and have significant qualitative and quantitative advantages over existing methods.

Advantage
1. Uniform procedure
Using tissue array technology, each tissue sample is treated in an identical manner. Like conventional formalin-fixed paraffin embedded material, tissue microarrays are amenable to a wide range of techniques including histochemical stains, immunologic stains with either chromogenic or fluorescent visualization, in situ hybridization (including both mRNA ISH and FISH), and even tissue micro-dissection techniques. For each of these protocols, conventional sections can have substantial slide to slide variability associated with processing 300 slides (for example, 20 batches of 15 slides). The tissue microarrays allow the entire cohort to be analyzed in one batch on a single slide. Thus reagent concentrations are identical for each case, as are incubation times and temperatures, wash conditions, and antigen retrieval, if necessary.

2. Saving the assay volume
Only a very small amount of reagent is required to analyze an entire cohort using tissue array. This advantage raises the possibility of use of tissue microarrays in screening procedures (for example in hybridoma screening), a protocol that is impossible using conventional sections. It also saves money when reagents are costly.

 

What are the advantages?

There are numerous advantages to this technology including:

1 .Amplification of a scarce resource
2. Experimental uniformity
3. Decreased assay volume
4. Does not destroy original block for diagnosis

  • A standard histologic section is about 3-5mm thick, with variation depending on the submitting pathologist or tech. After use for primary diagnosis, the sections can be cut 50-100 times depending on the care and skill of the sectioning technician. Thus, on average, each archived block might yield material for a maximum of 100 assays. If this same block is processed for optimal microarray construction it could routinely be needle biopsied 200-300 times or more depending on the size of the tumor in the original block (Theoretically it could be biopsied 1000's of times based on calculations of area, but empirically, 200-300 is selected as a conservative estimation) Then, once tissue microarrays are constructed, they can be judiciously sectioned in order to maximize the number of sections cut from an array. The sectioning process uses a tape-based sectioning aid (from Instrumedics Inc.) that allows cutting of thinner sections. Optimal sectioning of arrays is obtained with about 2-3 µm sections. Thus instead of 50-100 conventional sections or samples for analysis from one tissue biopsy, the microarray technique could produce material for 500,000 assays (assuming 250 biopsies per section times 2000 2.5 µm sections per 5mm array block) represented as 0.6 mm disks of tissue. Thus this technique essentially amplifies (up to 10,000 fold) the limited tissue resource.
  • Using this technology, each tissue sample is treated in an identical manner. Like conventional formalin-fixed paraffin embedded material, tissue microarrays are amenable to a wide range of techniques including histochemical stains, immunologic stains with either chromogenic or fluorescent visualization, in situ hybridization (including both mRNA ISH and FISH), and even tissue micro-dissection techniques. For each of these protocols, conventional sections can have substantial slide to slide variability associated with processing 300 slides (for example, 20 batches of 15 slides). The tissue microarrays allow the entire cohort to be analyzed in one batch on a single slide. Thus reagent concentrations are identical for each case, as are incubation times and temperatures, wash conditions, and antigen retrieval, if necessary.
  • Another significant advantage is that only a very small (a few µl) amount of reagent is required to analyze an entire cohort. This advantage raises the possibility of use of tissue microarrays in screening procedures (for example in hybridoma screening), a protocol that is impossible using conventional sections. It also saves money when reagents are costly.
  • Finally, there are occasions where the original block must be returned to the patient or donating institution. In these cases the block may be cored a few times without destroying the block. Then upon subsequent sectioning, it is still possible to make a diagnosis, even though tissue has been taken for array-based studies. This is illustrated in the adjoining figures.

 

References

  1. Yale Cancer Center/Pathology Tissue Microarray Facility Home http://tissuearray.org/yale/tisarray.html
  2. Schoenberg Fejzo, M. and D. J. Slamon (2001). "Frozen tumor tissue microarray technology for analysis of tumor RNA, DNA, and proteins." Am J Pathol 159(5): 1645-50.
  3. Frozen tissue microarray technology for analysis of RNA, DNA, and proteins. United States Patent. Slamon et. al.