關(guān)鍵詞： 芯片分析

Tissue microarrays are 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. This technology should not be confused with DNA microarrays where each tiny spot represents a unique cloned cDNA or oligonucleotide. In tissue microarrays, the spots are larger and contain small histologic sections from unique tissues or tumors.

Figure 1 shows an example of a tissue microarray and its construction. The arrays are assembled by taking core needle 揵iopsies?from specific locations in pre-existing paraffin-embedded tissue blocks and re-embedding them in an arrayed 搈aster?block, using techniques and an apparatus developed by Konenen et al. In this way, tissue from 600 specimens can be represented in a single paraffin block.

Figure 2 shows an overview of a completed colon cancer array and higher magnification views of spots from this array and a melanoma array. Stains shown in these examples include hematoxylin and eosin and DAB based-immunoperoxidase. The current Beecher Instruments arraying device is designed to produce sample circular spots that are 0.6mm in diameter at a spacing of 0.7-0.8mm. The surface area of each sample is 0.282 mm2, or in pathologists?terms, about the size of 2-3 high power fields. The number of spots on a single slide is variable depending on the array design, the current comfortable maximum with the 0.6 mm needle is about 600 spots per standard glass microscope slide. New technologies are under development that may allow as many as 2000 or more sections per slide. Using this method, an entire cohort of cases can be analyzed by staining just one or two master array slides, instead of staining hundreds of conventional slides, yet each spot on the array is similar to a conventional slide in that complete demographic and outcome information is maintained for each case so that rigorous statistical analysis can be done as rapidly as the arrays are analyzed.

         
General questions:
What about tumor heterogeneity and tissue arrays ? How many punches do you need to get a good representation ?
Depends on tissue type and the purpose of the study. As a rule of the thumb, making tissue arrays from normal breast epithelium, normal kidney and cancers with high degree of regional heterogeneity probably require 2-4 punches per tissue whereas most cancers are well represented with 1-2 punches. See Camp et al, and Torhorst et al, for technology validation papers discussing the use of tissue arrays in evaluating prognostic markers.

How many specimens can you include per array block ?
Number of specimens per array depends on the size of the punches and the desired array density. Using 0.6 mm punches you can construct tissue arrays with 500 or more specimens per block using regular tissue cassettes. Using 2 mm punches allows construction of tissue arrays with about 50-100 specimens.

How long does it take to make tissue arrays ?
Making tissue arrays should be considered as a project that involves many steps. Array construction itself is relatively fast as compared to the other, necessary preparatory steps that include marking of the representative tissue areas for punching. It is realistic to reserve a couple of weeks for completing tissue array construction project for an array consisting of 400 specimens. Because setting up an arraying project takes a considerable amount of time, it often makes sense to construct multiple replicas of the array blocks simultaneously. Making multiple replicate tissue array blocks is faster and more convenient with the automated arrayer than with the manual arrayer. See Arrayer comparison table for estimated core transfer rates for the automated and manual instruments.

How do you cut sections from the array block ?
Some users prefer to cut sections using regular microtome sectioning techniques, some prefer to use adhesive-coated slides from Instrumedics. The advantage of using coated slides is that even beginners can make good sections with the system and you have precise control over the orientation of the tissue array sections on the microscope slides. If you decide to apply conventional sectioning techniques, briefly heating the block at 35-37 degrees C for about 20-30 minutes (and letting the block to cool back to room temperature) before sectioning improves the section quality.

How many sections can you cut from typical tissue arrays ?
Depending on the height of the original donor tissue blocks you can cut 100-300 sections from tissue arrays. 150 sections is a reasonable expectation for a typical tissue array.

Can you make frozen tissue arrays with Beecher's instruments ?
It is possible to make small frozen tissue arrays with the manual instrument. See Fejzo et al for description. However, this approach is relatively difficult and keeping the tissue array quality high is challenging. Beecher is currently working on a prototype frozen tissue arrayer device.This instrument is not yet available for sale, but you can contact us regarding custom arraying.

Questions about the manual arrayer:
How to align punches in the manual arrayer ?
First, to verify punch alignment, place a blank paraffin block in the block holder and put this complex in position on the arrayer as if it were a recipient block. Press the smaller punch down until it leaves a mark in the paraffin surface. Move the turret to switch the larger sampling punch in position, and again make a mark on the paraffin surface. Adjustment is needed if the marks are not coincidental. Punch holes that appear one in front of the other need front to back adjustment; holes to the side of each other require left-to-right alignment. The blue arrow points to the left-to-right setscrews, the orange arrow indicates the location of front-to-back setscrews.