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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 3  |  Page : 304-309
Making and using inexpensive manually constructed tissue micro-array: Experience of a tertiary care hospital in India


Department of Pathology, G B Pant Hospital, New Delhi - 110 002, India

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Date of Web Publication12-Aug-2009
 

   Abstract 

Background: Tissue micro-array enables the analysis of a large number of tissues simultaneously. Widespread use of this technology is hampered by the high cost of commercial array instruments. We describe our experience of constructing tissue micro-array in a simple method using easily available and inexpensive instruments. Materials and Methods: We used an 11-19 gauge (G) bone marrow trephine biopsy needle/ small sized slotted screwdriver to punch holes in the wax blocks. Cores were taken from donor tissue blocks using a bone marrow trephine biopsy needle and arrayed into host paraffin wax blocks. A detailed database was constructed for each array constructed. Results: The array blocks were used over a period of one year as internal control for immunohistochemistry (IHC), quality control and research. It took about 10 minutes to construct a nine-dot array and about one hour for a 56-dot array. During IHC, the average loss of control dots was less than one per cent. We did not see any loss of antigenicity in the control sections even after four weeks storage. Discussion: Tissue array construction by the technique described here is inexpensive and reliable alternative to automated instruments. Because it is easy to modify the arrays by varying the core size, it is easy to adapt this to individual labs and requirements. We recommend using blocks with cores in 3 × 3 to 5 × 4 grids as controls in IHC and for standardizing antibodies and array blocks with a larger number of cores for research.

Keywords: Controls, immunohistochemistry, India, tissue micro-array

How to cite this article:
Singh DK, Sakhuja P, Gondal R. Making and using inexpensive manually constructed tissue micro-array: Experience of a tertiary care hospital in India. Indian J Pathol Microbiol 2009;52:304-9

How to cite this URL:
Singh DK, Sakhuja P, Gondal R. Making and using inexpensive manually constructed tissue micro-array: Experience of a tertiary care hospital in India. Indian J Pathol Microbiol [serial online] 2009 [cited 2019 Oct 23];52:304-9. Available from: http://www.ijpmonline.org/text.asp?2009/52/3/304/54980



   Introduction Top


The "multi-tissue sausage block" was first described by Battifora et al [1] in 1986 and later modified into a "checkerboard block'. [2] The tissue micro-array, as we know today, was developed by Kononen et al [3] in 1998.The basic premise of a tissue array is relocating tissue from many archival histological paraffin block, on to one recipient block, making it possible to study many different tissues quickly and efficiently thus saving time, effort and reagents. The multitissue arrays can be used for quality, internal control in immunohistochemistry (IHC) and in research.

Quality control in IHC is one of the major problems and challenges of diagnostic pathology. To ensure accuracy of procedure, it is essential to have a strict quality control program.[4],[5] In most IHC labs which use a good quality control program, one separate slide with a tissue known to be positively immuno-reactive with the test antibody is used as a control (external control) for each immunostained slide. It is a costly method, increases the processing time and doubles the amount of antibody used. Another method commonly used is use of one control slide (external control) with a whole batch of immunostained slides. Both these methods do not guarantee correct working of the antibody worked because the test section and control section do not undergo exactly the same procedure. Using separate positive control tissue for test cases is fraught with technical and logistic difficulties of maintaining a record of positive cases, getting sections cut and storing the unstained slides. Use of multi-tissue blocks in IHC solves these problems and makes quality control an easy and efficient task. The control arrays used for IHC are smaller and have lesser number of cores than those used for research. [1]

However useful the technology of tissue micro-array might be, its widespread use is hampered by high cost of commercial array instruments. We developed an easy method for constructing tissue micro-arrays which was modified from the method described by Pan et al, [6] and have been using these arrays since one year in our pathology department. These arrays are used as positive internal controls in IHC and for standardization of new antibodies. We have also used these arrays in research where more than 50 tissues from different cases are mounted on the same slide and immunostained using a tiny fraction of the amount of antibody normally required. These arrays can be made in any department using easily available and inexpensive instruments.


   Materials and Methods Top


To construct tissue micro-array blocks we collected tissues retrospectively from gross specimens kept in our records and prospectively from new specimens received in our daily practice over a period of 15 days. Some tissues/tumors infrequently seen were retrieved from paraffin blocks from our records. The tissues were selected on the basis of their known immunoreactivity. The tissues selected for constructing arrays and the antigens expressed by them is given in [Table 1]. A piece of tissue with maximum dimensions 20 20 0.4 mm was cut (smaller size if the tissue available was small) and embedded in paraffin block. If it was not possible to get one large piece of tissue, smaller pieces were cut and embedded in one block. The blocks with the tissues were stored in an air tight container till the micro-array was constructed. For taking tissue from archived paraffin blocks in our record, the hematoxylin eosin stained slide was overlaid on the donor block and the area of interest was marked. To construct a micro-array, a wax block without any tissue was made by routine procedure. Then we used an 11-19 gauge (G) bone marrow trephine biopsy needle to punch cylindrical holes in the wax block in a grid like manner. Hidalgo et al [7] used bone marrow aspiration needles modified by a grinding machine for punching holes and taking tissue cores. We found that instead of bone marrow trephine biopsy needles, a set of small sized slotted screwdrivers purchased from a local hardware shop were better at punching holes in the wax block. The holes were 3 mm in height and one to three mm in diameter depending on the size of array and number of cores needed. Core needle biopsies with a diameter ranging from one to three mm were taken from donor tissue blocks using a bone marrow trephine biopsy needles of the appropriate gauge. The tissue cores were arrayed into host paraffin wax blocks which had a surface area of 25 25 mm. Depending on the use of the array being constructed the number of tissue cores in one block measuring 25 mm 25 mm ranged from 9-56 cores. In a 45 25 mm block about 100 cores of 1 mm and 77 cores of 2 mm diameter could be arranged. The tissues were arrayed in3 3 to 8 7 dots in smaller block and 12 9 to 11 7 dots in larger block [Figure 1]. A pointer core was embedded on one corner for easy orientation. To combine the wax from donor cores with wax form recipient blocks the block was reheated in an oven at 60 o C (sixty degree centigrade) for 10 minutes. [7],[8],[9] We calculated the approximate number of sections from each block which were needed for one month. Four micron thick sections were cut from the array blocks using a rotatory microtome and the slides were stored in a dry environment till use. The exposed surface of the array blocks was dipped in wax to cover the surface and the blocks were also stored in airtight containers till further use. We also tried covering the exposed cut surface of array blocks with one sided sticky cellotape to prevent tissue oxidation and we found that this method was as efficient as covering the surface with wax. For IHC, the section from the test tissue was lifted on the slide which already had a micro-array and both were stained together. Antigen retrieval was done in buffered citrate solution at pH 6.0 in a microwave. The retrieval time was as per manufacturer's recommendations, the most common being two minutes at 50% power followed by two minutes at full power. A detailed database was constructed using MS Excel for each different array constructed. The excel document had four worksheets. The first worksheet, [Figure 2], documented the exact position of each tissue core as in the array with name of the tissue, the second worksheet, [Figure 3], had a list of all the tissues in the array and the antigen expressed by them, the third worksheet, [Figure 4], had the photograph of each tissue core stained by hematoxylin and eosin stain (HE), the fourth worksheet, [Figure 5], had patient data which included the histopathology reference number, age and gender of the patient in the same layout as the array, and [Figure 6] gives a flow chart for TMA construction.


   Results Top


The array blocks were used as internal control during IHC over a period of one year for 1200 sections. [Figure 7],[Figure 8],[Figure 9] One paraffin block containing prospectively collected tissue measuring 20 20 0.4 mm yielded 50 cores of one mm each and 25 cores of two mm each for making micro-array blocks. Building one array block with nine cores took about 10 minutes. About 150 sections could be cut from one array block however the number varied from 100-200. The limiting factor was the tissue cores taken from the stored paraffin blocks in our records because these blocks had been sectioned many times in the past. Loss of dots during sectioning of the array was prevented by chilling the block on ice before cutting and using fresh disposable blades. The loss during antigen retrieval was prevented by keeping the slides on hot-plate for at least one hour before staining and in some cases by using poly-L-lysine coated slides. The average loss by using these methods was less than one per cent. The positive control dots stained brightly as shown in Figure 7],[Figure 8],[Figure 9]. Because most of the arrays were constructed in such a way that there were two control tissues for each antibody, non-specific staining and failure of staining was easily noticed. Cases in which the control tissue and the test sections both did not show staining the IHC were repeated.

Another use of array blocks in our lab has been to standardize new antibodies. By using the array blocks we were able to test up to 50 different tissues displaying a range of antigen densities at the same time, quickly and without using much reagent. The staining reaction ranged from very strongly positive to negative staining. This reduced the task of searching for positive control; getting sections cut and significantly reduced the processing time and antibody consumption during standardization. A modification of this method which we have also tried successfully is that section from a known positive control tissue is also mounted on the same slide as the micro-array. This ensures that at least two known positive controls one in the array and one in the section from positive tissue are tested at the same time. This provides a very stringent test for the antibody during standardization procedure. This method was especially useful when constructing lesion-specific array blocks which had controls for a panel of antibodies used for diagnosing specific lesions like GIST (array block having controls for CD 117, SMA, CD 34, S-100 and Desmin), melanoma, soft tissue tumors etc.


   Discussion Top


The use of an internal positive control is the best method for quality control in IHC. The multitissue controls are the most effective internal controls. Using external control sections for quality control in IHC or for standardizing antibodies is time consuming, laborious, increasing the processing time and does not ensure that the procedure for staining was performed correctly or the reagents were dispersed in the correct order on the test case. The multi-tissue array as an internal control solves these problems. The technique described here is inexpensive and reliable alternative to automated instruments for constructing tissue micro-arrays. The diameter of the control dots can be varied from one to three mm to meet specific requirements.

The cost of array machine is one factor which is and will be a strong deterrent to its use in general practice in India for quiet some time. The array machines form Beecher Instruments, San Prairie, Wisconsin, USA costs anywhere from 480,000 INR (12,000 USD) to 1,700,000 INR (42,000 USD). Even a manual tissue array system such as that available form IHC World costs upward of 48,000 INR (1200 USD) with recurring costs of disposable parts. The array constructed by these will remain unaffordable except to some research institutes in our country. Some reports have described simpler methods of array construction as an alternative to commercial machines. [7],[8],[9],[10] We tried to find a method which did not require specialized instruments like cutting and grinding machines, which is inexpensive to implement, without recurring costs and was flexible enough be modified as per the requirements of the lab with respect to the number of cores in one block. The method we use in our lab produces excellent arrays at a fraction of the cost of other machines. The whole array set we used had a total price of 1200 INR (30 USD). We agree that a limitation of our arrays is the maximum number of tissue cores that can be manually fitted in one block of 25 mm 25 mm (1 mm cores - 56). This is less as compared to commercial machines which can fit up to 200, 1 mm cores in the same area. Another limitation is time to construct the array. It takes about 10 minutes to construct a nine-dot array and about one hour for a 56-dot array which is about twice that of commercial machines. But we think that these will be minor issues compared to the advantages of having an array when finances are important concerns or where limited number of samples needs to be studied, till the time the labs can afford a commercial array machines.

Majority of labs, when standardizing a new antibody, use a known positive control tissue and by varying the dilutions of the antibodies and retrieval time, if the control gives a positive reaction it is assumed that the antibody is working correctly. While standardizing a new antibody by varying the dilutions and time of antigen retrieval it is important that a known positive control gives a positive reaction. However, it is also important that tissues which are supposed to be negative are indeed non-reactive. Also there might be some tissues which stain with less intensity but are positive for the antigen. Testing many different tissues with varying antigen densities is rarely practiced as it is a cumbersome and time consuming task and uses a large amount of antibody. The tissues embedded in the array blocks we constructed had a positive control for the test antibody and other tissues with varying range of antigen densities, which made standardizing new antibodies an easy and efficient task using minimal amount of antibody.

When using tissue micro-array blocks an important question is whether a 1 mm or 2 mm or 3 mm dot be representative of the entire tissue/tumor? While using tissue micro-arrays it is important to remember that this technology is not used for diagnosing cases but to study parameters of many different cases simultaneously. Studies using tissue arrays have confirmed all clinico-pathologic correlations established previously by examining large tissue sections. [11],[12] In a study, Camp et al . showed that just two 0.6 mm cores from one block provided the same information on estrogen receptor indices as that provided by whole sections. [12] In some cases where it is essential to sample the case extensively, more than one dot from the same block can be sampled. Also with our technique the diameter of the core in the array block can be varied from one to three mm or more which can give a good representation of the entire tumor and will also make sure that the original block still preserves the morphology of the case.

Another cause for concern was loss of antigen staining in sections stored for a long time as reported in previous studies. [13],[14],[15],[16] It is said that oxidation of the antigens in the tissue on prolonged storage leads to loss of immunogenicity. To minimize loss of antigenicity we cut the sections required as controls once each month and the exposed surface of the array blocks was then coated with paraffin before being stored. The unused sections at the end of one month were discarded. A time period of one month was chosen based on previous studies which state that storage of unstained sections for up to four to six weeks gives adequate staining. These studies report that beyond six weeks some loss of antigenicity is seen. [13],[14],[15],[16] Other reasons for choosing four weeks was also based on the reporting schedule in our department, TMA construction time, and manpower available in our department. Because we have been collecting tissue for use in array blocks regularly and maintaining a record, there is abundant availability of tissues for constructing micro-arrays. In some cases if it is found that a known positive control is giving negative staining it will be a prompt to repeat the staining procedure for the antigen and if the negative staining of the known positive control persistent the block should not be used any more. [16]

Because it is easy to modify the number of cores in the array, it is easy to adapt this to individual labs and requirements. This technique can also be used to construct micro-arrays for use in research where more than 50 different tissues/tumors in one section can be stained together using one to two drops of the antibody. To illustrate the point, if we want to study 50 cases with five different antibodies one option is to get five sections cut from each of the 50 tumor blocks and stain 250 sections individually. Other option is to construct an array with tissues from 50 tumors which will take about one hour and stain one section with each of the five antibodies which is five sections in total. This tremendously reduces the time, effort and the antibody used and all the cases undergo same procedure ensuring uniformity of staining. We recommend using blocks with cores arranged in 33 to 54 grids for use as controls in IHC and for standardizing antibodies. This number of cores (nine-20) is enough to have controls for most antibodies and controls for lesion-specific antibody panels. When using these array blocks for research the number of cores in one block can be increased and a larger block can also be used. We used 100 cores of one mm and 77 cores of two mm diameter in one block. Using this number of cores can accommodate from 30 (three cores per case) to 100 (one core per case) cases in one block.

To conclude, the construction of array by the technique described here is easy, inexpensive, takes a reasonable amount of time and reduces antibody consumption. It is a viable alternative to extremely expensive commercial array instruments. The array blocks can be used as internal controls in IHC and can also be used to standardize antibodies. This technique can be adapted for use in research where tissue from up to 100 cases can be arrayed on a single slide and processed simultaneously by H and E, special stains or IHC.


   Acknowledgments Top


We sincerely thank Mrs. R. Gopinathan, Mrs. Deepa Arora, Mr. Chandermohan Kumar, Ms. Hina Shareef Quraishi and Mr. Jai Bhagwan Verma for helping in grossing of specimens, tissue processing, cutting of paraffin tissue blocks, staining of sections and performing immunohistochemistry of cases.

 
   References Top

1.Battifora H. The multitumor (sausage) tissue block: Novel method for immunohistochemical antibody testing. Lab Invest 1986;55:244-8.  Back to cited text no. 1  [PUBMED]  
2.Battifora H, Mehta P. The checkerboard tissue block. An improved multitissue control block. Lab Invest 1990;63:722-4.  Back to cited text no. 2    
3.Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, et al . Tissue micro-arrays for high-throughput molecular profiling of tumor specimens. Nat Med 1998;4:844-7.  Back to cited text no. 3    
4.Maxwell P, McCluggage WG. Audit and internal quality control in immunohistochemistry. J Clin Pathol 2000;53:929-32.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Rhodes A, Jasani B, Balaton AJ, Miller KD. Immunohistochemical demonstration of oestrogen and progesterone receptors: Correlation of standards achieved on in house tumours with that achieved on external quality assessment material in over 150 laboratories from 26 countries. J Clin Pathol 2000;53:292-301.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Pan CC, Chen PCH, Chiang H. An easy method for manual construction of high density tissue arrays. Appl Immunohistochem Mol Morphol 2004;12:370-2.  Back to cited text no. 6    
7.A Hidalgo, P Piρa, G Guerrero, M Lazos, M Salcedo. A simple method for the construction of small format tissue arrays. J Clin Pathol 2003;56:144-6.  Back to cited text no. 7    
8.Wang L, Deavers MT, Malpica A, Silva EG, Liu J. Tissue micro-array: A simple and cost-effective method for high-throughput studies. Appl Immunohistochem Mol Morphol 2003;11:174-6.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Datta MW, Kahler A, Macias V, Brodzeller T, Kajdacsy-Balla A. A simple inexpensive method for the production of tissue micro-arrays from needle biopsy specimens-examples with prostate cancer. Appl Immunohistochem Mol Morphol 2005;13:96-103.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Pires AR, Andreiuolo Fda M, de Souza SR. TMA for all: A new method for the construction of tissue micro-arrays without recipient paraffin block using custom-built needles. Diagn Pathol 2006;1:14.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Avninder S, Ylaya K, Hewitt SM. Tissue micro-array - A simple technology that has revolutionized research in pathology. J Postgrad Med 2008,Vol. 54;Isssue 2.  Back to cited text no. 11    
12.Camp RL, Charette LA, Rimm DL. Validation of tissue micro-array technology in breast carcinoma. Lab Invest 2000;80:1943-9.  Back to cited text no. 12  [PUBMED]  
13.Wester K, Wahlund E, Sundstrφm C, Ranefall P, Bengtsson E, Russell PJ, et al . Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p. 53 protein and MIB1 antigen. Appl Immunohistochem Mol Morphol 2000;8:61-70.  Back to cited text no. 13    
14.Bertheau P, Cazals-Hatem D, Meignin V, de Roquancourt A, Verola O, Lesourd A, et al . Variability of immunohistochemical reactivity on stored paraffin slides. J Clin Pathol 1998;51:370-4.  Back to cited text no. 14    
15.Van den Broek LJ, Van de Vijver MJ. Assessment of problems in diagnostic and research immunohistochemistry associated with epitope instability in stored paraffin sections. Appl Immunohistochem Mol Morphol 2000;8:316-21.  Back to cited text no. 15  [PUBMED]  
16.Packeisen J, Buerger H, Krech R, Boecker W. Tissue micro-arrays: A new approach for quality control in immunohistochemistry. J Clin Pathol 2002;55:613-615.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]

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Correspondence Address:
Puja Sakhuja
Department of Pathology, G. B. Pant Hospital, Jawahar Lal Nehru Marg, New Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.54980

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

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