Central Pathology Review and Tissue MicroArrays. Dr Lisa Storer Children s Brain Tumour Research Centre Nottingham

Transcription

1 Central Pathology Review and Tissue MicroArrays Dr Lisa Storer Children s Brain Tumour Research Centre Nottingham

2 What is a tissue microarray? Tissue microarrays (TMAs) consist of paraffin blocks in which up to 1000 separate tissue cores are assembled to allow multiplex histological analysis

3 Why should we use TMAs? Limitations in molecular clinical analysis on FFPE Small patient sample size Limited availability/expense of diagnostic reagents Cumbersome nature of procedures TMAs use novel sampling approach to produce samples of regular size and shape Allow high throughput analysis

4 Advantages of using TMAs Allow the analysis of many patient samples simultaneously Conservation of precious tissue samples Improved internal experimental control Reduced consumption of antibodies and other reagents Wide applicability to many molecular techniques including RNA and protein expression analysis, IHC, ISH, FISH, in situ PCR Cheaper and easier to perform technique on 1 slide containing 100 samples than 100 separate slides

5 Disadvantages Need accurate record keeping to prevent misplacing of cores Small cores sampled may not be representative of the whole tumour Particularly true in heterogenous cancers e.g. prostate adenocarcimoma and Hodgkin lymphoma Metastatic Medulloblastoma c ) d e ) highly positive regions Patches of positivity negative regions

6 Tissue Microarray Procedure A hollow needle used to remove 0.6mm diameter core from region of interest 1.0, 1.5 and 2.0mm diameter punches available Insert core into a recipient paraffin block in a precisely spaced array pattern using co-ordinates Section TMA block on microtome Mount on microscope slide Stain and analyse H&E IHC FISH

7 Building TMAs a Donor block b c Spotted H&E Recipient block

8 Importance of Central Pathology Review All cases submitted to a study should under go central pathology review by a pathologist Review of diagnostic slides H&E Any IHC Clinical information Other supporting techniques e.g. EM, FISH Once diagnosis has been confirmed, regions of interest should be spotted onto H&E by pathologist

9 Important considerations Always perform a central pathology review Spot multiple cores of representative areas label each core e.g. a, b, c Design TMA block before starting procedure Record co-ordinates of cores to enable each core to be tracked back to the original spot on a H&E Always include a reference core to allow orientation of block and sections Don t make your block symmetrical Always leave enough material in the donor block to allow use for further diagnostic testing

10 Manual Have to line up block and slide Punch core Move co-ordinates of stage for next sample Semi-automated Touch screen for set up of spacing, positioning Automated stage 4 recipient blocks at a time Automated Types of TMAers Automatic record keeping so no misplacing of cores

11 Automated Tissue Microarrayers I Spot region of interest on donor block with an oil pen Insert donor blocks into tray in order Load blocks into machine Pen marking is recognised by camera Puncher acquires sample and moves it to recipient block

12 Automated Tissue Microarrayers II Scan donor block and corresponding H&E Overlay images and mark on donor block image where to core Scan block and produce image on screen Mark on donor block image where to take core from Back fill donor block with paraffin to prevent collapse of small blocks

13 Case Study Nottingham have consented 2 families for PM examination, collection and storage of tissue for CCLG registration Both had been receiving treatment at NUH Both CCLG consented whilst alive: biopsy, debulk and bloods CCLG registered Performed limited PM for specific tissue collection Dependent on clinicians relationship with family Family must sign CCLG PM consent form as well as hospital PM consent form Consent received from the living does not cover the deceased