On slide controls for immunohistochemistry via tissue micro array (TMA) cores Institute for Pathology, University Hospital Erlangen Dr. Carol I. Geppert Feb. 2017

Contents Introduction – why TMA? vs. Insight Erlangen – TMA projects On-Slide controls On-Slide techniques Summary

Introduction – why TMA ? New level in quantity (>2000 cores/day) High quality punching by digital TMA annotations Statistics in automated & arbitrary tables Available for a lot of different issues (and tissues) Economical & time-efficient (~6 cores/min) Little tissue needed Barcodes (QM) Creation of clones (same layout and sampling)

Introduction – why TMA ? + in combination with digital image analysis (DIA): Space-efficient storage over longer periods of time No bleach-out (e.g. FISH and CISH) Independent of microscope and observer Portable and easy to share (www)

Introduction – why TMA ? Hardware liability, sensitivity and lack of speed Big data (up to 10GB/slide) Digital pathology still not common yet No use in daily routine! Expensive hardware, software, maintenance/support Special requirements for thickness and fixation Vulnerability to artifacts (bubbles)

Insight Erlangen: Scanner and ngTMA Flash 250 GrandMaster Analysis 250 slides 60 D, 12 R blocks 80-550 Spots/TMA 0.6 - 2 mm Ø 0.6 1.0 1.5 2.0

ngTMA interface HE bunny alignment annotation

Insight Erlangen: Projects using TMAs ngTMA for colon cancer >1100 patients; >12.000 cores, DIA: >600 pat. analyzed in IHCs: >10.000 cells/pat. The SAPE project: ngTMA for breast cancer, >5000 patients qualified; >10.000 patients planed) The 2nd Immunoscore project in colon cancer (>400 patients; digital analyzed for CD3 & CD8 …) Immune response in bladder cancer (>300 patients; IHC: GATA3, CD45, CD3, CD8)

Insight Erlangen: Colon cancer TMA: 391 spots and 132 pat Insight Erlangen: Colon cancer TMA: 391 spots and 132 pat. (triplets)/block

Insight Erlangen: Colon Cancer TMA targeting heterogeneity with high accuracy TMA

ngTMA + digital image analysis Colon cancer: CDX2 ~2000 cells /spot tc triple punches in 640 pat. ~ 3.8 mio. 2 stains (CDX1, CDX2) in tc, im, no (x6) ~ 23,04 mio. ! individual counted & qualified cells

On-Slide controls – what for? In diagnostics there is an increasing pressure of time and cost QM increases in routine (QM pressure) On slide controls are needed for certified and accredited institutes of pathology (DAKKS) For thousands of IHC stainings new solutions are needed to avoid explosion of cost & time Erlangen: >70.000 IHCs/year in routine

On-Slide controls – what is needed in certified laboratories? On slide controls are necessary and must be cross checked with all antibodies before using Depending on IHC staining system (autostainer) “controls by run” will not pass QM standards There will be a transition period to improve laboratory workflow  TMA techniques provide a solution for those issues

On-Slide controls – How does it work? What is the case mix, mostly used IHCs? Field of diagnostics? Do we have consultant diagnostics? (Caution: Paraffin! Fixation!) Which autostainer? Do we use drop zones? Do we have all tissues in the house? Do we have to perform cross checks? Design “the perfect multi-block” for your IHCs:  E.g.: Liver, Placenta, ovary, testis, colon, skin, lymph node a/o spleen, lung, breast, kidney, (prostate and urothel, melanoma, etc.)

On-Slide controls – How does it work? Cross check all antibodies Unusual reaction/ No reaction Economic impact: QM! Use your routine FFPE workflow ! Maybe 20 blocks for each tissue sample 3 punches from each block ~60 cores Procedure for each tissue (e.g. 10 different tissues) = 600 cores  2 TMA blocks vs. 200 blocks for cross check (e.g. 40 Antibodies) More economical with fewer runs (autostainer is always the bottleneck in the lab) Normal Reject tissue Use tissue

On-Slide controls – How does it work? Producing multiple clone blocks with control tissue Useable tissue Depending on donor thickness ~150 slides/block x(20 D x25 R clones=500) = 75.000 “on slides”) + QM: daily feedback from the pathologists

On slide controls – techniques special molds were designed “to act from necessity”

On slide controls – techniques special molds -12 mm feed size, 16 cores max. -paraffin brink for secure sectioning 5 mm thick for any donor thickness By now: no special on slide-software tool available for GTMA

On slide controls – techniques Puncher for carrier/recipient tissue, 10mm feed size

On slide controls – techniques the “on slide multi block” (OSMB) Punch and create recipient tissue (not only as matrix!) (e.g. liver or spleen) Punch controls by GTMA OSMB Caution: US patents! Create it for your lab or for others (non profit) Separate the controls by new embedding (4 times faster, thanks to Dr. Scheel!) 48 blocks by 1x GTMA run !

On slide controls – techniques Big cover slip needed (caution: drop zones!) On slides need optimal conditions for temperature & paraffin polymer! Enough space even for step sectioning on one slide Optional carrier tissue (16 cores max.) no melting loss, re-embedding possible Prof. A. Jungbluth, NY

Summary …… what’s coming next ? …… Research: With GTMA & DIA huge numbers of tissue samples can be provided very fast Diagnostic: On slide controls can be produced in an economic way saving tissue, time and resources Especially interesting for certified & accredited institutes of pathology with a lot of IHC stainings (DAKKS) The “on slide multi-block” offers many possible combinations for control tissue regarding high QM standards Future aspects: QM pressure will increase and on slide tools are needed! An upgrade for the GTMA for OSMB would be nice to have! Funding: On slides are the best way to get the GTMA back on the market and into routine labs! …… what’s coming next ? ……

The Institute for Pathology, Erlangen

Thank you for your attention! Mont Blanc, ascent from southwest, near summit before sunrise, 09/2014