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

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

3. 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)

4. 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)

5. 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)

6. Insight Erlangen: Scanner and ngTMA
Flash 250 GrandMaster Analysis
250 slides
60 D, 12 R blocks
Spots/TMA
mm Ø

7. ngTMA interface
HE bunny
alignment
annotation

8. Insight Erlangen: Projects using TMAs
ngTMA for colon cancer
>1100 patients; > cores,
DIA: >600 pat. analyzed in IHCs: > cells/pat.
The SAPE project: ngTMA for breast cancer, >5000 patients qualified; > 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)

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

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

11. 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

12. 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: > IHCs/year in routine

13. 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

14. 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.)

15. 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

16. 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) = “on slides”)
+ QM: daily feedback from the pathologists

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

18. 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

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

20. 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 !

21. 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

22. 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 ? ……

23. The Institute for Pathology, Erlangen

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