1. The value of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection E RIC S ELVA *, V ERONIQUE H OFMAN *$, F REDERICK B ERTO *, S ANDRA M USSO $, L AURENT C ASTILLO %, J OSE S ANTINI %, P IERRE D ELLAMONICA # AND P AUL H OFMAN * $ * INSERM 02-15, IFR 50, $Department of Pathology, %Department of Otorhinolaryngology, and #Department of Infectious Disease, University of Nice, Nice, France Pathology （ February 2004 ） 36 （ 1 ）, pp. 77-81

2. Introduction Mycobacterium tuberculosis in 2004 High infection Drug resistant Methods for detecting mycobacteria Cultural identification Ziehl-Neelsen （ ZN ） stain PCR Ile de France Provence Alpes Côte d’Azur

3. Introduction Cultural identification Standard method Antibiotic sensitivity Fresh specimen Take up to 6 weeks Ziehl-Neelsen （ ZN ） stain Acid-fast bacilli Not sensitive Not identification species

4. Introduction Polymerase chain reaction （ PCR ） Perform rapidly High sensitivity High specificity >20 μ m in thickness False positive False negative

5. Introduction LCM ┼ PCR on FFPE Increase efficiency Overcome problem FFPE Formalin fixed paraffin embedded

6. Materials Lymph nodes from 49 case of inflammatory necrotizing granuloma Department of pathology of Pasteur Hospital, University of Nice, France 1990 – 1998 Ziehl-Neelsen stain Cultural identification Positive2549 Negative240

7. Methods Laser capture microdissection 5 μ m tissue section Deparaffinised and H&E stain Microdissect using LCM microscope （ PixCell Ⅱ, Arcturus Engineering, Alphelys, Paris, France ） DNA extraction Clean with xylene

8. Before microdissection

9. After microdissection

10. Image on plastic cap

11. Methods Whole section DNA extraction （ 1 ） 10 whole cross 5 μ m tissue sections Deparaffinised by 1mL xylene, 20 minutes, 65 ℃, then 14000 rpm, 2 minutes, remove supernatant, twice Washing 1mL 100 ％ ethanol, 5 minutes, then 14000 rpm, 5 minutes, twice Dried in a speed vacuum

12. Methods Whole section DNA extraction （ 2 ） Digestion buffer ： Distilled water Proteinase K ； 100 μ g/ μ l ； Sigma, Paris Tris HCl ； 20mM ； pH8.0 EDTA ； 20mM ； Sigma SDS ； 2 ％ Digestion 72 hours, 55 ℃

13. Methods Whole section DNA extraction （ 3 ） 14000 rpm, 5 minutes The supernatant ＝ template for PCR DNA concentration and purity ： Absorbency at 260 and 280 λ （ Ultraspec 2000, USA ）

14. Methods PCR （ 1 ） 1 μ l DNA template ； 50mM KCl ； 10mM Tris HCl, pH8.3 ； dH 2 O ； 1.5mM MgCl 2 ； dNTPs （ 200 μ M ； Invitrogen, Paris, France ）； primer 1 ； primer 2 ； Taq Polymerase （ 1.25U ； Invitrogen ）； final 50 μ l

15. Methods PCR （ 2 ） Sequence of primer ： Primer 1 （ IS6110 5 ’ ）： 5 ’ – 3 ’ ： CCTGCGAGCGTAGGCGTCGG Primer 2 （ IS6110 3 ’ ）： 5 ’ – 3 ’ ： CTCGTCCAGCGCCGCTTCGG 16S ribosomal RNA gene of M. tuberculosis

16. Methods PCR （ 3 ） Condition for PCR ： 94 ℃ 5 minutes 67 ℃ 1 minutes 72 ℃ 40 seconds 35 cycles Perkin – Elmer Thermal Cycler ； CA, USA

17. Methods Electrophoresis 8 μ l PCR product 2 μ l bromophenol blue （ Ficoll ） 1.5 ％ agarose, ethidium bromide – stained gel 135 V 30 minutes

18. Methods DNA isolation control ： Amplification of β -actin gene β -actin 5 ’ ： 3 ’ – AGCGGGAAATCGTGCGTG β -actin 3 ’ ： 5 ’ – CAGGGTACATGGTGGTGC Invitrogen, Paris, France

19. Methods PCR control ： 5 cases of sarcoidosis 5 cases of lymph nodes from HIV patients who presented with atypical mycobacteriosis （ with positive culture for M. avium intracellulare ） Paraffin block without tissue

20. Results PCR from microdissected granulomas and correlation with PCR from whole sections （ 1 ） Microdissected granulomas Whole sections Positive4545 Negative44

22. Results PCR from microdissected granulomas and correlation with PCR from whole sections （ 2 ） ※ 4 cases were negative both ※ The signal obtained from microdissected granulomas was weaker than the signal obtained from whole sections ※ All negative controls were negative both ※ The latter case had been embedded more than 8 years

23. Results Correlation of PCR from microdissected granulomas with acid fast stained tissue PCR positive PCR negative ZN stain positive 250 ZN stain negative 204

24. Results Correlation of PCR from microdissected granulomas with culture PCR positive PCR negative Culture positive 454 Culture negative 00

25. Discussion ※ Infectious diseases ＝＞ Granulomas ※ Detection of granuloma ＝＞ Diagnosis of Tuberculosis ※ Determination the causative agent ＝＞ Special stain ＊ Sensitivity ＊ Specificity

26. Discussion ※ Cultural identification ： ＊ Mycobacterial infection may not have been clinically suspected and specimens not obtained for culture. ※ Molecular diagnosis of M. tuberculosis ＝ ＞ PCR （ whole section ） ※ PCR positive rate ＝＞ DNA quantity ※ PCR false negative ＝＞ 2 ％～ 19 ％ （ whole section ）

27. Discussion ※ The PCR performed from whole sections may present some disadvantages ： ＊ An excessive material-consuming method on small specimens. ＊ Increasing the effect of tissue inhibitors or contamination. ※ Lesion ＜＝＞ PCR result ＊ Relatively low amount of specimen ＊ PCR-based analysis for amplification

28. Discussion ※ LCM ：

29. Discussion ※ PCR detection of M. tuberculosis ： ＊ Sensitivity ＝＞ 92 ％ ＊ Specificity ＝＞ 100 ％ ※ LCM ＋ PCR ： ＊ Useful on small specimen ＊ Reduce false positive due to contamination ※ Successful confirmation of M. tuberculosis ＊ (×) Tissue DNA ＊ (o) Digestion for obtain M. tuberculosis DNA

30. Discussion IS6110 ： CCT GCG AGC GTA GGC GTC GG CTC GTC CAG CGC CGC TTC GG TB11/12 ： ACC AAC GAT GGT GTG TCC AT CTT GTC GAA CCG CAT ACC CT TB2A/2B ： GAG ATC GAG GTC GAG GAT CC AGC TGC AGC CCA AAG GTG TT

31. Discussion IS6110TB11/12TB2A/2B 94 ℃ 5 min 94 ℃ 30 sec 67 ℃ 1 min 60-56 ℃ 1 min 72 ℃ 40 sec 72 ℃ 1.5 min 35 cycles 36 cycles 60-57 ℃ ： 6 ； 56 ℃ ： 12

32. END