MOLECULAR DIAGNOSTICS OF INFECTIOUS DISEASES: STABILITY OF SPECIMENS DURING PREANALYTICS

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1 MOLECULAR DIAGNOSTICS OF INFECTIOUS DISEASES: STABILITY OF SPECIMENS DURING PREANALYTICS

2 OVERVIEW (1) Background (2) Sample collection vials useful for different types of specimens (3) Stability of nucleic acids: data from studies (4) Summary and take home message

3 PRE-ANALYTICS Sample collection Transport (stabilization of DNA/RNA) Sample storage Pretreatment (prior to start of extraction)

4 AVOIDANCE OF PCR INHIBITORS Direct interaction of inhibitors with DNA Direct binding to single-stranded or double-stranded DNA preventing amplification and facilitating co-purification of the inhibitor and the DNA Interference of inhibitors with DNA polymerase Direct interaction with DNA polymerase blocking enzyme activity Interference with co-factors (e.g., reduction of Mg 2+ availability or interference with binding of Mg 2+ to the DNA polymerase)

5 TYPES OF SPECIMENS & POSSIBLE INHIBITORS Type of specimen Blood Urine Feces Possible inhibitors Heme, hemoglobin, heparin Urea Bile salts

6 OVERVIEW (1) Background (2) Sample collection vials useful for different types of specimens (3) Stability of nucleic acids: data from studies (4) Summary and take home message

7 SAMPLE COLLECTION VIALS: BLOOD, CSF EDTA whole blood tubes Sterile 1.5-mL polypropylene (PP) tube with screw-cap for collection of CSF

8 SAMPLE COLLECTION VIALS: SWABS Swab container for viruses and chlamydia, mycoplasma and ureaplasma specimens* Swab container for aerobic and anaerobic bacteria* *Viability of the organisms for at least 48 h at room temperature guaranteed

9 SAMPLE COLLECTION VIALS: SWABS Swab container for endocervical and vaginal swab specimens; e.g., for Chlamydia trachomatis (female patients)* Cell collection device for HPV** *Specimen stable at 2 30 C up to 12 months **Specimen stable at C up to 6 weeks

10 SAMPLE COLLECTION VIALS: URINE, STOOL, ETC. Sterile collection vessel with screw-cap for collection of urine, BAL, nasal secretion, induced sputum, joint aspirate, bone marrow, amniotic fluid Sterile collection vessel with screw-cap for collection of stool

11 OVERVIEW (1) Background (2) Sample collection vials useful for different types of specimens (3) Stability of nucleic acids: data from studies (4) Summary and take home message

12 STABILITY OF HCV RNA Kessler HH et al., JCM, 2001 COBAS AMPLICOR HCV Monitor Test, v2.0 Storage at RT Vacuette EDTA Vacutainer EDTA Vacuette NAST

13 STABILITY OF HIV-1 RNA Amellal B et al., HIV Med, 2008 Blood collected in EDTA tubes processed within 6 h of collection plasma samples investigated Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v1.0 Storage for 7 days

14 STABILITY OF HBV DNA Baleriola C et al., JCM, 2011 Plasma samples A, storage at M20 C B, storage at M70 C

15 STABILITY OF HCV RNA Baleriola C et al., JCM, 2011 Plasma samples A, storage at M20 C B, storage at M70 C

16 STABILITY OF HIV-1 RNA Baleriola C et al., JCM, 2011 Plasma samples A, storage at M20 C B, storage at M70 C

17 STABILITY OF HIV-1 RNA Schulze TJ et al., Transfusion Med, 2011 Blood collected in EDTA tubes (Monovette, Sarstedt) processed within 2 h of collection (spiked) plasma samples investigated COBAS AmpliPrep/COBAS TaqMan HIV-1 Test Detection limits with upper confidence intervals

18 STABILITY OF HIV-1 RNA Bonner K et al., PLOS ONE, 2014 HIV-1 RNA degradation over time and temperature Meta-analysis of 9 studies

19 OVERVIEW (1) Background (2) Sample collection vials useful for different types of specimens (3) Stability of nucleic acids: data from studies (4) Summary and take home message

20 SUMMARY: STABILITY OF HBV DNA, HCV RNA Specimen/sample Temperature ( C) Stability assured EDTA whole blood 25 6 h EDTA plasma 25 1 d EDTA whole blood 4 1 d EDTA plasma 4 7 d EDTA plasma M20 5 yrs (HBV), 1.2 yrs (HCV) EDTA plasma M70 4 yrs (HBV), 9 yrs (HCV) NOTE: Data may be insufficient

21 SUMMARY: STABILITY OF HIV-1 RNA Specimen/sample Temperature ( C) Stability assured EDTA plasma 30 4 d EDTA whole blood 25 7 d EDTA whole blood 4 7 d EDTA plasma 4 14 d EDTA plasma M yrs EDTA plasma M yrs NOTE: Data may be insufficient

22 SUMMARY: STABILITY OF SWABS Specimen Temperature ( C) Stability assured Swabs in tubes containing liquid media Especially designed cell collection device (for HPV) Especially designed swab container for endocervical and vaginal swab specimens 25 2 d wks yr NOTE: Data may be insufficient

23 TAKE HOME MESSAGE Samples are usually stable for at least 24 hours For certain types of specimens, transportation at 4 C may be preferable For swabs, especially designed, commercially available collection devices guarantee stability of nucleic acids without freezing over a longer period (up to 1 year) Nucleic acid stability data sparse (or even none) for several types of specimens Further studies needed

24 ISMD2014 May 29 31, 2014 >250 participants (>25 countries)

25 ISMD2016 May 26 28,

26 THANK YOU FOR YOUR ATTENTION!

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