Sample management in clinical trials.pptx

In this presentation sample management in Clinical trials has been explaned.

1. Sample management in clinical trials Mohammad Shafiul Alam, PhD Scientist Emerging Infections and Parasitology Laboratory icddr,b    

2. Clinical Trial A clinical trial is a type of  clinical research study . A clinical trial is an experiment designed to answer specific questions about possible new treatments or new ways of using existing (known) treatments. Clinical trials are done to determine whether new drugs or treatments are safe and effective.

3. Good Clinical Practices (GCP) IRB approved protocol Informed consent prior to collect any information and sample SOP and trained people

4. Informed consent elements Sample collection procedure Volume/number of sample (s) collected Duration of the participation Types of diagnostic tests to be done Future use of sample and storage Sample shipment to other country (if required) Anonymity, right to withdraw and refusal Compensation Future use of information Signed by the volunteer or legal parents (if minor 11-17 years, take verbal assent)

5. Sample requirements Documentation Date and time of each step Collection method Processing method Volume or other metric Refrigerator/freezer logs Shipping records Chain of custody Sample type Blood urine Sputum/saliva stool Collection Method Timing Tube/microtainer Media Processing Storage conditions Shipping materials Shipping methods Sample tracking (identification/codes)

6. Types of Lab in Clinical Trials

7. Sample processing workflow (If we perform any test before storage) Fresh EDTA blood sample (500µl) Centrifuge at 2200 RPM for 5 mins Removal of plasma & buffy coat & replacement with same volume of additive Store at 4 º C (for maximum 21 days) Wash out the additive from RBC with 0.9% saline, centrifuge at 1,000/g for 5 min, and aspirate the supernatant (wash until supernatant become clear) Addition of two volumes of Glycerolyte to one volume of RBC Store at -80 º C

8. Sample processing workflow (Only for storage) Fresh EDTA blood sample (500µl) centrifuge the fresh EDTA blood at 1000/g for 5 mins Removal of plasma & buffy coat Wash with 0.9% saline, centrifuge at 1,000/g for 5 min, and aspirate the supernatant (wash until supernatant become clear) Addition of two volumes of Glycerolyte to one volume of RBC Transfer glycerolyte -treated red blood cells into cryovials and store at −80°C

9. Slide Preparation

10. Staining Number of Slides Giemsa stock solution (mL) Final Volume, made up with Buffered water pH7.2 (mL) 1 0.5 5 2-3 1 10 4-6 2 20 Cover smear by 10% v/v Giemsa for 45 – 60 mins Rinse the slide by flooding Tap water/Drinking water/buffered water Dry slide For screening follow standard procedures. After enrolment prepare one study slide. Follow same procedures for every visit

11. Good quality Bad quality Slide quality

12. Sample collection Day 0 and day of recurrence: 7.5 ml venous blood (EDTA) All other days 450µl capillary blood (EDTA) Avoid squeezing the finger Invert container at least 10x

13. All samples need to be stored in the fridge until processing Processing must happen as soon as possible Sample storage

14. Sample processing Time point after Enrolment D0 D1 D2 D3-6 1 7 14 21 28 35 42 49 56 63 Day of recurrence Treatment                             Schizontocidal treatment X X X                       Primaquine treatment 1 X X X X                     Procedures                             Symptom Questionnaire X X X X X X X X X X X X X X Medical Examination X X X x X X X X X X X X X X Pregnancy test X                           Capillary blood collection (450 µl) X 5 X X X 3 X X X X X X X X X X 5 Venous blood collection (7.5 ml) 6 X                         X G6PD biosensor X   X X 4 X     X         X   Malaria microscopy X X X   X X X X X X X X X X Hb X X X X 3 X X X X X X X X X X Cell free Hb X                           Biomass X                         X Drug levels X       X                 X Parasite PCR/Genotyping X                         X Host genotyping & RBC polymorphism X                           Serology X       X     X         X  

15. Sample processing Item Days Amount Aliquots Comments Storage Source Dried Blood Spot All days 25 µ l 1 Only if sufficient sample is available With desiccant at +4 ° C to -20 ° C Whole Blood Cell Free Hb Day 0 and recurrence Aliquot 150 µ l, spin, store 100 µ l 1 Spin 2x at full speed for 5 minutes each -20 ° C initially, then -80 ° C Plasma Biomass Day 0 200 µ l 1   -20 ° C Drug levels Day 6 1ml 1 Only intervention arm -20 ° C Serology Day 0 and recurrence Any left over sample 1-2   -20 ° C Parasite PCR Day 0 and recurrence 1.5ml 2   - 20 ° C RBC pellet Host PCR Day 0 1.5ml 2   - 20 ° C

16. Venous samples 7.5 ml EDTA vacutainer On day 0 and day of recurrence

17. Venous samples Cell free Hb Biomass Drug levels Serology Host genotyping Parasite genotyping Microscopy slide SD Biosensor Dried Blood spot Centrifugation, top speed/ 5min .

18. Cell free Hb Needs to be centrifuged 2x : Centrifuge Vacutainer at top speed for 5 minutes Aliquot the top 150µl plasma into an Eppendorf Centrifuge Eppendorf at top speed for 5 minutes Dispense the top 100µl into a Cryovial

19. Capillary samples 450µl in EDTA microtainer Every day unless venous blood is collected

20. Capillary samples Drug levels Serology Host genotyping Parasite genotyping Microscopy slide SD Biosensor Dried Blood spot Centrifugation, top speed/5 min.

21. Reducing the risk of P. vivax after falciparum infections in co-endemic areas - a randomized controlled trial Blood processing procedures Mohammad Golam Kibria

22. Age groups >17 years 11-17 years 1-10 years Follow up Enrollment(D0), Day- 1 to 7, 14, 21, 28, 35, 42, 49, 56, 63 and Day of recurrence

23. Time point after Enrolment D0 D1 D2 D3-6 1 7 14 21 28 35 42 49 56 63 Day of recurrence Treatment                             Schizontocidal treatment X X X                       Primaquine treatment 1 X X X X                     Procedures                             Symptom Questionnaire X X X X X X X X X X X X X X Medical Examination X X X X 2 X X X X X X X X X X Pregnancy test X                           Capillary blood collection (450 µl) X 5 X X X 3 X X X X X X X X X X 5 Venous blood collection X                         X G6PD biosensor X   X X 4 X     X         X   Malaria microscopy X X X   X X X X X X X X X X Hb X X X X 3 X X X X X X X X X X Cell free Hb X                           Biomass X                         X Drug levels X       X                 X Parasite PCR/Genotyping X                         X Host genotyping & RBC polymorphism X                           Serology X       X     X         X   1 Intervention arm only 2 Day 3 only 3 Only if clinically warranted 4 Only if blood sample is collected for measurement of Hb 5 Only if no venous blood is collected

24. Pregnancy testing Pregnancy testing (please check manual for exact procedures):

25. Biosensor and HB Preparation Specimen Collection Operation

26. Biosensor and HB

27. Biosensor and HB

28. Biosensor and HB_ QC

29. Biosensor and HB_ QC

30. Blood Smear Prep. Use reverse pipetting to transfer blood Dry slide Fix thin film with methanol Thick film must not be fixed

31. Slide Preparation

32. Staining Number of Slides Giemsa stock solution (mL) Final Volume, made up with Buffered water pH7.2 (mL) 1 0.5 5 2-3 1 10 4-6 2 20 Cover smear by 10% v/v Giemsa for 45 – 60 mins Rinse the slide by flooding Tap water/Drinking water/buffered water Dry slide For screening follow standard procedures. After enrolment prepare one study slide. Follow same procedures for every visit

33. Slide quality Good quality Bad quality

34. Sample collection Day 0 and day of recurrence: 7.5 ml venous blood (EDTA) All other days 450µl capillary blood (EDTA) Avoid squeezing the finger Invert container at least 10x

35. Venous samples 7.5 ml EDTA vacutainer On day 0 and day of recurrence

36. Venous samples Cell free Hb Biomass Drug levels Serology Host genotyping Parasite genotyping Microscopy slide SD Biosensor Dried Blood spot Centrifugation, top speed/ 5min .

37. Cell free Hb Needs to be centrifuged 2x : Centrifuge Vacutainer at top speed for 5 minutes Aliquot the top 150µl plasma into an Eppendorf Centrifuge Eppendorf at top speed for 5 minutes Dispense the top 100µl into a Cryovial

38. Capillary samples 450µl in EDTA microtainer Every day unless venous blood is collected

39. Capillary samples Drug levels Serology Host genotyping Parasite genotyping Microscopy slide SD Biosensor Dried Blood spot Centrifugation, top speed/5 min.

40. Sample Processing Workflow: Heterozygous Females Fresh EDTA blood sample (≤7.5 mL) Centrifuge at 2,200 rpm for 5 min Removal of plasma & buffy coat & replacement with equal volume of additive = 2.5% glucose, 0.9% sodium chloride, 0.027% adenine, 0.75% mannitol Store and transfer at 4 º C (for a maximum of 24 hours) Wash out the additive from RBC with 0.9% saline, centrifuge at 1,000 x g for 5 min and aspirate the supernatant (wash until supernatant becomes clear) Addition of two volumes of Glycerolyte to one volume of RBC [see details in Menzies SOP 01] Transfer Glycerolyte -treated red blood cells into cryovials and store at −80°C

41. Sample Processing Workflow: All Other Fresh EDTA blood sample Centrifuge the fresh EDTA blood at 1000 x g for 5 min Removal of plasma & buffy coat Wash with 0.9% saline, centrifuge at 1,000 x g for 5 min, aspirate the supernatant (wash until supernatant becomes clear) Addition of two volumes of Glycerolyte to one volume of RBC Transfer Glycerolyte -treated red blood cells into cryovials and store at −80°C

42. Sample storage All samples need to be stored in the fridge until processing Processing must happen as soon as possible

43. THANK YOU