1. HOPE (MTN-025) Laboratory Training Edward Livant MT (ASCP), MPH University of Pittsburgh Medical Center

2. Goals For this Presentation. HOPE Lab Testing Menu Describe processing for different specimen types. Logistical issues Generate questions and identify areas that require attention. Note: HIV testing and Hair Collection covered seperately

3. SSP Lab Section This will be your best resource for MTN 025 Lab questions. Make sure you have the most current version It is available at www.mtnstopshiv.orgwww.mtnstopshiv.org

4. Schedule of Lab Testing

5. HOPE Blood Testing HIV testing-covered in depth in separate presentation Rapid Tests Geenius RNA Viral Load-algorithm, seroconverters HIV related testing CD4 Count-seroconverters

6. HOPE Blood Testing Syphilis serology RPR Treponemal Confirmation (TPHA or other) Hematology FBC-with 5 part differential Chemistry ALT AST Creatinine

7. HOPE Plasma Storage HIV testing Confirmatory and QA testing at the MTN LC Resolution of ambiguous endpoints Resistance Testing among seroconverters PK testing is still to be determined. Future research

8. HOPE Urine and Pelvic Test Urine hCG Urine NAAT for GC/CT Rapid Trichomonas test Vaginal Wet Mount (saline and KOH) Pap Smear

9. Other Specimens Self collected vaginal swab Used rings-may be collected by clinician or participant Hair for PK-same collection procedure as VOICE

10. Urine Specimen Processing Urine Collect urine specimens before collection of any pelvic specimens Collect first specimen-not mid stream If performing NAAT and hCG Separate urine first and hCG Refrigerate urine for NAAT Collect a separate sample for urine culture if indicated

11. Urine Specimen Processing (cont.) To prevent GC/CT Contamination Change gloves between specimens Open one specimen at a time Use sterile screw top containers Do not introduce non-sterile items (such as pipettes) into the sample Contact the LC if any unexpected increase in positives noted.

12. Urine Specimen Processing (cont.) Pregnancy Must use Quidel QuickVue Products-validated for use with product

13. Pelvic Specimen Processing Pelvic Specimens (in order of collection; each specimen requires a separate swab; use only swab type listed.) SampleSwab Type Trichomonas Rapid TestDacron-provided with kit Vaginal Swab for Wet MountDacron or Cotton Pap SmearLocally Defined

14. Pelvic Specimen Processing (cont.) Specimen collection: VERY IMPORTANT-get enough sample on the swabs. Take the time to roll the swab several time and allow the swab to become saturated. X3 full turns for vaginal wall collections. Always follow collection order. The first swabs are used for tests that require the most specimen (Trichomonas test)

15. Pelvic Specimen Processing (cont.) OSOM Trichomonas Test Detection of Trichomonas protein antigen A capillary flow dipstick Takes approximately 12 minutes to perform Collect vaginal swab and place in a clean tube with no additives until testing can be performed. Swabs can be held at room temperature for 24 hours or refrigerated for 36 hours before testing.

16. Pelvic Specimen Processing (cont.) Wet Prep Rotate the swab over lateral vaginal wall several time to collect sufficient specimen Place swab in glass or plastic tube with 6 drops of saline for transport to testing area Use swab to transfer specimen to 2 slides Add 1 drop of KOH to one slide, sniff immediately for amine odor. Add coverslip to view yeast Add coverslip to second slide to view for clue cells

17. Pelvic Specimen Processing (cont.) Note: if motile trichomonads are noted on the saline wet prep, these can be reported to the clinician. If Trichomonas vaginalis is seen on the wet mount but the OSOM Rapid Trichomonas test is negative, report as positive by wet mount only

18. Other Specimen Processing: Used IVR Ring Collection for Residual Drug Analysis Same process as ASPIRE, except addition of “ring code” to track order of use (i.e. 1.0, 2.0, 3.0, etc.) Participants will be asked to make their best effort to use rings in order and place in the return bag with appropriate ring code Illiterate participant may require modified system – e.g. colors in addition to codes It is expected that some ring codes will be unknown at the time of ring return – participants will not be reprimanded for this!

19. Other Specimen Processing: Used IVR Collect all used ring(s) from the participant Make note of rings inserted at time of collection on RCI CRF If no ring inserted at time of collection, note date last inserted on RCI CRF For each ring returned, capture information from participant on use (qualitative/quant) on ring tracking log Confirm that any rings returned have the labeling of PTID, date and visit returned, and appropriate ring code. If the ring code is not known for a given ring(s), clinic staff should line through the Ring Code field and write “UKN” on the label on the bag.

20. Other Specimen Processing: Used IVR Ring Collection for Residual Drug Analysis Rings can be rinsed with water only Make sure they are dry before storage Place in a clean bag for storage Use 99.9 for unknown ring codes

21. Other Specimen Processing: Used IVR Enter ring code in the time field in LDMS:

22. Other Specimen Processing: Used IVR Shipping Residual drug analysis results expected TAT Rings returned at M1→Results back by M3 Rings returned at M2, M3→Results back by M6 Rings returned at all other quarterly visits→ Results back by next quarterly visit

23. Other Specimen Processing: Used IVR Shipping This will require all stored rings to be shipped weekly starting with first M1 visit Store rings so that they can be easily shipped Create new containers in LDMS each week Mark to ship in LDMS to reduce sorting

24. Other Specimen Processing: Self Collected Vaginal Swabs One Dacron swab self collected by participant (see section 6 of SSP for collection procedures) Swab must be collected while old ring is still in place. Exception: at enrollment, collect swab before insertion. Stored with no additive Freeze within 8 hours of collections If specimens come in late, will need to be processed the same day Store on site until notification from study team or Network Laboratory

25. Self Collected Vaginal Swabs: Guide found on MTN-025 Study Implementation Materials Page: http://www.mtnstopshiv.org/node/7330

26. Blood Specimen Processing Most testing of blood specimens done onsite for HOPE Are not specified methods-sites choose and validate methods. In these cases, there will be a review of SOP’s and oversight of the testing by the Network Laboratories and SMILE Specimen Handling requirements are locally defined Plasma storage specimens that are shipped will have mandated handling criteria.

27. Blood Draw Volumes Refer to Blood Draw Volume Tables-these are approximate volumes Sites must determine tubes to be drawn that will satisfy local testing requirements and yield adequate volumes for testing done at Network Laboratories Volumes must be consistent with Informed Consent Process

28. Specimen Quality is Key Problems with Specimen Quality Include: Hemolysed Serum Affects many chemistry tests Can cause false elevation of AST, ALT etc… Clotted EDTA Tubes Affect numerous hematology parameters Underfilled EDTA Tubes May dilute specimens for hematology

29. Specimen Quality is Key (con’t) Phlebotomy technique and handling will affect specimen quality Proper training (and retraining when problems are noted) is key. Some issues Trauma caused by technique-too much needle movement during draw, etc… Proper needle gauge Allow alcohol to dry Properly filled tubes (use appropriate size) Properly connected phlebotomy equipment Syringes-do not draw back too hard Hemolysis may also occur during transport- handle specimens with care

30. Flow Cytometry Flow Cytometry (Seroconversion) CD4 Positive T-Lymphocytes EDTA Whole Blood Testing done per site SOP’s Generally done within 48 hours or per site SOP

31. Chemistry Chemistry (Serum) Liver Function: AST+ALT Kidney Function: Creatinine Performed per local SOP Testing done same day or as allowable per site SOP

32. Hematology Hematology (FBC or CBC) (EDTA Whole Blood) Hemoglobin Hematocrit Mean Corpuscular Volume Platelets White blood cell count with differential Absolute neutrophil count Percent neutrophils Absolute lymphocyte count Absolute monocyte count Absolute eosinophil count Absolute basophil count Per Site SOP Testing done same day of collection or as acceptable by site SOP

33. Syphilis Serology RPR for screening Very Non specific Treponemal Confirmation TPPA, TPHA or other Testing done per local SOP on serum or plasma These are batched per local SOP-usually at least weekly

34. EDTA Plasma Storage Kept at room temp: freeze within 4 hours Refrigerated: freeze within 24 hours We recommend that sites routinely refrigerate these samples All plasma storage will be 1 mL aliquots Note-the term “archive” will only apply to baseline enrollment samples. All other follow up samples will be referred to as “storage”.

35. EDTA Plasma Storage (cont.) Plasma archive samples from failed enrollment visits Once it is confirmed that a participant is not going to be enrolled, the plasma archive should not be stored. The sample can be discarded without NL approval. If another enrollment visit is successful, the plasma archive will be collected at that visit.

36. Specimen Storage and Shipment To simplify shipping procedures, MTN 025 will identify four types of specimen storage in LDMS in the “other SPEC ID” field for plasma. “RPS” for Enrollment and Routine Plasma Storage “CON” for HIV Seroconversion Confirmation Plasma “SER” for Seroconverter Plasma

37. Specimen Storage and Shipment All sites will use LDMS to track specimen storage and shipments Specimen shipment schedules to follow Specimens stored in LDMS Plasma Vaginal Swabs Used Rings Hair

38. Specimen Storage and Shipment LDMS reconciliation criteria will be set before study starts and will not change mid-study. The NL will work with site labs and clinic staff to resolve issues each month Resolving these issues is crucial to maintaining the integrity of specimen storage

39. LDMS Reconciliations Sites should have monthly meetings between Lab and Clinic data team. These meetings should address any pending SCHARP QC reports. Lab team should export LDMS, as soon as changes are made. Clinic Data team should re-fax CRF’s as soon as corrections are made.

40. LDMS Reconciliations (cont.) LDMS reconciliations will check the following (other criteria may be added before first report): No LDMS match: CRF states storage, no specimen found in LDMS No CRF match: specimen found in LDMS does not have corresponding entry on CRF Visit code and dates do not agree Errors with specimen codes Storage information