ASPIRE (MTN-020) Laboratory Training Edward Livant MT (ASCP), MPH University of Pittsburgh Medical Center Yaw Agyei MT (ASCP) Johns Hopkins University HPTN/MTN Network Laboratory Protocol Training- MRC Durban.

SSP Lab Section This will be your best resource for MTN 020 Lab questions. Make sure you have the most current version It is available at

Blood Specimen Processing and Testing

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

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

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

ASPIRE Blood Testing - HIV HIV testing Rapid Tests Western Blot RNA Viral Load-algorithm, seroconverters DNA Viral Load-algorithm-only as instructed by NL HIV genotypic Resistance Testing- seroconverters HIV related testing CD4 Count-seroconverters

Some Lessons Learned from VOICE and HEROES HIV algorithm “Sample 2” eliminated. More closely mimic local guidelines. Simplifies counseling messages Eliminates need for repeat visits. Improves completion of algorithm. Less burden on staff/participants without compromising quality.

Some Lessons Learned from VOICE and HEROES (cont.) HIV algorithm Make sure all relevant staff understand every aspect of the algorithm and review it frequently. Keep on top of rapid test and WB kit inventories. Send the NL all your questions-we will respond as soon as possible. (Only mark s as urgent that are truly urgent.)

ASPIRE Plasma Storage PK analyses HIV testing Confirmatory (QA) testing at the MTN NL Resolution of ambiguous endpoints Resistance Testing among seroconverters Future research

Specimen Storage and Shipment Plasma SpecimenDraw volume Minimum Plasma Required Enrollment archive; Routine storage (quarterly, semi-annual, PUEV, Termination Visit) ~10 mL4 mL Follow-up HIV testing algorithm Storage ~15 mL6 mL Post-seroconversion Months 1, 3, 6 and every 6 months thereafter date of a positive rapid(s) ~15 mL6 mL

EDTA Plasma Storage (cont.) Plasma archive samples from failed enrollment visits Once it is confirmed that a participant is not going to be randomized at enrollment visit, 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.

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

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

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

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

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

ASPIRE Urine and Pelvic Tests Urine hCG Urine SDA for GC/CT Rapid Trichomonas test Vaginal Wet Mount (saline and KOH) Vaginal pH Vaginal Gram stain Endocervical Swab for storage Pap Smear

Urine Specimen Processing Urine Collect urine specimens before collection of any pelvic specimens Collect first specimen-not mid stream mL of urine If performing SDA and hCG Separate urine first and hCG Refrigerate urine for SDA

Urine Specimen Processing (cont.) To prevent 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 NL if any unexpected increase in positives noted.

Urine Specimen Processing (cont.) Pregnancy Must use Quidel QuickVue Products-validated for use with product Notify the NL of any concerns with the kit.

Pelvic Specimen Processing Pelvic Specimens (in order of collection; each specimen requires a separate swab; use only swab type listed.)

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. X2 full turns for Endocervical wall collections. Always follow collection order. The first swabs are used for tests that require the most specimen (Trichomonas test)

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

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

Pelvic Specimen Processing (cont.) Vaginal pH Swab vagina and apply to pH paper. Do not insert pH paper into the vagina. pH Indicator Strips (pH range 3.6 to 6.1) These Brands are all acceptable-sites will be asked to purchase directly. Contact NL if this is not possible. Machery-Nagel Baker SP

Pelvic Specimen Processing (cont.) Vaginal Gram Stain for Storage Roll the swab across two slides and pressing the swab as you roll. Allow to air dry (no heat fixing) Do not stain Periodic shipments during the study will be made One slide shipped to Network Lab, one retained onsite in case of problem-make sure to keep two identical duplicate boxes. This will facilitate shipping.

Pelvic Specimen Processing (cont.) Endocervical Swabs One swab from endocervix Stored in 400 µL PBS immediately after collection. 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

Pelvic Specimens Collected before Enrollment Gram Stains and endocervical swabs will be collected during the screening pelvic exam. Specimens from failed screening attempts are destroyed and removed from LDMS. Sites do not need NL approval to destroy samples from participants failed screening attempts.

Pelvic Specimens Collected before Enrollment (cont.) Suggest sites review this monthly and remove samples from failed screening attempts. These samples can be destroyed once confirmed failed screening attempt by clinic records or stored until end of the study. Samples cannot be shipped to the NL.

Other Specimens Self collected vaginal swab Used rings-may be collected by clinician or participant

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. 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

Self-Swab Instructions

Other Specimen Processing Ring Collection for Biofilms Rings will be fixed for 1 to 8 hours in paraformaldehyde then cut into 4 pieces and stored in PBS Shipped to NL for staining with fluorescent in situ hybridization (FISH) and scanning electron microscopy (SEM)

Supplies needed for processing rings for biofilms

Immediately place ring in container with 2.5% paraformaldehyde after removing from vagina Cap tightly and transport the day of collection to the lab for processing and storage

Other Specimen Processing: Used Rings Rings are stored at Month 3, semi-annual visits, when there is an HIV positive rapid If the participant is on product and missed the visit for ring collection, the VR intended for collection/storage should be collected when the participant is provided with a new VR (either at the next scheduled visit, or at an off-site or in-clinic interim visit). If the participant is off product at the time of a visit scheduled for ring collection, do not collect a VR until the next visit where ring collection is required in the protocol.

MTN 005 ring stained with FISH Epithelial cell nucleus: stained with DAPI Lactobacillus stained with lectin

SEM of ring at 3500X Epithelial cells Lactobacillus

SEM of MTN 005 ring at 10,000X

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 Gram Stains Vaginal Swabs Endocervical Swabs Used Rings

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

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.

LAB and CLINIC QA/QC Procedures. Sites should have procedures in place to address queries generated between Lab and clinic ( Chain of Custody -COC) This SOP, should indicate TAT for resolving queries, as well as the contact people. A results COC document should be in place to track all outstanding results.

Any Questions for Network Lab?