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IMPAACT 2010 (VESTED) Laboratory Considerations Laboratory Technologist: Sikhulile Moyo IMPAACT Lab Center Rep: William Murtaugh QUICK REVIEW OF STUDY ACTIVATION: LABORATORY REQUIREMENTS 2 IMPAACT Laboratory Center (LC) approval of local laboratory site readiness, based on confirmation of the following: All laboratories • Confirmation of maternal and infant HIV testing algorithms • Confirmation of access to Abbott Real-Time HIV-1 RNA PCR assay at local VQA-certified laboratory • Confirmation of current IATA training certificates for at least two individuals 3 IMPAACT Laboratory Center (LC) approval of local laboratory site readiness, based on confirmation of the following: Additionally for non-US site laboratories • DCLOT approval of laboratory readiness based on: – Completion of relevant IMPAACT Critical Items from DAIDS Audit Action Plan, if applicable – Approval of protocol analyte list (PAL) – Confirmation of acceptable EQA status (pSMILE, VQA, IQA) for protocol analytes for both primary and backup laboratories – Submission of current maternal and pediatric reference ranges (must be reviewed, approved, and signed by Laboratory Medical Director) – Have made significant progress toward obtaining required permits and M/STAs for shipping specimens to the University of Washington 4 Accessing Laboratory Related Documents Protocol Specific Webpage: • Laboratory Processing Chart (LPC) • Manual of Procedures (MOP) Email Communication with ILC Rep: • Protocol Analyte List (PAL) IMPAACT Laboratory Center webpage: http://impaactnetwork.org/aboutus/LabCenter/LabGuidance.htm • HIV NAT Assay requirements 5 REVIEW OF THE LABORATORY PROCESSING CHART (LPC) 6 LPC includes information on… • • • • • • Blood volumes tube types LDMS test codes Relevant Case Report Forms (eCRF) Collection, processing, storage requirements Shipping details 7 LPC Sections • Protocol Required Non-Standard Reagents and Supplies • Section A (1a-4a): Maternal Laboratory Processing Instructions – – – – 1a: Antepartum, Delivery and Postpartum SoE 2a: Safety/Clinical Laboratory Evaluations 3a: Specimen Processing 4a: Evaluations by Visit • Section B (1b-4b): Infant Laboratory Processing Instructions – – – – 1a: Delivery and Postpartum SoE 2a: Safety/Clinical Laboratory Evaluations 3a: Specimen Processing 4a: Evaluations by Visit • Section 5: Shipping Instructions • Section 6: Revision History 8 Protocol Required Non-standard Reagents and Supplies • There are no unusual reagents and supplies required for this study. • However, sites should always ensure that they have all the required tube volumes and tube types by the time of study activation. – It is essential to use the correct tube volume and tube type so as not to compromise sample integrity and laboratory results. • If you have difficulty obtaining tube supplies, contact the IMPAACT Laboratory Center Representative (William Murtaugh) 9 MATERNAL LABORATORY EVALUATIONS 10 Prioritization of Blood Collection and Processing for Short Draws: MATERNAL IMPAACT 2010 complies with NIH recommendations to limit adult blood collection to 10.5 mL/kg in a single day and 550 mL in any 8-week period. In the event that maternal blood collection must be limited, refer to the blood draw priority list below. Visit Order of blood collection Purpose 2 SST or NON Confirmatory HIV testing (if needed at or EDTA Screening Visit) EDTA HIV-1 RNA 3 SST or NON Chemistries (ALT, AST, creatinine) 4 SST or NON Hepatitis B surface antigen (required at Entry Visit only) 5 EDTA Hematology (complete blood count) 6 EDTA CD4+ cell count 7 EDTA Storage (plasma, cell pellets, and serum) 1 Screening through Week 50 Tube type 11 Section 2a: Safety/Clinical Laboratory Evaluations – MATERNAL VISITS Defer to local clinical specimen collection guidelines for tube types and collection volumes as needed. Evaluation Tube Type Confirmatory HIV testing SST or NON or EDTA Hepatitis B Surface Antigen (HBsAg) testing Hematology SST or NON EDTA Chemistries SST or NON CD4+ cell count EDTA OR DPE Urine Pregnancy Testing Urine Serum Pregnancy Testing SST or NON HIV-1 RNA PCR (Abbott RealTime Viral Load Assay) EDTA Tests eCRF(s) Complete HIV testing if needed to meet criteria specified in protocol Section 4.1.2 N/A Laboratory Test Results (Chem/Hem) Hepatitis B surface antigen Complete blood count (CBC; to include red blood cell count, hemoglobin, hematocrit, white blood cell count, white blood cell differential, absolute neutrophil count, and platelet count) ALT, AST, creatinine Note: Creatinine Clearance (CrCl) may be calculated by clinic or laboratory staff, and should be calculated based on creatinine results using the Cockcroft-Gault formula CD4+ absolute cell counts HCG (pregnancy test) Test used must have sensitivity of 25mIU or less. HCG (pregnancy test) Test used must have sensitivity of 25mIU or less. The total blood volume listed in the SoE at the Screening and Week 50 Visits accommodates collection of 1mL of blood if needed for serum pregnancy testing. HIV-1 RNA using the Abbott platform in a VQA-certified lab (all sites). 12 Laboratory Test Results (Chem/Hem) Laboratory Test Results (Chem/Hem) Lymphocyte Subsets Pregnancy Test Pregnancy Test Specimen Tracking HIV-1 RNA Plasma Viral Load Safety/Clinical Lab Considerations Confirmation of HIV Infection (if required at screening): • Testing algorithm must adhere to protocol section 4.1.2 and be approved by the IMPAACT Lab Center Chemistries: • Creatinine Clearance (CrCl) may be calculated by clinic or laboratory staff, and should be calculated based on creatinine results using the CockcroftGault formula Serum pregnancy testing (if required at screening): • The total blood volume listed in the SoE at the Screening and Week 50 Visits accommodates collection of 1mL of blood if needed for serum pregnancy testing. Maternal HIV RNA Testing: • Real-time testing is required to be performed on the Abbott platform (Real-Time HIV-1 viral load assay) by a VQA-approved laboratory • Will be confirmed on Protocol Analyte List (PAL) 13 Confirmation of Maternal Infection Sample #1 Sample #2 • Two different rapid antibody tests • Rapid antibody test for a total of three different rapid tests • One EIA or WB or immunofluorescence or chemiluminescence test • One EIA or WB or immunofluorescence or chemiluminescence test • One HIV DNA PCR • One HIV DNA PCR • One quantitative HIV RNA PCR • One quantitative HIV RNA PCR (result above LOD) (result above LOD) • One qualitative HIV RNA PCR • One qualitative HIV RNA PCR • One total nucleic acid test • One total nucleic acid test 14 Confirmation of Maternal Infection • If both samples are tested using antibody tests, at least one must be tested in a laboratory setting that operates according to GCLP guidelines and participates in an appropriate EQA program • If nucleic acid testing is used, at least one test must be performed in a CLIA-certified (US sites) or VQA-certified (non-US sites) laboratory • For tests performed in other settings, adequate source documentation including the date of specimen collection, date of testing, test performed, and test result must be available • FDA-approved methods should be used when possible, and when a combination of three rapid tests are performed, at least one must be FDA approved 15 MATERNAL SPECIMEN PROCESSING 16 Section 3a: Specimen Processing – MATERNAL VISITS Refer to Section 4 for collection volumes. Evaluation Plasma For HIV-1 RNA (Abbott RealTime Viral Load Assay) Perform in real time, store residual plasma Plasma storage for HIV-1 RNA at centralized lab Plasma storage for ARV drug resistance testing Tube Type EDTA EDTA EDTA Special eCRF(s) Collection Notes Invert 8-10 times gently. Invert 8-10 times gently. Invert 8-10 times gently. Processing Shipping Spin blood at 400 x g for Send to VQA certified lab. 10 min. Remove plasma Specimen Store residual plasma at and respin at 800 x g for Tracking site until requested by 10 min. Freeze residual protocol team. plasma at -70°C or colder. Spin blood at 400 x g for Specimen 10 min. Remove plasma Tracking and respin at 800 x g for 10 min. Freeze plasma at -70°C or colder. Spin blood at 400 x g for Specimen 10 min. Remove plasma Tracking and respin at 800 x g for 10 min. Freeze plasma at -70°C or colder. Store at site until requested by protocol team for batch testing at centralized virology lab. Refer to Section 5 for shipping details. Store at site until requested by protocol team for batch testing at centralized virology laboratory. Refer to Section 5 for shipping details. 17 Screening Visit (window: up to -14d) LDMS VISIT CODE = 0/SCR Evaluation Specimen eCRF(s) Aliquots LDMS Code Special Notes Confirmatory HIV testing SST or NON or EDTA 0-6 mL N/A N/A N/A Complete HIV testing as needed per protocol Sections 4.1.2 and 6.1. Hematology (complete blood count) EDTA 3 mL Laboratory Test Results (Chem/Hem) N/A N/A Send to local lab . Send to local lab. Chemistries (AST, ALT, creatinine, CrCl) Stored plasma for ARV drug resistance testing Confirmatory serum or urine pregnancy test SST or NON 4 mL Laboratory Test Results (Chem/Hem) N/A 2 x 1.0 mL EDTA 6 mL EDTA 1 mL Urine 5 mL Specimen Tracking Pregnancy Test Store residual: 1 x 0.5 – 1.0 mL N/A N/A BLD/EDT/PL2 (BLD/DPE/PL2) Creatinine Clearance (CrCl) may be calculated by clinic or laboratory staff, and should be calculated based on creatinine results using the CockcroftGault formula Specimens collected and stored at screening for participants who do not subsequently enroll should be destroyed. For enrolled participants, store aliquots on site until requested by protocol team. Refer to Section 5 for shipping details. N/A If needed per protocol Sections 4.1.6 and 6.1. Send to local lab. 18 Maternal Specimen Processing Plasma: Residual plasma should be stored wherever possible. • Abbott RealTime HIV-1 viral load – Local VQA-approved laboratory • Plasma storage for centralized HIV-1 viral load testing – Store until requested to batch ship to U. Washington • Plasma storage for ARV Drug Resistance testing – Store until requested to batch ship to U. Washington – If participant is not enrolled, stored plasma sample collected at screening should be destroyed • Plasma Storage for markers of adverse pregnancy outcomes – Store at site until requested by protocol team 19 Maternal Specimen Processing Cell Pellet (non-viable PBMCs): • Storage for markers of adverse pregnancy outcomes • Site should follow the Cross-Network PBMC Processing SOP 20 Maternal Specimen Processing Serum: • Zika diagnostic testing (if indicated) • Store at site until requested by protocol team 21 Maternal Specimen Processing Hair Storage: • PK testing • Collection instructions can be found in the Manual of Procedures (MOP) • Hair sample should be stored at room temperature without exposure to light • Ship at request of protocol team to University of California, San Francisco 22 Maternal Specimen Processing Breast Milk: • Store at site until requested for batch testing at centralized pharmacology lab (University of California, San Diego) – Whole Milk – Skim Milk – Dry cell pellet 23 Breast Milk Collection and Processing • Collected in 50mL conical centrifuge tube • Keep breast milk as cold as possible through ALL processing steps. Send to local processing lab on wet ice or cold packs. • Complete processing within 6 hours of collection. If processing occurs outside this window, note this in the LDMS. 24 Breast Milk Collection and Processing Whole Milk: • Vortex 50 mL conical centrifuge tube • Making at least 6x1mL aliquots in 2 mL cryovials. • Freeze at -70°C or colder. 25 Breast Milk Collection and Processing Skim Milk: • Transfer remaining whole milk to a 15mL conical centrifuge tube. • Centrifuge sample at 400xg for 20 min. Use sterile spatula or wide tip pipette to remove lipid layer. • Aliquot 4-6 x 1mL of skim milk, leaving approximately 0.5mL skim milk remaining. Do not disturb the pellet. • Freeze skim milk aliquots at -70°C or colder. 26 Breast Milk Collection and Processing Cell Pellet: • Add 1.0mL PBS to remaining skim milk • Resuspend the pellet. • Transfer to to 2mL centrifuge tube and spin at 2000xg for 3 minutes. • Remove liquid without disturbing the pellet. • Store dry cell pellet at -70°C or colder. 27 Breast Milk Processing Lab Processing Clinic 20mL Breast Milk Tube Type BMK collected in 50mL conical centrifuge 20mL Breast Milk Store at 4°C within 10 min of collection Send to local lab on wet ice or cold packs. Whole Milk: Vortex (5-10s) 6 x 1.0mL cryovials (Store at -70C) Centrifuge remaining Sample at 400xg for 20 mins. Use Sterile spatula or wide tip pipette to remove lipid layer Skim Milk: 4-6 x 1.0mL cryovials (Store at -70C) Approx. 0.5mL skim milk remaining Add 1.0mL PBS and resuspend the pellet Transfer to 2mL centrifug e tube and spin at 2000xg for 3 minutes Remove liquid without disturbin g the pellet and store at -70C or colder Keep breast milk as cold as possible through ALL processing steps. Process within 6 hours of collection (>6 hrs. Note in LDMS) 28 Maternal Specimen Processing Urine storage: • Markers of Renal Toxicity • Keep specimen as cold as possible and complete processing within 6 hours of collection. If processing occurs outside this window, note this in the LDMS. • Send to local processing lab on wet ice or cold packs. • Aliquot and freeze at -70C or colder • Store until requested by protocol team to ship to designated testing lab 29 INFANT LABORATORY EVALUATIONS 30 Prioritization of Collection of Blood Collection and Processing for Short Draws: INFANT IMPAACT 2010 complies with NIH recommendation to limit infant blood collection to 5 mL/kg in a single day and 9.5 mL/kg in any 8-week period. In the event that infant blood collection must be limited, refer to the blood draw priority list below. Order of Blood collection Tube type Purpose 1 EDTA HIV NAT 2 NON or SST Chemistries (ALT, creatinine, ALT) 3 EDTA Hematology (Complete Blood Count) 4 NON or SST Serum storage 31 INFANT SPECIMEN PROCESSING 32 Infant Specimen Processing HIV Nucleic Acid Testing: • Performed locally for infants with unconfirmed infection or who are uninfected • Processing will be based on blood product required for lab specific assay (two options): – Whole blood (BLD) – Whole blood pellet (PER) – Plasma (PL2) • All remaining aliquots and remnant blood products should be frozen and stored until requested by the protocol team 33 Infant Specimen Processing HIV Nucleic Acid Testing: • At Infant Week 6 Visit, an additional 1mL of blood is drawn for plasma PK testing (4mL total blood draw). 34 Infant Specimen Processing HIV Nucleic Acid Testing: • For infants with confirmed HIV-1 infection: – No additional diagnostic HIV tests will be performed – Sample volume specified in the SoE will still be collected – Sample will be processed, and stored for ARV Drug Resistance testing • Plasma storage • Cell pellet storage 35 Infant Specimen Processing Serum: • Zika diagnostic testing (if indicated) • Store at site until requested by protocol team 36 Infant Specimen Processing Hair Storage: • PK testing • Collection instructions can be found in the Manual of Procedures (MOP) • Hair sample should be stored at room temperature without exposure to light • Ship at request of protocol team to University of California, San Francisco 37 ACTG/IMPAACT Laboratory Manual, Shipping Information and other useful information: http://www.hanc.info/labs/labresources/Pages/informationActgImpaactLabs.aspx NOTE: Please contact the laboratories prior to shipment to ensure they can receive the samples at the designated time. Do not ship until you receive approval from the receiving lab. Ensure that all import permits are up to date and accurate prior to shipping samples. VIROLOGY Centralized HIV Viral Load testing University of Washington Retrovirus Lab Attn: Joan Dragavon and Socorro Harb HMC / R&T BLDG 300 Ninth Ave. RM 726 Seattle WA 98104 Email: [email protected] Phone:206-897-5210 Fax: 206-897-5237 LDMS lab code: 15 PHARMACOLOGY Plasma and Breast Milk for ARV Drug Levels IMPAACT Pharmacology Specialty Laboratory at University of California, San Diego (UCSD) Steven S. Rossi, Ph.D. or Rowena Espina MT Pediatric Pharmacology and Antiviral Assay Laboratory ATTN: Steve Rossi, Ph.D or Rowena Espina MT Centralized ARV drug resistance 212 Dickinson Street testing BLDG CTF-B, Room 112 Seattle Children’s Research Institute San Diego, CA 92103-0808 Attn: Ingrid Beck or Sheila Styrchak Phone: 619-543-5293 1900 9th Ave, Seattle WA 98101 Fax: 619-543-5422 email: Email: [email protected] frenkellabshipments@seattlechildrens Laboratory Contact: Rowena .org Espina Phone:206-884-7201 Phone: 619-543-5293 Fax: 206-884-7311 Email: [email protected] LDMS lab code: 238 LDMS lab code: 196 PHARMACOLOGY Hair for ARV Drug Levels Dr. Monica Gandhi Attention: Karen Kuncze c/o VESTED study (Shahin Lockman) Room S-907 513 Parnassus Ave Medical Sciences Building, S864 San Francisco, CA 94143,USA Email: [email protected] Email: [email protected] Phone: (415) 502-1446 Phone: 415 476 9960; Fax number: 415-476-6770 LDMS lab code: 607 MARKERS OF ADVERSE PREGNANCY OUTCOMES PASS THROUGH SHIPPING Biomedical Research Institute 9410 Key West Avenue First Floor Rockville, MD 20850 USA Phone : (301) 881-7636 Fax: (301) 770-9811 Email: [email protected] LDMS lab code: 999 38 Section 5: Shipping Instructions • All shipping to occur upon request of the protocol team • Laboratories should ship specimens directly to specialized testing laboratories whenever possible. • If your country or institution requires S/MTA, begin this process as soon as possible to avoid shipping delays. – It is the responsibility of the shipping and recipient laboratories to complete paperwork and execute an agreement. – Copies of signed MTAs must be made available to IMPAACT Lab Center Representative • For sites that are unable to ship directly, follow Cross Network BRI pass through shipping instructions 39 Shipping Instructions (continued) • Hair PK shipments must include certification statement document from UCSF • Document is available on the protocol specific webpage under “Study Implementation Documents” 40 Section 6: Revision History Version & Date (dd/mmm/yy) Comments 41 Questions? Overall support for the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Network was provided by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) under Award Numbers UM1AI068632 (IMPAACT LOC), UM1AI068616 (IMPAACT SDMC) and UM1AI106716 (IMPAACT LC), with co-funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) and the National Institute of Mental Health (NIMH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. 42