ASPIRE (MTN-020) Laboratory Training Edward Livant MT (ASCP), MPH University of Pittsburgh Medical...
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Transcript of ASPIRE (MTN-020) Laboratory Training Edward Livant MT (ASCP), MPH University of Pittsburgh Medical...
ASPIRE (MTN-020) Laboratory Training
Edward Livant MT (ASCP), MPH University of Pittsburgh Medical Center
Yaw Agyei MT (ASCP) Johns Hopkins UniversityHPTN/MTN Network LaboratoryProtocol 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 www.mtnstopshiv.org
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 emails 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 Specimen Draw volumeMinimum
Plasma Required
Enrollment archive; Routine storage (quarterly, semi-annual, PUEV, Termination Visit)
~10 mL 4 mL
Follow-up HIV testing algorithm Storage
~15 mL 6 mL
Post-seroconversion Months 1, 3, 6 and every 6 months thereafter date of a positive rapid(s)
~15 mL 6 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
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 15-60 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 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
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)
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
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.