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 www.mtnstopshiv.org
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:
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Hemolysed Serum
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Clotted EDTA Tubes
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Affects many chemistry tests
Can cause false elevation of AST, ALT etc…
Affect numerous hematology parameters
Underfilled EDTA Tubes
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May dilute specimens for hematology
Specimen Quality is Key (con’t)
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Phlebotomy technique and handling will affect
specimen quality
Proper training (and retraining when problems
are noted) is key.
Some issues
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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 transporthandle specimens with care
ASPIRE Blood Testing - HIV
 HIV testing
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Rapid Tests
Western Blot
RNA Viral Load-algorithm, seroconverters
DNA Viral Load-algorithm-only as instructed
by NL
HIV genotypic Resistance Testingseroconverters
 HIV related testing
 CD4 Count-seroconverters
Some Lessons Learned from
VOICE and HEROES
 HIV algorithm
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“Sample 2” eliminated.
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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
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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
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Confirmatory (QA) testing at the MTN NL
Resolution of ambiguous endpoints
Resistance Testing among seroconverters
 Future research
Specimen Storage and Shipment
Plasma Specimen
Draw volume
Minimum
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
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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)
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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
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FBC-with 5 part differential
 Chemistry
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ALT
AST
Creatinine
Syphilis Serology
 Syphilis Serology
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RPR for screening
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Treponemal Confirmation
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Very Non specific
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)
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Hemoglobin
Hematocrit
Mean Corpuscular Volume
Platelets
White blood cell count with differential
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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
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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
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Separate urine first and hCG
Refrigerate urine for SDA
Urine Specimen Processing (cont.)
 To prevent Contamination
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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
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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
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Rotate the swab over lateral vaginal wall
several time to collect sufficient specimen
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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
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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.
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Machery-Nagel
Baker
SP
Pelvic Specimen Processing (cont.)
 Vaginal Gram Stain for Storage
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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
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One swab from endocervix
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Freeze within 8 hours of collections
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Stored in 400 µL PBS immediately after
collection.
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.
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Stored with no additive
Freeze within 8 hours of collections
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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
Self-Swab Instructions
Other Specimen Processing
 Ring Collection for Biofilms
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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
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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?