Laboratory QC in the ART - Ph.D. Lars Johansson
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QC in the ART
Laboratory
Ph.D. Lars Johansson,
Clinical & Scientific Advisor,
Director of Embryology,
ljohansson@origio.com
+46 709 925460
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
AIM
• Basic Requirements
• Environment
• Quality Management
• Quality of Service
• Optimization of Outcome
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Basic Considerations
•Allocate Rooms & Functions
• Evaluations vs Treatments?
• Building Materials, Furniture & Equipment’s
• HVAC (constant temp, particles, VOCs)
• Gas Support (Pure CO2, mix & N2)
• Liquid Nitrogen
• UPS – Outlets (Height & Numbers)
• Transport & Deliveries – Easy Access
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Seasonal Variations in
VOCs, TQE & CPR
MEDICULT HUMAGEN MIDATLANTIC
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Contaminations Affecting Outcome
•Components form Outside Ambient Air
•Laboratory Recirculated Air
• Tissue Culture Ware
• Equipment Off-Gassing
• HVAC Specific Organisms
• Gas Cylinders - contaminations
• Personnel Bioburden
VOCs MOST HARMFUL!!
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
VOCs vary in their polarity, molecular
weight and biochemical structure
Require different mechanisms of removal.
Must target all biochemical families.
Increase the effectiveness.
Resonance(removal) and dwell(hold) time.
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Analysis of TVOC Loading & CPR
within the IVF Laboratory
Clinical Pregnancy Rate (%)
100 0.0
0.1 0.1 0.1 0.1 0.1
90
TVOC Levels (ppb)
80 0.5
70 64.7%
1.0
60 55.5%
52.4% 54.5% 51.6%
50.0%
50 41.6%
41.6% 41.6% 1.5
40 37.5%
37.5%
33.3%
33.3% 29.4%
29.4%
2.0
30
2.2
2.2
20 2.5
16.0%
16.0% 2.7
2.7
10 13.3%
13.3%
0 TQ1 TQ2 TQ3 TQ4 TQ5 TQ6 TQ7 TQ8 TQ9 TQ10 TQ11 TQ12
Testing Quarters (TQ)
The presence of 2.2 ppb VOCs
severely compromised clinical outcomes
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Third Party Independent Testing:
2 CODA Towers + HEPA Final Filters
IVF
Source Air
Laboratory
TVOC 1324 ppb 1372 ppb
Biologicals:
469 cfu/m3 1778 cfu/m3
Spores
Particulate
2,318,663 11,642
0.3 um
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
LIFE-Aire VOC Free Ventilation System
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
LIFE-Aire VOC Free Ventilation SystemMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Cleanliness: Tightness Factor
- Clean Air – In/Outlets
- Exchange: >20/h
- Over-Pressure
- Lab: 30 PA
- Pre-room: 15 PA
- Pharmacy Hatches &
Pressurized DoorMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Total Quality Management (TQM)
•Quality Manager & Quality Manual
•Covers ALL Aspects
•Clinic (Financial, Facility, Safety, Patient Care, Staff etc)
•Treatments Offered
•Team Work - All Staff Involved & CommittedMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Quality Management
• QC: Quality Control
• Provide routine and consistent checks to ensure
performance meets predetermined set criteria
• Identify and address errors and neglected items
• Documented and archived (paper & wireless)
• QA: Quality Assurance
• Verify that quality criteria has been met.
• Financial, management and service satisfaction.
• Key Performance Indicators (KPI)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
24 hours Equipment Surveillance Network
(Planer - T, CO2, pH, Humidity, Gas, Particle Count)
relay mains
output
Alarm!
Audible / SMS
visual alarming
alarming
Data visualization / management via LAN / web interfaceMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
QA & International KPI
•Routine, robust and reliable
• Laboratory, but not limited to the laboratory
•Facilitate Auditing
•Benchmarking against other programs
•Exchange Ideas / Studies
•Customer SatisfactionMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Laboratory KPI – Timely Evaluations
David Mortimer, ALPHA 2012MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
QA & Factors
•Factors affecting Outcome & Interpretation
• Facility & Laboratory Function
• Patients
• Demographics & Selection
• Batch Treatments
•Expertise / skills (Education, New Staff)
•Time of evaluation (Suitable Intervals)
•Products (Hormones, Disposables, Media)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Control Charts for Different
Parameters (KPI)
• Plot mean of variables each month (KPI)
• Average of monthly means expected value
• Benchmark value
• Control limits = s.dMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
QA charts
70 20
15
60
10
50
5
40 0
100
100
98
96 90
94
80
92
90 70MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
But use prospectively…Log Book
70
60
50
40
20
15
10
5
0 monthMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Quality Improvements (QI) –
Continuously Performed
FOCUS +
The Devil is in the DetailsMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Education & EQA in Folliculometry,
Andrology & Embryology
•Samples or via Internet
•SIRT, www.eqasrm.com.au
•AAB, www.americanmedicalacademy.org
•FertAid – www.fertaid.com, QAPonline
•Swiss Clinical Embryologists
•UK NEQAS/ACE EQAMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Total Quality Management (TQM)
• Risk Management – Adverse Events
• Complaint Handling
• Analyse (Root Cause), identify, eliminate & avoid
• Management System
• Describe Processes & Procedures
• Flow Charts – Process Mapping
• Standard Operational Procedures (SOP)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Standard Operational Procedures (SOPS)
•Written Instructions
•Increase in Standardization
•Efficiency
•Transparency
•Traceability
• Controlled Activities
• Reduce Complaints & Failures
•Facilitates Trouble-Shooting (Auditing)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Total Quality Management Manual
• Organisational Hierarchy
• Clinics Mission, Vision & Quality Statement
• Staffing
• Position within the Organization
• Job Description
• Duties, Responsibilities
• Education & Competency (Written Plan)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Pro-Active Actions (SOPs)
1) Cleaning of Premesis and Equipment’s
2) Dress Code / Behavior
3) Hand Hygene
4)Preparation of dishes
5)Selection of disposables
6) Waste HandlingMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Media Products & Disposables - QC
• Specific for Human ART?
• Certified Quality?
• CE marked (93/42/EEC)
• FDA 510k
• TGA
• Quality Tested with several tests
• MEA
• Sperm survival
• Endotoxin (LAL)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
VOC Levels: Dishes
VOC Levels in Culture Dishes
7,0
6,0
VOC (ppm)
5,0
4,0
3,0
2,0 NUNC
4-Well Dish
#144444
1,0 NUNC (1.4 ppm)
Petri Dish
#50255
0,0 (0.7 ppm)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
VOC Levels: Tubes
VOC Levels in Culture Tubes
9,0
8,0
VOC (ppm)
7,0
BD FALCON
6,0 5ml Tube
#352058
5,0 (6.8 ppm)
4,0
3,0
2,0
NUNC
1.8 ml
1,0 CryoTube
#177280
0,0 NUNC
(1.2 ppm)
11ml MEA
Tube
#137860
(0.6 ppm)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Eliminate MisIdentification –
Adverse Effects
• Double Identification
• Labelling of dishes
• Barcodes
• Stickers (www.fertgms.com)
• Laser Etching (www.ankdatasystems.com)
• Electronic Witnessing
• Increase TraceabilityMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
QC - Standard Semen Analysis
• Concentration
• Total number of sperm per ejaculate
• Motility
• Deletions within mitochondrial DNA is associated with
reduced motility (Ozmen et al., 2007)
• Morphology “THE FORM OF THE SPERM
HEAD IS MORE IMPORTANT
THAN ITS DIMENSIONS”MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Sperm DNA damage with ageMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
DNA Packing, IR & CPR
Group Implantation Rate CPR/ET
Per ET Per Cycle
CMA3MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Preparation of Sperm &
DNA FragmentationMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
GM-CSF, Sperm Activation & Time
• Ejaculated Sperm (Zambrano et al., 2001)
• Tail Region & Acrosome of Ejaculated Sperm (Naz.,2006)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Reduce the Effects of the Paternal
Genome on Embryo Development
•Objective Selection of Competent Sperm
•Less Fragmentation (D3)
•Higher Blastocyst Conversion Rate
•Improved Blastocyst Quality
•Higher CPR
•Lower Miscarriage Rate
•IVF!!!!!MEDICULT HUMAGEN MIDATLANTIC MEDIA PIPETS DEVICES N= 537 patients
MEDICULT HUMAGEN MIDATLANTIC MEDIA PIPETS DEVICES N= 537 patients
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Egg Collection - QC
• Anasthesia
• Stimulation Protocols
• Dysmorphism – short & high doses
• Temperature Control
• Tubal Length – Needle to test-tube
• Needle size (gauge): Bleeding & pain
• Single Lumen
• Test-Tube Warmer (36.5-36.9)
• Speed & Handling
• Pump Pressure
• Calibration & ServiceMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Evaluation & Scoring (Istanbul Consensus, 2011)
•Timing of Scoring Important
•What can/Should we score?
• Oocyte Quality
• Aggregation or Clustering of SER
• Imprinting Disorders?
• High Biochem & Miscarriage Rates (Montag)
• Large Polar Body (Aneuploidy)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Class II Cabinet or LAF
51MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
QC - Handling of COCs
•Media for rinsing of the COC
• AA, ”goodies”
•Control of temp, pH, osmolarity & oxygen
• Meiotic spindle, embryo development & IR
•Do not cut COCs
• Destroy TZP
•Transfer to Mini-IncubatorMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Incubators – Recovery,CO2, pH,O2&Temp.MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
DAY 0/2-3 and 2/3-5/6 DAY 0 - 5/6
Fertilization
w/wo GMCSF
SAGE 1Step
Medium
Cleavage
w/wo GMCSF 2PN-Blast
5 Days
Blastocyst
w/wo GMCSF
UTM/GMCSF ?MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
The ORIGIO® Sequential Series™ = 3 media
ORIGIO® Sequential
For the fertilisation of oocytes.
Fert™
ORIGIO® Sequential
For the culture of 2PN embryos to the 2-8
Cleav™ cell-stage (up to day 3).
ORIGIO® Sequential
For the culture of 4-8 cell-stage embryos to
Blast™ the blastocyst stage (from day 2/3).
TRANSFER
57MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Formulation Focus Areas
• Robustness - Use of pharmacopeia graded products
• Revised components:
- Optimised ionic composition
- Optimised energy substrate composition
- Optimised amino acid concentrations
- Addition of vitamins to Fert™ and Cleav™
- New glutamine source (L-alanyl-glutamine)
• Introduced components: Hyaluronic Acid
58MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
New Sequential Media
New Seq.
Control
Fertilization Rate (%) 76 73
Cleavage Rate (%) 88 87
Good quality embryos (%) 54 49
Blastocyst Rate (Day 5, %) 53 51
Good quality blastocysts (%) 49 51
CPR per patient (%) 48 62MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Sage Single-Step Medium
• Same Results as the Competition
• Culture Techniques
- No need for changing media
- Culture from 2PN – Blastocyst (Day 5)
60MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Precipitates
Precipitates of Calcium Carbonate occurs when
•Vials/flasks are frequently opened which
increases the pH – Use small vials
•If you store aliquots of media under 5% CO2
at low temperature (2-8C).
•Do not aliquot!!!MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Recommended pH of Commercial Media
Swain RBM Online 2010 21:6-16MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Courtesy of Kathy Miller
100
90
80
70 7.1
60 7.15
50 7.2
40 7.25
30 7.3
20
10
0
Optimum pH for Cleavage Medium% 8 cell embryos
7.18-7.25 N= 96, 431 embryosMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Courtesy of Kathy Miller
Optimum pH for Blastocyst Medium 7.28-7.35 N= 39, 567 embryosMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Reducing the negative effects on/off oil
• Mineral oil is a mixture of various hydrocarbons.
• Unsaturated hydrocarbons are more susceptible to
peroxidation (POVs), which is embryo-toxic.
• Light & UV light increase the peroxidation.
• Store in dark.
• Heat and prolonged storage increases peroxidation.
• Store at low temperatures (2-8°C).
• Companies pre-wash oil with water or mediumMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Low Oxygen (5-6%) = Low ROS
• Reduce chromosomal
mosaicism:
- Blastomeres
- Sex Chromosomes
• Cleavage Pattern
• Blastocysts
• Higher Cell Numbers
• Lower Apoptosis
• On time development
Bean et al., 2002, Dunn et al., 2012MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Other considerations
QC Micromanipulation
Which ICSI? Heating, Anti-Vibration, Location.MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
QC Equipment’s –
Particles, Temp, CO2, O2, pH, VOCsMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
NON-CUMULASE® ICSI-CUMULASE®
• All are testicular enzymes • Recombinant
from slaughterhouse animals human enzyme (rHuPH20)
• Purity not ensured • Extremely high purity
many unknown substances > 99% pure
Contents of typical products
Bovine Ovine Human
(Type IV-S) (Hyase) (Cumulase)
??
Cumulase
(rHuPH20)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Regulation of pHi during Oogenesis,
Embryogenesis & DenudationMEDICULT HUMAGEN MIDATLANTIC MEDIA PIPETS DEVICES
MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Spindle Dynamics and ICSI
Spindle Bridge
MI > MII
70 - 90 min
MII
40- 60 min
Montag 2012 ALPHAMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Culture Techniques
•Open vs Micro-drops?
• Small drops (20 – 40 ul)
• Airflow & High temperature
• Higher osmolarity
• Bad Embryo Development (Swain et al., 2012)
•Single vs Group Culture
• Sequential Evaluation
• Paracrine & autocrine & pH?
• Day 1-3, Better Outcome (Ebner et al., 2010)
• After Compaction, single (Lane, FSA 2008)MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Direct cleavage (DC) from 1 to 3
Cleavage from 1-blastomere to 3-blastomeres in less than 5 hrs
Rubio et al 2012, Fertil & SterilMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Blastomere Size and Genetic Health
photomicrographs courtesy of
Dr S PickeringMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
De-Selection Cleavage Patterns
t2 t3 t4 t5
cc1
cc2
cc3
t2 ≈ t3
< 5 hrs t4 ≈ t5 ≈ t6
cc1
cc2 cc3MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Aneuploidy / Morphokinetics: PGDMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Factors affecting
morphokinetics…
Stimulation ICSI Media
Etiology
Age Handling
Embryo Development
t2 t3 t4 t5 t8
cc1 cc2 cc3
s2 s3
Maturity
Oxygen
Temperature pH Biopsy Aneuploidy Viability?MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Day 3 vs Day 5 Biopsy
Age D3 IR D5 IR D3 AR D5AR
Group
30 – 34 51.6 85.3 50.5 41.2
35 – 39 38.1 63.5 65.1 48.1
40 - 42 31.2 76.3 77.5 60.5
Harton et al., ASRM 2011MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
ID Monitoring – Risk Reduction
•Electronic witnessingMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Lasers: Octax
- AHA on Day 3/4/5MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Assisted Hatching with Laser
• Hands not removed from manipulators.
• Laser fired with foot pedal
• Easier to target cell junctions
• Allows aspiration during ablation
• Movable and no calibrationMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Uterine Transfer Medium (UTM) / GMCSF
Effect of hyaluronan-enriched transfer medium
Urman et al 2008
60
Hyaluronan
50
Conventional
40 “…beneficial effect was more
30 prominent in women >35 years
20 of age, and in women who had
10
poor-quality embryos”
0
Clinical pregnancy ImplantationMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Vitrification - QC
Toxicity of components reduced by
• Speed of handling
• Short exposure to final vitrification solution
• Rapid warming
Survival Rates & Developmental Competency
• Selection of high quality gametes & embryos
• Whole vitrification process optimizedMEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
FET better than fresh?
Kato et al., 2012MEDICULT HUMAGEN MIDATLANTIC
MEDIA PIPETS DEVICES
Thank You!
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