Post on 02-Apr-2018
QC in the ART
Laboratory
Ph.D. Lars Johansson, Clinical & Scientific Advisor,
Director of Embryology,
ljohansson@origio.com
+46 709 925460
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
• Basic Requirements
• Environment
• Quality Management
• Quality of Service
• Optimization of Outcome
AIM
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
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!!
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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.
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Analysis of TVOC Loading & CPR
within the IVF Laboratory
Cli
nic
al P
reg
na
nc
y R
ate
(%
)
Testing Quarters (TQ)
2.7 2.7
2.2 2.2
16.0% 16.0%
37.5% 37.5% 33.3% 33.3%
41.6% 41.6%
13.3% 13.3%
29.4% 29.4%
41.6%
2.7
2.2
16.0%
37.5% 33.3%
41.6%
13.3%
29.4%
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5 TV
OC
Le
ve
ls (
pp
b)
51.6%
TQ12 0
10
20
30
40
50
60
70
80
90
100
TQ11 TQ10 TQ9 TQ8 TQ7 TQ6 TQ5 TQ4 TQ3 TQ2 TQ1
54.5% 50.0% 52.4%
0.1 0.1 0.1 0.1 0.1
64.7%
55.5%
The presence of 2.2 ppb VOCs
severely compromised clinical outcomes
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Third Party Independent Testing:
2 CODA Towers + HEPA Final Filters
Source Air IVF
Laboratory
TVOC 1324 ppb 1372 ppb
Biologicals:
Spores 469 cfu/m3 1778 cfu/m3
Particulate
0.3 um 2,318,663 11,642
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Cleanliness: Tightness Factor
- Clean Air – In/Outlets - Exchange: >20/h
- Over-Pressure - Lab: 30 PA
- Pre-room: 15 PA
- Pharmacy Hatches &
Pressurized Door
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
•Quality Manager & Quality Manual
•Covers ALL Aspects
•Clinic (Financial, Facility, Safety, Patient Care, Staff etc)
•Treatments Offered
•Team Work - All Staff Involved & Committed
Total Quality Management (TQM)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Alarm!
Audible /
visual
alarming
SMS
alarming
Data visualization / management via LAN / web interface
relay
output
mains
24 hours Equipment Surveillance Network (Planer - T, CO2, pH, Humidity, Gas, Particle Count)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
•Routine, robust and reliable • Laboratory, but not limited to the laboratory
•Facilitate Auditing
•Benchmarking against other programs
•Exchange Ideas / Studies
•Customer Satisfaction
QA & International KPI
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Laboratory KPI – Timely Evaluations
David Mortimer, ALPHA 2012
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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.d
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
0
5
10
15
20
QA charts
40
50
60
70
70
80
90
100
90
92
94
96
98
100
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
month 0
5
10
15
20
But use prospectively…Log Book
40
50
60
70
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Quality Improvements (QI) – Continuously Performed
FOCUS +
The Devil is in the Details
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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 EQA
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Standard Operational Procedures (SOPS)
•Written Instructions
•Increase in Standardization
•Efficiency
•Transparency
•Traceability
• Controlled Activities
• Reduce Complaints & Failures
•Facilitates Trouble-Shooting (Auditing)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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 Handling
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
• Specific for Human ART?
• Certified Quality?
• CE marked (93/42/EEC)
• FDA 510k
• TGA
• Quality Tested with several tests
• MEA
• Sperm survival
• Endotoxin (LAL)
Media Products & Disposables - QC
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
VOC Levels: Dishes
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
VO
C (
pp
m)
VOC Levels in Culture Dishes
NUNC
Petri Dish
#50255
(0.7 ppm)
NUNC
4-Well Dish
#144444
(1.4 ppm)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
9,0
VO
C (
pp
m)
VOC Levels in Culture Tubes
BD FALCON
5ml Tube
#352058
(6.8 ppm)
VOC Levels: Tubes
NUNC
11ml MEA
Tube
#137860
(0.6 ppm)
NUNC
1.8 ml
CryoTube
#177280
(1.2 ppm)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Eliminate MisIdentification –
Adverse Effects
• Double Identification
• Labelling of dishes
• Barcodes
• Stickers (www.fertgms.com)
• Laser Etching (www.ankdatasystems.com)
• Electronic Witnessing
• Increase Traceability
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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”
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
DNA Packing, IR & CPR
Group Implantation Rate CPR/ET
Per ET Per Cycle
CMA3<44%
43 40 32
44<CMA3<60% 35 33 28
CMA3>60% 29 21 18
Esterhuizen et al, 2000
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
GM-CSF, Sperm Activation & Time
• Ejaculated Sperm (Zambrano et al., 2001)
• Tail Region & Acrosome of Ejaculated Sperm (Naz.,2006)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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!!!!!
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
• 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 & Service
Egg Collection - QC
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
•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)
Evaluation & Scoring (Istanbul Consensus, 2011)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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-Incubator
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
DAY 0/2-3 and 2/3-5/6
UTM/GMCSF ?
DAY 0 - 5/6
Fertilization w/wo GMCSF
Cleavage w/wo GMCSF
Blastocyst w/wo GMCSF
SAGE 1Step
Medium
2PN-Blast
5 Days
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
57
The ORIGIO® Sequential Series™ = 3 media
TRANSFER
ORIGIO® Sequential
Cleav™
ORIGIO® Sequential
Fert™
ORIGIO® Sequential
Blast™
For the fertilisation of oocytes.
For the culture of 2PN embryos to the 2-8
cell-stage (up to day 3).
For the culture of 4-8 cell-stage embryos to
the blastocyst stage (from day 2/3).
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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
58
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Control New Seq.
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 62
New Sequential Media
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Sage Single-Step Medium
• Same Results as the Competition
• Culture Techniques
- No need for changing media
- Culture from 2PN – Blastocyst (Day 5)
60
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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!!!
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Recommended pH of Commercial Media
Swain RBM Online 2010 21:6-16
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
0
10
20
30
40
50
60
70
80
90
100
% 8 cell embryos
7.1
7.15
7.2
7.25
7.3
N= 96, 431 embryos
Courtesy of Kathy Miller
Optimum pH for Cleavage Medium 7.18-7.25
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
N= 39, 567 embryos
Courtesy of Kathy Miller
Optimum pH for Blastocyst Medium 7.28-7.35
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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 medium
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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., 2012
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Other considerations QC Micromanipulation
Which ICSI? Heating, Anti-Vibration, Location.
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
QC Equipment’s – Particles, Temp, CO2, O2, pH, VOCs
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
ICSI-CUMULASE® • Recombinant
human enzyme (rHuPH20)
• Extremely high purity
> 99% pure
NON-CUMULASE® • All are testicular enzymes
from slaughterhouse animals
• Purity not ensured
many unknown substances
Contents of typical products
Bovine
(Type IV-S)
Ovine
(Hyase)
Human
(Cumulase)
? ? Cumulase
(rHuPH20)
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Regulation of pHi during Oogenesis,
Embryogenesis & Denudation
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
70 - 90 min
40- 60 min
Spindle Bridge
MI > MII
MII
Montag 2012 ALPHA
Spindle Dynamics and ICSI
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
•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)
Culture Techniques
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Direct cleavage (DC) from 1 to 3
Rubio et al 2012, Fertil & Steril
Cleavage from 1-blastomere to 3-blastomeres in less than 5 hrs
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Blastomere Size and Genetic Health
photomicrographs courtesy of
Dr S Pickering
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
De-Selection Cleavage Patterns
t2 t3 t4 t5
cc1 cc2
cc3
t2 ≈ t3 < 5 hrs t4 ≈ t5 ≈ t6
cc1
cc2 cc3
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Factors affecting
morphokinetics…
t2 t3 t4 t5 t8
cc1 cc2 cc3
s2 s3
Age
ICSI Media
pH Oxygen
Aneuploidy Viability?
Etiology
Temperature
Handling
Embryo Development
Stimulation
Maturity
Biopsy
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Day 3 vs Day 5 Biopsy
Age
Group
D3 IR D5 IR D3 AR D5AR
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 2011
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
•Electronic witnessing
ID Monitoring – Risk Reduction
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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 calibration
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC DEVICES
Uterine Transfer Medium (UTM) / GMCSF
Effect of hyaluronan-enriched transfer medium Urman et al 2008
0
10
20
30
40
50
60
Clinical pregnancy Implantation
Hyaluronan
Conventional
“…beneficial effect was more
prominent in women >35 years
of age, and in women who had
poor-quality embryos”
HUMAGEN PIPETS
MEDICULT MEDIA
MIDATLANTIC 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 optimized