Transcript Hanabi PIII validation at Guy’s

Validation of the
Hanabi-PIII Robotic
Metaphase Cell Harvester
Juliana Groisman, Richard Hall
ACC Spring Conference
Liverpool, March 2008
Overview
 Introduction and context
 Hanabi-PIII Robotic Harvester
 Validation, data analysis and preparations
 Considerations and limitations
 Oncology trial
 Conclusions and further work
Introduction

Manual harvesting is physically demanding, time consuming
and prone to operator variability.

Automation of this process should increase efficiency and
reduce manual routine work, leaving experienced staff
free to concentrate on other important tasks.

Robotic harvesters have been around for a number of
years; however, there has been relatively little use of
these machines in cytogenetics labs in the UK.
- There is little evidence available to prospective
laboratories regarding the effectiveness of these machines;
- Machines have not been fully automated and require
operator interaction.
 Hanabi harvesters were the first harvesters to offer
complete automation of the process. The Hanabi PII
harvesters, which have a capacity of 24 samples, have been
used for some years in Europe, USA and Japan.
 We had the opportunity to validate the first Hanabi PIII
robotic harvester, which has a sample capacity of 64 tubes.
 For a period of two weeks the Hanabi-PIII robotic
harvester was trialled in our laboratory.
 Our main aims were to evaluate and compare the
preparations obtained with the automated Hanabi-PIII
robotic harvester against our manual harvest of blood
lymphocytes.
Criteria assessed
The robotic harvester should:
 Produce preparations suitable for diagnostic use;
 Increase sample throughput;
 Be efficient and require no intervention;
 Show high reproducibility - removing operator
variability;
 Reduce manual routine work
 Be robust and easy to use
Hanabi-PIII Robotic Harvester
 The Hanabi-PIII automated suspension harvester
(ADStec – ADScience Technologies, Japan) is unique
in its mode of action and sample throughput
capability:
– It includes a centrifuge and a vortex for agitation
– It is capable of harvesting 64 specimens
simultaneously
– Once the specimens have been loaded no operator
interaction is required until the harvest is complete
HEPA filter
Touch screen menu
Validation
Technical aspects
Setting the
programme using
the touch-screen
menu was easy; to
start with, we
changed the
settings to suit our
current protocol.
Good results
obtained with the
first run
Slide making
Validation
Technical aspects
We carried out experimental runs to optimise
the settings
•
•
•
•
•
•
Pre-fixation step (G step)
Second hypotonic step
Different lengths of exposure to hypotonic
solution
Different vortex speeds
Changed the centrifuge times
Changed aspiration levels (which determine
how much supernatant should aspirated)
After assessing how much supernatant
Changing
the
level towe
12 had a
was left in
ouraspiration
manual harvest,
When
weat
tried
aspiration
we
positive
effect,
preparations
clean
started
aspiration
levellevel
14 were
10,
but
found
that
our
yields
affected
and
mitotic
index
was were
good
found
that preparations
were
slightly
cytoplasmic
Validation
Technical aspects
Our settings for the
validation:
A. Centrifuge for 5 mins (1,000
rpm);
B.
Aspirate to level 12
C.
Start the vortex; add 5.5ml KCl
(hypotonic solution);
D. Incubate for 6 mins;
H. Centrifuge for 5 mins (1,000
rpm);
I.
Aspirate the supernatant to
level 12;
J.
Start the vortex; add 5ml
fixative
K.
slidemaking
3X
Centrifuge for 5 mins (1,000
rpm);
Harvest time was comparable to our manual
harvest
1.
2.
3.
4.
5.
Switch on the harvester
Prepare fix and hypotonic solutions, top-up distilled
water
Carry out reagent exchange – flushes the system
replacing water with reagents (fixative and hypotonic)
Select saved protocol (at this point parameters can be
changed if required)
Set sample number (the number of tubes to be
loaded). Dummy tubes containing water must be used
to balance the centrifuge.
Preparation
takes approx
5 mins to
prepare and
2-10 mins to
load
depending on
sample size
6.
7.
Take samples to Class II hood and add colcemid; start
the timer.
Load the samples. The air temperature inside the
robotic harvester can be set at 37°C – samples can be
incubated in colcemid whilst inside the harvester. The
lid is removed (and discarded) and each sample is
placed in its allocated slot – sensors detect that
loading is carried out correctly.
8.
Harvest
takes
50mins-1h58
depending on
sample size
9.
10.
11.
Washing
takes approx
10mins
12.
13.
14.
When the time in colcemid is up (15 minutes at
Guy’s), press main start button and walk away.
When the harvest is finished, remove tubes and
use fresh lids. Samples are ready for
slidemaking.
Carry out reagent exchange – flushes the system
with water
Carry out tube washing (soaking of aspiration
probes for 3 mins), remove tubes and allow pump
to dry (~5 mins)
Swab surfaces with virkon
Empty waste containers
Switch off the harvester
Quality Assurance
 There is no sample transfer; samples are harvested in the same
tubes used for culturing
 For consistency, hypotonic solution is warmed up to 37˚C during
injection
 The program ensures that each step is carried out correctly
before proceeding to the following step
- Reagent exchange must be carried out before loading
- After adding sample size, loading positions are indicated.
Sensors detect that samples have been loaded in their
correct positions to ensure that the centrifuge is balanced
- Only then it is possible to start the harvest
Validation
1. 53 patients were processed for the validation.
2. For each patient, test and control cultures were set up and
processed exactly the same apart from the harvest;
3. Blind study – All slides were coded prior to data collection;
4. Slides were scanned on Metasystems and the 30 best metaphases
were selected;
5. The following parameters were assessed:
Quantitative
Qualitative
Yield
Total number of cells obtained
Mitotic index
Calculated by Metasystems for the
whole slide
Chromosome length
Spreading
A total of
3,000
metaphases
were assessed
Results
Validation results
Data analysis
Yield (total cells/culture)
Mitotic index
Quality (%QA 6)
Spreading (% well spread)
Robotic harvester n=53
average variance
179,603 14,445,419,924
14.07
4.44%
52.97% 539.94
68.96% 427.47
Manual harvest n=53
average variance
170,257 8,959,414,637
42.5
6.17%
588.73
51.72
72.91% 551.91
Yield
Comparable
Found to be slightly higher in the robotic harvester;
Comparable
Comparable
Comparable
Mitotic Index
Found to be lower with the robotic harvester but more consistent;
Quality
Possibly more consistent with the robotic harvester
Spreading
Slightly lower with the robotic harvester but possibly less variable
Preparations
Specimen 07/12308
Robotic harvester
Manual harvest
Specimen 07/12304
Robotic harvester
Manual harvest
Operator variability
 In our validation we found that some manual harvests
were better than others;
 Harvest 4 (n=13, operator 2) in the validation was a poor
manual harvest, with some broken and cytoplasmic
preparations; cultures harvested with the robotic
harvester were considerably better.
 This is likely to be due to operator error, which is one of
the factors we aim to remove using the robotic harvester.
Operator variability
Poor Manual harvest (harvest 4)
Manual harvest
Robotic harvester
Specimen 07/12516
Considerations/Limitations
 Culture tubes
Conical tubes made of polystyrene are not suitable for the robotic
harvester. Polypropylene tubes (15ml) are needed.
 We tested polypropylene BD Falcon tubes for toxicity and found that
they had no adverse effect on our cultures and Falcon tubes turned out
to be cheaper than our current culture tubes;
 The lids are slightly bigger than our current lids, although they still
fit into our racks and centrifuge buckets; they are not as transparent
our current polystyrene tubes.
 Lids must be removed and stored during the harvest; an additional
mechanism needs to be in place to return lids to the correct tubes;
furthermore any material still on the lid would not be fixed and may
affect the quality of preparations. We decided to discard the lids and
use fresh lids after the harvest. Using a fresh lid currently means using
two tubes per culture - we have been in contact with the manufacturer
to purchase packs of lids.
Considerations/Limitations
 Pre-fixation
 The harvester can incorporate a G step, or pre-fixation step, for
the addition of small volumes of fixative whilst the sample is in
hypotonic.
 The G step must be applied to the whole run; it cannot be limited to
certain sample types (e.g. newborn babies).
 Centrifuge r.p.m. is not readily adjustable
 It can be changed by one of the engineers. We did not need to
change the setting; we found that 1,000 r.p.m. was a suitable setting.
 Cannot use a third reagent
 We currently use 5% acetic acid for one of our two cultures; it is
not possible to use a third reagent with the harvester.
Considerations/Limitations
 Underspreading and “clumping” with current settings
 Some cultures on the robotic harvester had a tendency to be
underspread and require more effort during slide preparation; further
optimisation of the protocol might improve spreading
 Vortex settings
The speed of vortex is fully adjustable;
 However it can only be set at two different
speeds per harvest.
There are
two vortex
speeds
Oncology trial
 The oncology team have not yet carried out a validation trial;
they only processed a few samples.
 Comments from our oncology team:
– Initial results indicate that Hanabi preparations for
standard oncology bone marrow samples are equivalent in
quality to cultures harvested manually;
– Aspiration level cannot be changed throughout the harvest.
Tests so far suggest that it may not be possible to include
bone marrows with low counts in a routine Hanabi harvest as these generally have large pellets following centrifugation,
which would require individual adjustment of aspiration
volumes;
– There has been no evidence that culturing bone marrows in
the Falcon tubes is detrimental.
Fulfilling the criteria
The robotic harvester should:

- Produce preparations suitable for diagnostic use;

– Increase sample throughput;

– Be efficient and require no intervention during harvest;

– Show high reproducibility - no operator variability;
• 24 x 2 operators  64 cultures
• Takes ~1h58m to process 64 cultures;
• Easy to load, approx 15-20 mins hands-on time for preparation,
tube loading and cleaning of the robot
• Validation results confirm that preparations obtained with the
harvester are more consistent, and for MI this difference is
statistically significant (p =0.0004)

- Be robust and easy to use
• There are protocols available for recovering in case of
malfunction
Conclusions
from the Validation
 Our trial only lasted two weeks in October 2007- there is still
scope to optimise the robotic harvester settings and further
improve the quality of preparations.
 The preparations obtained, even prior to optimizing the
protocols, are comparable to our routine preparations and
perfectly suitable for diagnostic use.
 The robotic harvester is user-friendly, simple to programme,
straightforward to load and requires no supervision or
intervention.
Since then…
 We decided to purchase the harvester and successfully applied
for a grant from the Evelina Appeal Charity.
 We have been using the robot routinely for one of our two
cultures since January; to date, we have carried out over 50
harvests using the Hanabi PIII and harvested over 600
samples.
 Chromosome preparations are consistently good quality and
often significantly better than our second cultures harvested
manually.
 We aim to fully automate the harvest of blood lymphocytes in
the next few months.
 Settings have not been changed since the validation; we will be
experimenting with the settings to improve preparations
further.
Acknowledgements
Guy’s
Anne Bergbaum
Ian Kesterton
Sally Walsh
Paul Stevens
Shiply Begum
Sara Kadir
Kamal Uddin
Liz Allan
Helen Geoghegan
Paul Scriven
Shehla Mohammed
Transgenomic
Ben Nouri
Paul Hornsby