1. Validation of the Hanabi-PIII Robotic Metaphase Cell Harvester
Juliana Groisman, Richard Hall
ACC Spring Conference
Liverpool, March 2008

2. Overview Introduction and context Hanabi-PIII Robotic Harvester
Validation, data analysis and preparations
Considerations and limitations
Oncology trial
Conclusions and further work

3. 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.

4. 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.

5. 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

6. 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

7. HEPA filter
Touch screen menu

8. 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

9. 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 was left in our manual harvest, we started at aspiration level 14  but found that preparations were slightly cytoplasmic
Changing the aspiration level to 12 had a positive effect, preparations were clean and mitotic index was good
When we tried aspiration level 10, we found that our yields were affected

10. Validation Technical aspects
Our settings for the validation:
Centrifuge for 5 mins (1,000 rpm);
Aspirate to level 12
Start the vortex; add 5.5ml KCl (hypotonic solution);
Incubate for 6 mins;
Aspirate the supernatant to level 12;
Start the vortex; add 5ml fixative
slidemaking 3X
Harvest time was comparable to our manual harvest

11. 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.
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.
Preparation
takes approx 5 mins to prepare and 2-10 mins to load depending on sample size

12. Carry out reagent exchange – flushes the system with water
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
Harvest
takes 50mins-1h58 depending on sample size
Washing
takes approx 10mins

13. 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

14. A total of 3,000 metaphases were assessed
Validation
53 patients were processed for the validation.
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
Yield
Total number of cells obtained
Mitotic index
Calculated by Metasystems for the whole slide
Qualitative
Chromosome length
Spreading
A total of 3,000 metaphases were assessed

15. Results

16. Validation results Data analysis
Yield
Found to be slightly higher in the robotic harvester;
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
Comparable
Comparable
Comparable
Comparable

17. Preparations

18. Specimen 07/12308
Robotic harvester
Manual harvest

19. Specimen 07/12304
Robotic harvester
Manual harvest

20. 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.

21. Operator variability Poor Manual harvest (harvest 4)
Specimen 07/12516
Robotic harvester

22. 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.

23. 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.

24. 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

25. 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.

26. Fulfilling the criteria
The robotic harvester should:
- Produce preparations suitable for diagnostic use;
Increase sample throughput;
24 x 2 operators  64 cultures
Be efficient and require no intervention during harvest;
Takes ~1h58m to process 64 cultures;
Easy to load, approx mins hands-on time for preparation, tube loading and cleaning of the robot
Show high reproducibility - no operator variability;
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     

27. Conclusions from the Validation
Our trial only lasted two weeks in October 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.

28. 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.

29. 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