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

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.

Introduction

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

Slide making

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

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

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. Centrifuge for 5 mins (1,000 rpm);

3X

slidemaking

Validation Technical aspects

Harvest time was comparable to our manual harvest

1. Switch on the harvester2. Prepare fix and hypotonic solutions, top-up distilled

water3. Carry out reagent exchange – flushes the system

replacing water with reagents (fixative and hypotonic)

4. Select saved protocol (at this point parameters can be changed if required)

5. Set sample number (the number of tubes to be loaded). Dummy tubes containing water must be used to balance the centrifuge.

6. Take samples to Class II hood and add colcemid; start the timer.

7. 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

8. When the time in colcemid is up (15 minutes at Guy’s), press main start button and walk away.

9. When the harvest is finished, remove tubes and use fresh lids. Samples are ready for slidemaking.

10. Carry out reagent exchange – flushes the system with water

11. Carry out tube washing (soaking of aspiration probes for 3 mins), remove tubes and allow pump to dry (~5 mins)

12. Swab surfaces with virkon13. Empty waste containers 14. Switch off the harvester

Harvest

takes 50mins-1h58 depending on sample size

Washing

takes approx 10mins

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

Validation1. 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:

QuantitativeYield

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

Results

Validation resultsData analysis

Robotic harvester n=53 Manual harvest n=53 average variance average variance

Yield (total cells/culture) 179,603 14,445,419,924 170,257 8,959,414,637

Mitotic index 4.44% 14.07 6.17% 42.5

Quality (%QA 6) 52.97% 539.94 51.72 588.73

Spreading (% well spread) 68.96% 427.47 72.91% 551.91

Yield

Found to be slightly higher in the robotic harvester;

Mitotic IndexFound 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

Preparations

Manual harvestRobotic harvester

Specimen 07/12308

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.

Robotic harvester

Manual harvest

Operator variabilityPoor Manual harvest (harvest 4)

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 criteriaThe 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 15-20 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

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.

Conclusions from the Validation

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.

Since then…

AcknowledgementsGuy’s

Anne BergbaumIan KestertonSally WalshPaul StevensShiply Begum Sara KadirKamal UddinLiz Allan

Helen Geoghegan

Paul Scriven

Shehla Mohammed

TransgenomicBen NouriPaul Hornsby