Roche cobas s 201 system User manual

Brand: Roche

Model: cobas s 201 system

Type: User manual

Roche cobas s 201 system is a fully automated sample preparation and PCR amplification system for molecular diagnostic testing. It processes samples in batches, with each batch consisting of samples and controls. The system can pipette large primary pools of up to 96 donor samples, and it also supports 2D pooling for retesting samples from reactive large primary pools. The cobas s 201 system is designed for use with Roche-manufactured external controls (RMECs) and allows up to five user-defined external controls (UDECs) to be assigned to each test.

07/2008, version 1.0 2.1

Pipetting 2

Batch Concept

The cobas s 201 system is designed to process samples in batches. A batch

is a collection of samples and controls that are pipetted, extracted, and

amplified and detected together according to the rules for the associated

test specification.

A batch consists of all of the samples and controls in one SK24 rack.

A batch is tracked from pooling through results review using the SK24

rack ID plus a unique batch ID assigned during pipetting.

Samples and controls in the batch are tracked by associating their scanned

barcode IDs with the unique barcode clips that hold S-tubes (during

pooling and sample preparation) and K-tubes (during amplification and

detection).

Figure 2.1

Batch

SK24 Rack ID

Unique S-tube

Barcode Clip

2.2 07/2008, version 1.0

Roche-Manufactured External Controls (RMECs)

Each batch requires Roche-manufactured external controls (RMECs).

The number of RMECs required is test-specific.

MPX testing encompasses five analytes. Five positive RMECs plus one

negative RMEC must be pipetted for each batch. During pipetting, an

aliquot of the negative control is transferred to the S-tube in position 19 of

each SK24 rack. Then, aliquots from each positive control are transferred

to S-tubes in positions 20 through 24 of each SK24 rack (Figure 2.2).

Pipetting of RMECs always occurs before sample pipetting. This

allows the operator to correct any control pipetting errors before

sample pipetting begins.

RMECs are always placed in the last positions in each SK24 rack so

that the entire testing process, from extraction through

amplification and detection, is monitored by control samples.

User-Defined External Controls (UDECs)

The cobas s 201 system allows up to five user-defined external controls

(UDECs) to be assigned to each test. UDEC requirements, including the

control name, barcode pattern, lot number, expiration date, and position

of the UDECs in the SK24 rack, are specified by the laboratory

administrator.

Once UDECs are assigned for a particular test, the operator can determine

whether to include them in a pipetting run.

If included, UDECs are always pipetted in the first SK24 rack.

UDECs are identified in the Roche PDM Pooling Manager and Roche

PDM Data Manager screens and reports.

Figure 2.2

MPX RMECs in an SK24 Rack

Negative

Positive

Controls

Control

Pipetting

07/2008, version 1.0 2.3

Deep-Well Plates

Identical deep-well plates are used as Library Plates and Intermediate

Plates during pipetting of large Primary Pools (Figure 2.3).

Every deep-well plate has a unique barcode label.

Library Plate

A Library Plate can be prepared during Primary Pooling to save an aliquot

from each donor tube in case Secondary Pooling is required.

Secondary Pooling can be performed directly from the donor tubes

if a Library Plate is not prepared or if a particular well in the

Library Plate is unusable.

Use of a Library Plate is an option that is configured during

installation.

The well position that a sample occupies in the Library Plate is

dependent on the number of samples in the run and the type of

pooling that is being performed.

Intermediate Plate

An Intermediate Plate is required when pipetting large Primary Pools.

The Intermediate Plate holds interim 12-specimen pools that are then

combined to form a large Primary Pool.

Details of pipetting large Primary Pools are described in the

following section.

Figure 2.3

Library Plate and Intermediate Plate

2.4 07/2008, version 1.0

Primary Pools

A large

Primary Pool

is a multi-specimen (

, or

donor samples)

pool that is created for initial sample testing.

Pipetting large Primary Pools is a two-step process consisting of 1) a Plate

Run and 2) a Batch Run. Each is a separate pipetting run.

Plate Run

During a Plate Run, aliquots from groups of donor samples are combined

in Intermediate Plate wells to create 12-

specimen

interim pools.

The number of donor samples that are loaded must be a multiple of the

final pool size (24, 48, or 96). The maximum number of donor samples

that can be pipetted depends upon the pipettor that is used:

•Up to 864 donor samples can be pipetted when the Hamilton

Microlab STAR IVD Pipettor is used

•Up to 384 donor samples can be pipetted when the Hamilton

Microlab STARlet IVD Pipettor is used

Pipetting a Plate Run for a 96-specimen pool is summarized below.

Pipetting a Plate Run for a 24- or a 48-specimen pool is similar.

• 1 mL of each donor sample is aspirated from the first group of

donor sample tubes and dispensed into wells in a Library Plate. The

process is then repeated with 700 μL aliquots from the same donor

sample tubes, resulting in the transfer of 1.7 mL aliquots of donor

samples to corresponding wells of the Library Plate (Figure 2.4).

• 135 μL is aspirated from the Library Plate wells and dispensed into

the first column of Intermediate Plate wells (used to store the

interim pools) (Figure 2.4).

If a Library Plate is not prepared, 135 μL aliquots are pipetted

directly into the Intermediate Plate from the donor sample tubes.

Figure 2.4

Pipetting of the First Group of Donor Samples During the Plate Run

(Example of a Plate Run for a 96-Specimen Primary Pool)

Library Plate

Intermediate Plate

32-Position Donor Tube Carrier

1 mL +700 μL

135 μL

Position 1

Position 32

Pipetting

07/2008, version 1.0 2.5

• The process is then repeated with the next group of donor samples.

1.7 mL of each donor sample in the group is transferred to the next

available wells of the Library Plate, and 135 μL is then aspirated

from those Library Plate wells and dispensed into the same column

of Intermediate Plate wells as the first group of donor samples

(Figure 2.5).

• The process continues until all of the donor samples that are

included in the first large Primary Pool have been pipetted and the

wells in the first column of the Intermediate Plate contain aliquots

of twelve donor samples

• The entire process is then repeated to create interim pools for each

additional large Primary Pool, using additional wells in the

Intermediate Plate for the additional interim pools.

At the end of the Plate Run, the Library Plate(s) contain 1.565 mL aliquots

from each of the donor sample tubes, and the Intermediate Plate wells that

are used each contain pooled 135 μL aliquots from twelve donor sample

tubes.

Figure 2.5

Pipetting of the Next Group of Donor Samples During the Plate Run

(Example of a Plate Run for a 96-Specimen Primary Pool)

Library Plate

Intermediate Plate

32-Position Donor Tube Carrier

1 mL +700 μL

135 μL

Position 1

Position 32

2.6 07/2008, version 1.0

Batch Run

During a Batch Run, aliquots are pipetted from Intermediate Plate wells

into S-tubes to create the large Primary Pool (Figure 2.6):

• 500 μL aliquots from two wells are pipetted into one S-tube for a

Primary Pool size of 24

• 250 μL aliquots from four wells are pipetted into one S-tube for a

Primary Pool size of 48

• 125 μL aliquots from eight wells are pipetted into one S-tube for a

Primary Pool size of 96

Intermediate Plates from more than one Plate Run can be loaded. The

maximum number of Intermediate Plates that can be pipetted depends

upon the pipettor that is used:

•Up to 5 Intermediate Plates can be pipetted when the Hamilton

Microlab STAR IVD Pipettor is used

•Up to 4 Intermediate Plates can be pipetted when the Hamilton

Microlab STARlet IVD Pipettor is used

Figure 2.6

Pipetting of Two 96-Specimen Pools During a Batch Run

Intermediate Plate

125 μL

Pipetting

07/2008, version 1.0 2.7

Repeat Batch Run

A Repeat Batch Run

creates another large Primary Pool to replace one with

invalid test result

The Repeat Batch Run uses the Intermediate Plate

that was created during the first step (the Plate Run) of the original

Primary Pooling run.

A Repeat Batch Run must be pipetted from the Intermediate Plate.

If an Intermediate Plate well is not available the samples are

scheduled for Resolution Pooling.

Load only one Intermediate Plate to preform a Repeat Batch Run.

During a Repeat Batch Run, aliquots from the same Intermediate Plate

wells used to create the original large Primary Pool (Figure 2.6) are

pipetted into S-tubes to create the replacement Primary Pool (Figure 2.7).

Figure 2.7

Repeat Batch Run for a 48-Specimen Primary Pool

Intermediate Plate

250 μL

2.8 07/2008, version 1.0

2D Pooling

2D Pooling (two dimensional pooling) is used to retest samples from

reactive large Primary Pools. During 2D Pooling, each sample is pipetted

into two different pools with a sample distribution (Figure 2.8) that allows

non-reactive sample(s) to be identified after a single run.

Samples from one Primary Pool can be processed during a 2D

Pooling run.

The number of 2D Pools that are created depends on the size of the

reactive large Primary Pool:

Figure 2.8

Sample Distribution in 2D Pools

(Example of 2D Pooling for a 48-Specimen Primary Pool)

Library Plate

Primary Pool 2

Primary Pool 1

Six-Specimen Pools

S-tube Positions 1-8

Eight-Specimen Pools

S-tube Positions 9-14

Pool Size Number of 2D Pools Aliquot of each Sample

24 4 pools of 6 specimens

6 pools of 4 specimens

167 μL

250 μL

48 8 pools of 6 specimens

6 pools of 8 specimens

167 μL

125 μL

96 Two batches each containing:

8 pools of 6 specimens

6 pools of 8 specimens

167 μL

125 μL

Pipetting

07/2008, version 1.0 2.9

Pipetting a 2D Pooling run for a 48-specimen pool is summarized below.

Pipetting a 2D Pooling run for a 24- or a 96-specimen pool is similar.

If a Library Plate is not used, if a Library Plate well is invalid, or if a

Library Plate well contains insufficient volume, aliquots can be

pipetted from donor sample tubes.

• 167 μL aliquots of donor samples are aspirated from four wells in

the first column of the Library Plate and dispensed into four

S-tubes, starting in position 1 of the SK24 rack (Figure 2.9).

• 125 μL aliquots of donor sample are then aspirated from the same

four Library Plate wells and dispensed into a single S-tube in

position 9 of the SK24 rack.

• 167 μL aliquots of donor samples are aspirated from four wells in

the next column of the Library Plate and dispensed into four

S-tubes, starting in position 5 of the SK24 rack.

• 125 μL aliquots of donor sample are aspirated from the same four

Library Plate wells and dispensed into the single S-tube in position

9 of the SK24 rack.

At this point, the S-tubes in positions 1 through 8 of the SK24 rack

each contain an aliquot of a single donor sample, and the S-tube in

position 9 contains an aliquot of eight donor samples (Figure 2.9).

Figure 2.9

Pipetting the First Eight Wells

(Example of 2D Pooling for a 48-Specimen Primary Pool)

Library Plate

Position 9

167 μL

125 μL

2.10 07/2008, version 1.0

• The process is then repeated for the next eight wells on the Library

Plate. 167 μL aliquots of donor samples are again dispensed into

the S-tubes in positions 1 through 8 of the SK24 rack, and 125 μL

aliquots are dispensed into a single S-tube in position 10 of the

SK24 rack (Figure 2.10).

• The process continues until all donor samples have been pipetted.

When the run is completed, the S-tubes in positions 1 through 8 of

the SK24 rack each contain aliquots of a six donor samples, and the

S-tubes in positions 9 through 14 each contain aliquots of eight

donor samples.

Figure 2.10

Pipetting the Next Eight Wells

(Example of 2D Pooling for a 48-Specimen Primary Pool)

Library Plate

SK24 Rack

167 μL

125 μL

Pipetting

07/2008, version 1.0 2.11

Confirmation Pooling

Confirmation Pooling is used to retest samples from reactive large

Primary Pools when there is additional evidence (e.g., a positive serology

test) that suggests one or more samples in the Primary Pool is reactive.

Confirmation Pooling is also used to retest Short Pools.

Samples that are suspected to be reactive are pipetted in individual pools.

The remaining samples are pooled in multi-specimen pools (Figure 2.11).

Samples from one Primary Pool can be processed during a

Confirmation Pooling run.

Up to four samples from a Primary Pool of 48 or 96 can be selected

for pipetting in single-specimen pools. Up to two samples from a

Primary Pool of 24 can be selected for pipetting in single-specimen

pools.

The multi-specimen pools are prepared as 12-, 11-, 6-, or 4-specimen

pools, depending on: 1) the number of samples in the Primary Pool and 2)

the number of donor samples selected for individual testing. The volume

of sample that is aspirated for each size multi-specimen pool is

summarized below:

Figure 2.11

Sample Distribution in Confirmation Pools

(Example of Confirmation Pooling for a 48-Specimen Primary Pool)

Library Plate

Primary Pool 2

Primary Pool 1

Multi-Specimen Pools

S-tube Positions 1-4

Single-Specimen Pools

S-tube Positions 5-8

Multi-Specimen Pool Size Aliquot of each Sample

12-specimen 92 μL

11-specimen 92 μL

6-specimen 167 μL

4-specimen 250 μL

2.12 07/2008, version 1.0

Pipetting during Confirmation Pooling is summarized below:

If a Library Plate is not used, if a Library Plate well is invalid, or if a

Library Plate well contains insufficient volume, aliquots can be

pipetted from donor sample tubes.

• Aliquots (92 μL, 167 μL, or 250 μL) of donor samples are aspirated

from wells in the first group of presumed non-reactive Library

Plate wells. Each sample aspiration is dispensed into an S-tube,

starting in position 1 of an SK24 rack.

• The process is repeated, skipping the wells (or donor tubes) for any

donor samples that are suspected of being reactive (Figure 2.12).

Figure 2.12

Pipetting Presumed Non-Reactive Confirmation Pools

(Example of Confirmation Pooling for a 48-Specimen Primary Pool)

Library Plate

SK24 Rack

92 μL

Pipetting

07/2008, version 1.0 2.13

• After all aliquots of presumed non-reactive donor samples have

been pipetted, 1 mL aliquots of the presumed reactive donor

samples are aspirated from their wells (or donor tubes) and

dispensed into individual S-tubes (Figure 2.13).

Figure 2.13

Pipetting Presumed Reactive Individual Pools

(Example of Confirmation Pooling for a 48-Specimen Primary Pool)

Library Plate

SK24 Rack

1 mL

2.14 07/2008, version 1.0

Resolution Pooling

A Resolution Pool is a single-specimen pool that is created for samples

that 1) have pipetting errors during the Primary Pooling run or 2) are

contained in a reactive Confirmation Pool or reactive 2D Pool. A

Resolution Pool is prepared by pipetting an aliquot from a Library plate

well into its own S-tube.

The laboratory administrator can also elect to use Resolution

Pooling to resolve invalid results.

The sample can be aspirated from the donor tube if a Library Plate

is not available or if the Library Plate well for that donor sample is

unusable.

Up to 36 samples can be processed during a Resolution Pooling

run.

During Resolution Pooling, 1 mL of a sample is aspirated from its well in

the Library Plate and dispensed into a single S-tube.

Figure 2.14

Pipetting Resolution Pools

Library Plate

SK24 Rack

1 mL

Wells selected for Resolution Pooling

Pipetting

07/2008, version 1.0 2.15

Handling of Pipetting Errors

Plate Run

During a Plate Run, the Hamilton Microlab STAR IVD / STARlet IVD

Pipettor combines equal aliquots from groups of donor samples to create

12-specimen interim pools in an Intermediate Plate well (see page 2.4).

A pipetting error during the Plate Run may result in one or more of the

Intermediate Plate wells having less than 12 specimens. An Intermediate

Plate well must contain 9 to 12 specimens to be valid. A well containing

less than 9 specimens is automatically invalidated by the system. Wells that

are invalidated by the system (and wells that are manually rejected by the

user) are not included in the Batch Run.

Batch Run

During a Batch Run, the Hamilton Microlab STAR IVD / STARlet IVD

Pipettor combines aliquots from the Intermediate Plate wells to create a

Large Pool in the S-tube (see page 2.6).

Intermediate Plate wells that are invalid are not pipetted during the Batch

Run. Instead, additional sample is pipetted from one (or more) valid

wells, so that the S-tube contains 1 mL, the volume required for successful

processing on the COBAS® AmpliPrep Instrument.

2.16 07/2008, version 1.0

Example 1 - Pipetting Error During a Pool of 96 Run

One donor tube (Rack 1, sample 16 in the example in Figure 2.15)

contains insufficient volume (or e.g., a clot) and is not successfully

pipetted during the Plate Run. Therefore, one Intermediate Plate well

(H1in the example) contains aliquots from 11 instead of 12 donor

samples.

An Intermediate Plate well containing 11 donor samples is valid. When

the Intermediate Plate is loaded for a Batch Run, the system pipettes

aliquots from all of the valid wells and creates a pool containing 95 donor

samples.

Figure 2.15

Example of Pipetting a Pool Containing 95 Donor Samples

First Group

Plate Run (one pipetting error)

Batch Run

Second Group

Third Group

Last Group

Pipetting

07/2008, version 1.0 2.17

Example 2 - Pipetting Error During a Pool of 96 Run

Several donor tubes (five in the example in Figure 2.16) cause pipetting

errors during the Plate Run. The result is that one Intermediate Plate well

(B1 in the example) contains 11 instead of 12 donor samples and one well

(H1 in the example) contains 8 instead of 12 donor samples.

The well containing 11 donor samples is valid and is included in the Batch

Run. The well containing 8 donor samples is not valid and is skipped. One

of the valid wells (A1) is pipetted twice to make up the required 1 mL

volume. The pool created during the Batch Run contains aliquots from 83

donor samples.

Figure 2.16

Example of Pipetting a Pool Containing 83 Donor Samples

First Group

Plate Run (five pipetting errors)

Batch Run

Second Group

Third Group

Last Group

2.18 07/2008, version 1.0

Pool Size

An Intermediate Plate well that contains 9 to 12 samples is

considered valid.

A minimum of one valid Intermediate Plate well is required to

perform a Batch Run.

During a Batch Run the system pipettes an additional aliquot from a valid

well when an Intermediate Plate contains an invalid well. For example, if

the Intermediate Plate contains one invalid well, one of the valid wells is

pipetted twice to create the S-tube pool. If the Intermediate Plate contains

two invalid wells, two of the valid wells are pipetted twice to create the

pool in the S-tube.

The maximum number of donors in a Large Pool is 24, 48, or 96,

depending on the system configuration. The minimum number of donors

in a Large Pool is 9.

When a pool contains a reduced number of donor samples, the sample

volume for some of the donors in the pool is increased. Donor sample

volume can vary from 1/96 (for a 96-specimen pool) to 1/9 of the total

volume.

Repeat Batch Run

The rules for pipetting samples from the Intermediate Plate for a Repeat

Batch Run are the same as the rules for pipetting in a Batch Run. Wells

containing 9 to 12 samples are included in the run. Wells containing less

than 9 samples are excluded. When an invalid well is excluded, a valid well

is pipetted in its place.

Secondary Pooling

Reactive short pools are not scheduled for 2D Pooling. Instead, reactive

short pools that contain 18 or more samples are scheduled for

Confirmation Pooling. Donor samples in reactive short pools that contain

fewer than 18 samples are scheduled for Resolution Pooling.

The samples that caused the pipetting error are either scheduled for

another Plate Run or they are scheduled for Resolution Pooling,

depending on when the pipetting error occurred.

Roche cobas s 201 system User manual

Roche cobas s 201 system User manual

Roche cobas s 201 system User manual

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