Immunohistochemical staining

automatical coverslipping scanner

iSaCS

Annamaria Csizmadia, 3DHISTECH Ltd.

Dig

ital

imag

es,

Macrosta

tion

Au

tom

ati

cla

bo

rato

ry

instr

um

en

t

Tissue/Sample fixation

Grossing

Embedding

Deparaffination

Sectioning

Antigen retrieval

IHC staining

Coverslipping

Scanning

Image Analysis

Digital methods in the histology workflow

Scanner

Stainer

Staining-Coverslipping-Scanning Unit

Pannoramic MIDI

Leica BOND MAX

Agilent technologies, DAKO Coverslipper

Coverslipper

Leica Bond Max

Dako Omnis VentanaBenchMark

ULTRA

iSaCS

Deparaffination + + + -

HIER + + + -

Blocking of endogenoous enzyme + + + +

Primary antobody + + + +

Secondery antibody + + + +

Chromogen substrate + + + +

Counterstain + + + +

Mounting - - - +

Coverslipping - - - +

Scanning - - - +

Image Analyis - - - +

Automated phase of different stainers

Leica Bond Max Dako Omnis Ventana BenchMark ULTRA iSaCS

Slide capacity 30(10 slides/rack)

45(5slides/rack)

30(Individual chamber)

36(72 slides/rack)

Runtime 3:30 2:30 2:30 5:45

Slides/day 90 165 90 108

Number of Reagents

36 36 35 40

Contonous staining yes yes yes no

Staining Principles Capillary Gap Staining Dynamic GapStaining

Liquid Overly Technology Open individualslide staining

Deparaffination;HIER

yes yes yes no

UsedAntibody/slide

100µl 100µl 100µl 47µl

First digital slide Min. 04:00 Min. 03:00 Min. 03:00 02:15

Comparison of different stainers

iSaCS - System architecture

1. Digital slide scanner

2. Slide preview module

3. Slide moving arm

4. Pipetting module

5. Reagent changer

6. Incubation module

7. Wash & rinse chamber

8. Air wiper module

9. Coverslipping module

Scanner, Preview imaging, Slide moving arm

1. Digital slide scanner:

•Improved Midi scanner

•Flash scanning technology

•Fluorescent modification

2. Preview imaging of unstained samples:

•Special high-contrast illumination

•Localization of tissue sample on the slide

•Reagent amount based on tissue size (µl/mm2)

•Dispensing trajectory covers tissue area exclusively

•Slide identification by 2D code

3.Slide moving arm:

•Transports slides between modules

Pipetting unit, Reagent changer

4. Pipetting unit:

•Liquid level sensor

5. Reagent changer:

•Reagent carrier chain: adjustable number of chain links (max. 40)

•Chain link: 8 ml reagent vial + disposable pipetting tip (200 µl)

•Reagent vial identification by 2D code, pipetting tip sensor

•Reagent tray with lid: removable, fits into the refrigerator

Coverslipping unit, Incubation tray

8. Coverslipping unit:

•Mounting medium (aqueous, aqueous with hardening)

•Disposable pipetting tip (200 µl)

•Glass coverslips (22x50 mm)

•Vacuum: pump,

•Pick-and-place suction cup

•Separation of coverslips

•Precise positioning eliminates bubbles

•Liquid level sensor

•Pipetting tip sensor

9. Incubation tray:

•72 slides in compact arrangement

•Chambers eliminate evaporation

Wash & Rinse unit, Airwiper

6. Wash & Rinse unit:

•11 parallel Wash chambers

•1 chamber only for rinsing

•Flooding/draining

•3 peristaltic pumps:

• Wash buffer, DI water

• Hazardous / Non-H waste

•4 removable 5l tanks: tank ID

•Tank cap: tubing, level sensor

7. Airwiper removes liquid film:

• Airblade avoids desiccation

• Suction tray avoids spilling

Prepared slide forstaining

PIS

/LIS

Recues

sta

inin

gpro

cedure

Slide recognation

Slide Barcodereading

ID recognationbased on

Label Area

Staining arearecognation based on

DFP

Protocolcreation

ProtocolAssignement

Extrenal request from Database User-defined System-defined

PREPARATION

STAINING

Staining of the definedtissue area

COVERING

Coverslipping of the stained

slide

IMAGE ANALYSIS

SCANNING

Scanning of the stained

area

Image analysis of whole slide

Trecking/Database

iSaCS sample detection

Active area for stain

iSaCS Protocol Editor

Structure of Protocol

Protocol

Protocol parts

Blocking Primery antibody Secondary antibody Labeling Finishing

Pro

toco

lste

ps

- Pipetting- Incubating

- Rinsing- Washing

- Covering- Scanning- Quantification

Reagent recognition

Undetermined vials

- The system recognized them as empty vials

- Manual Reagent attachment

Automatic reagent recognition

- Predefined reagent type to the vial

- Deliver with the system

Changes in the software of iSaCS

New failure management :

- Covering unit: we check the vacuum after the coverslip is taken up

- Scan : Continous communication between the staining and

scanning parts

Improuved reagent recognation:

Virtual reagent card shows the needed reagent type for the staining

Emergency STOP

Changes on the hardware

Covering unit:

- Opto sensor for X axis

- Keyence Sensor for tip detection

- Mounting medium level engraving

- Mounting medium level clamp

- Accessories gate

Moving arm:

• Belt guide

• Fixed vane

Changes on the hardware

• Pipetting unit:

- Opto sensor for X axis

• Accessories:

- Teflon coated reagent chamber

- Complete new tanks

- Black painted incubator

- Slide deflector for the incubator chamber

iSaCS – The main features

A single device integrating an immunostainer, a coverslipper and a scanner

Cost-effective open system, large capacity, bench-top construction saves lab-space

The patented automatic sample detection makes only the effective sample area

gets stained

Standardized and controlled sample treatment without human intervention

Parallel task execution, modular construction

First stained slides in CaseCenter can be available on-line right away:

speedy evaluation

iSaCS – The main features

Digital scanning and staining optimized using pre-staining preview images

Fluorescent IHC features: preview, scanning, archiving without degradation

Quick protocol definition, and easy handling of consumables

iSaCS scanning device can be used separately as individual scanner

Image analysis process will be run automatically on the whole slide

Slide and protocol recognition based on barcode

Easy to use software with hierarchical user interface

What will be new in 2018?

• New integrated scanner workflow into iSaCS software

• Operation log under the staining/covering/scanning process

• Auto Dilatation on the preview of tissue

Thank you for the attention