Oxford Chipcytometry Days

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The Chipcytometry User Meeting, Wednesday, December 2nd 2015

University of Oxford, Peter Medawar Building for Pathogen Research

Chipcytometry is a fairly new technology enabling deep immune cell phenotyping and biobanking of precious (clinical) samples. Chris Willberg’s group participates in Zellkraftwerk’s Early Access program and the team has gained first-hand experience using Chipcytometry since early 2015 for a range of projects. In a user-meeting kind of workshop first-hand information among researchers was shared.

[CLICK ON THE TITLES TO DOWNLOAD THE TALKS]

Later-on, attendees had the chance to have a look at the Zellscanner ONE at Chris Willbergs Lab:

OxfordScanner

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Zellkraftwerk public SOPs

Find below the links to our public SOPs for sample preparation:

SOP DATE
human PBMC 2013-11-08
human whole blood / erythrocyte lysis 2014-03-14
human CSF 2013-09-29
human BALF cells 2012-11-01
primary cells from short-term cell culture 2012-11-01
leukocytes from mouse lung cell culture 2012-11-01
Preparation and fixation of tissue cryosections on ZellSafe_T chips 2014-11-04
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Publications describing aspects of the technology


 

A versatile platform for comprehensive chip-based explorative cytometry.
Hennig C, Adams N, Hansen G. Cytometry A. 2009.

 

Publications using Chipcytometry


Gain-of-function STAT1 mutations are associated with intracranial aneurysms.
Dadak M, Jacobs R, Skuljec J, Jirmo AC, Yildiz Ö, Donnerstag F, Baerlecken NT, Schmidt RE, Lanfermann H, Skripuletz T, Schwenkenbecher P, Kleinschnitz C, Tumani H, Stangel M, Pul R. Clin Immunol. 2017.

 

Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice.
Skuljec J, Cabanski M, Surdziel E, Lachmann N, Brennig S, Pul R, Jirmo AC, Habener A, Visic J, Daluege K, Hennig C, Moritz T, Happle C, Hansen G. Eur J Immunol. 2016.

 

a-NAC-specific autoreactive CD8+ T cells in atopic dermatitis are of an effector-memory 2 type and secrete IL-4 and IFN-y.
Roesner LM, Heratizadeh A, Wieschowski S, Mittermann I, Valenta R, Eiz-Vesper B, Hennig C, Hansen G, Falk CS, Werfel T. Journal of Immunology. 2016.

 

Der p1 and Der p2-specific T cells display a Th2, Th17, and Th2/Th17 phenotype in atopic dermatitis
Roesner LM, Heratizadeh A,Begemann G,Kienlin P,Hradetzky S,Niebuhr M,Eiz-Vesper B,Hennig C,Hansen G,Baron-Bodo V,Moingeon P,Werfel T. Journal of Investigative Dermatology. 2015.

 

Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis
Happle C, Lachmann N, Skuljec J, Wetzke M, Ackermann M, Brennig S, Mucci A, Chari AJ, Groos S, Mirenska A, Hennig C, Rodt T, Bankstahl JP, Schwerk N, Moritz T, Hansen G. Science Translational Medicine. 2014.

 

Identification and quantification of basophils in the airways of asthmatics following segmental allergen challenge.
Dijkstra D, Hennig C, Hansen G, Biller H, Krug N, Hohlfeld JM. Cytometry A. 2014.

 

GATA1s induces hyperproliferation of eosinophil precursors in down syndrome transient leukemia.
Maroz A, Stachorski L, Emmrich S, Reinhardt K, Xu J, Shao Z, Kaebler S, Dertmann T, Hitzler J, Roberts I, Vyas P, Juban G, Hennig C, Hansen G, Li Z, Orkin S, Reinhardt D, Klusmann JH. Leukemia. 2013.

 
Distinct phenotypic features of neonatal murine macrophages.
Winterberg T, Vieten G, Meier T, Yu Y, Busse M, Hennig C, Hansen G, Jacobs R, Ure BM, Kuebler JF. Eur J Immunol. 2014.

 
High-content cytometry and transcriptomic biomarker profiling of human B-cell activation.
Hennig C, Ilginus C, Boztug K, Skokowa J, Marodi L, Szaflarska A, Sass M, Pignata C, Kilic SS, Caragol I, Baumann U, Klein C, Welte K, Hansen G. Journal Allergy Clin Immunolo. 2013.

 

Basophils from humans with systemic lupus erythematosus do not express MHC-II.
Dijkstra D, Hennig C, Witte T, Hansen G. Nature Medicine. 2012.

 

Lung function and inflammation during murine Pseudomonas aeruginosa airway infection.
Woelbeling F, Munder A, Kerber-Momot T, Neumann D, Hennig C, Hansen G, Tuemmler B, Baumann U. Immunobiology. 2011.

 

CD3+CD20+ T cells in peripheral blood: Proper flow cytometry is a prerequisite of exact phenotyping.
Wilk E, Witte T, Marquardt N, Hennig C, Hansen G, Schmidt RE, Jacobs R. Arthritis Rheum. 2010.

 

Successful treatment of autoimmune and lymphoproliferative complications of patients with intrinsic B-cell immunodeficiencies with Rituximab.
Hennig C, Baumann U, Ilginus C, Horneff G, Foell J, Hansen G. Br J Haematol. 2010.

 

Allergy prevention starts before conception: maternofetal transfer of tolerance protects against the development of asthma.
Polte T, Hennig C, Hansen G.J Allergy Clin Immunol. 2008.

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Mission accomplished: Longterm cellculture on ZellSafe chips works :-)

We often have been asked if it would be possible to seed cells on our chips and let them grow until confluency and perform assays followed by Chipcytometry on these cells. Well, in theory it should work since the chips are equipped with standard microfluidic connectors. So we gave it a try: We seeded 16 HDE cells on the chips, connected a microfluidic pump to the chip and allowed the medium to recirculate between chip and reservoir for 72h in an incubator.

This image shows the cells after seeding:

image

 

 

 

 

 

After 72h, the cells nicely formed an confluent cell layer:

livecell

 

 

 

 

 

So let’s use it for some fancy stuff…

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ZellExplorer – Quick Guide Datawizzard

ZellScanIconDatawizzard – How to…

Start Datawizzard

 

Select a marker

 

Exclude/include marker from analysis

 

Rename displayed marker caption

 

IMAGE VIEW

Select cell

 

Add cell

Click on any position on the image.

Change cell size

Place mouse pointer on the cell to change size.

Press and hold the [CTRL] key. Scroll mouse wheel while holding the [CTRL] key. This will change the cell size

Move cell

 

Cell eraser

Press and hold the [SHIFT] key. This activates the delete mode. Every cell the mouse pointer touches will be deleted permanently.

 

Display segmented cells / sub-segments / gates /

 

Copy image of selected cell  (only selected marker) to clipboard

 

Copy image of selected cell  (all markers) to clipboard

 

 

 

 

2D PLOT

 

Select markers to display by drag and drop

 

Generate Gate

 

Change High/Med Gate

 

Copy 2D plot to clipboard

 

STATISTICS

 

Calculate Statistics

 

Copy

 

Copy data to clipboard

 

INFO WINDOW

 

Marker/Scan

 

Histogram

Set High/Med and Med/Low Gates

 

Gates

Select Gate to display / calculate statisitics

Delete Gate

Change Gate name

Change Gate color

 

Segmentation/Values

Order Segmentation

Select Segmentation

Order Value Calculation

Select Value Calculation

Export csv/FCS

 

 

CHANNEL VIEW

 

Please note that only the 1st 150 positions will be displayed at the moment in the channel view

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ZellExplorer UI

ZellScanIcon ZellExplorer App – The User Interface

 

USER INTERFACES

  1. Login
  2. Main Window
    1. Project Management
    2. Store
    3. User Management
  3. Datawizzard
    1. Info-window
      1. Sample
      2. Segmentation/Value
      3. Gates
      4. Histogram
    1. Channel view
    2. Raw-Image
    3. 2D-Plot
    4. Statistics
  1. Order Markers
  2. Order Scans
  3. Chip batch editor
  4. Qualify sample

 

 

 

Start ZellExplorer

Enter Username

Enter Password

Press Enter or press the LOGIN button

 

TROUBLESHOOTING:

ERROR CODE: „***Anzahl der Lizenzen nicht ausreichend“

ERROR CODE: „**Keine Verbindung zur MongoDB“

ERROR CODE: „ ** Kann ZKW.config nicht laden“

 

USER MANAGEMENT

OWNER MANAGEMENT

DATAWIZZARD

PROJECT MANAGER

STOCK/STORE

 

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ZellScan App

ZellScanIcon

 

 

Start ZellScanner ONE

This is the start-up sequence:

1. Switch on the power supplies for the mercury lamp [A] and the microscope [B]

IMPORTANT: The mercury lamp takes about 15 minutes to heat-up and self-calibrate. You must not start a scan before the LED on the mercury lamp housing turns permanently green [C].

2. Switch on the microscope [D]

3. Switch on the computer [E] + monitor [F]

4. Login to the computer using the credentials provided by your system administrator.

5. Start the ZellScan App and login with your ZellScanner ONE user account (ask your administrator, if you don’t have one).

TROUBLESHOOTING:

  • ZellScan App cannot connect to database and does not start: The ZellScanner ONE server is down. Please ask your administrator or contact ZELLKRAFTWERK support (support@zellkraftwerk.com).

Calibrate your ZellScanner

If your ZellScanner has been switched off, you have to re-calibrate your ZellScanner (since inbetween, someone might have changed something on the system). Therefore, please use the provided calibration slide [G], insert it into the chip holder of your ZellScanner [VIDEO], move the stage to a position inside the calibration pattern [G1] and then

1. press ‘Adjust Axis’ within the ZellScan App.

2. MANUALLY focus.

3. Press OK when you are done with focussing.

 

TROUBLESHOOTING:

Scan a chip

Insert the chip properly into the chip holder [VIDEO]. The Barcode should face up and to the right. Click on the Barcode-reading field on the ZellScan App [H]. Scan or enter the barcode of the chip. If there is an open scan job for this chip, it will be displayed below. You can start this job by pressing ‘Start Scan’ [I].

TROUBLESHOOTING:

Tissue Cytometry: Define Area of interest (prior to first scan)

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ZellExplorer – Quick Guide Projects, Chips, Scans

ZellScanIconProjects, Chips, Scans – How to…

 

Introduction

The ZellExplorer App helps the user to keep an overview of running projects, results, but also supports stock management, biorepository functionality for chips and automatic generation of electronic lab book entries for each step during Chipcytometry analysis of samples – from sample to data. It follows the spirit of GLP to enable complete traceability, searchability and comparability of all involved items and processes.

Here are the main functionalities covered by ZellExplorer:

1. Stock Manager

All items that will be used in Chipcytometry (chips, antibodies, buffers) have to be declared and described in ZellExplorer’s stock manager. New items can be added and their properties like antibody’s clone and dye, amount of fluid, vendor or max. shelflife can be specified for optimal stock management.

2. Biorepository Manager

Each item (‘Ingredient’) and each storage place (‘Container’) in Chipcytometry has an unique ID. Additionally, it’s primary storage place has to be specified. This enables the tracking system to display for each item/container its storage path (e.g.: box – shelf – room – floor – building). Furthermore, the storage history is also traceable, so storage/shipping conditions over the complete lifecycle of an item can be monitored.

3. Project Manager

The project manager allow to group chips into chip groups that can be placed in one or multiple projects thus helping you not to loose the overview over hundreds of samples in parallel.

4. Electronic Lab book

All actions performed by a user in the wetlab (like adding a sample to a chip, staining a sample with an antibody or preparing an antibody working solution) have to be documented in a standardized way using the electronic labbook function of Chipcytometry to allow for comparability and searchability of all performed analyses in spirit of GLP.

Therefore each action that is linked to the electronic labbook will generate a standardized user interface asking for specification of the used items, startpoint, duration and involved executors (human, robot) of the action.

5. Datawizzard

The Datawizzard has been designed to support your exploration of the data generated by staining biomarkers using Chipcytometry. In the background,  a service on the ZKWServer is performing cell recognition and fluorescence value calculation (only on single cell chips)

… add a new stock antibody to the store:

1. Open the ‘Stock Manager’ tab

2. From the ACTION dropdown menu, select ‘Add item’

3. On the appearing treeview selection dialog, browse ‘Ingredient –> Solution –> Protein-Solution –> Antibody stock solution’ and click ‘>Generate new’

4. When asked for the number of item to add, type ‘1’.

5. Enter the name of the antibody as it should be displayed later (e.g., ‘anti-human CD4 PE’)

6. The item is generated in the database. Select the item from the stock list (left top) by clicking on the item

7. Enter the parameters (left – bottom) like Epitope, Clone, Amount…

IMPORTANT: Modification/Label, Amount and Aliquot Size MUST be specified!

… add a new box/drawer/ insert/… to the store and select its primary storage:

1. Open the ‘Stock Manager’ tab

2. From the ACTION list, select ‘Add item’

3. On the popup-menu, select in the ‘Container’ section the needed container (e.g. ‘Box’, ‘Drawer’…) and click ‘>Generate new’

4. Enter the name of the container as it should be displayed later (e.g., ‘box for mouse antibodies 1’)

5. The item is generated in the database. Select the item from the stock list (left top) by clicking on the item

6. Enter the maximum number of items that can be contained in this container

7. On the right (‘>Select Action’ list), click ‘Change primary storage’

8. On the popup-menu, select the primary container type (e.g. ‘Box’ for antibodies and click ‘> Select from database’

9. The Advanced Search window opens. Search for the right container, select by clicking within the search results (left-bottom) and click ‘-> Select’

… activate bought chips:

1. Open the ‘Project Manager’ tab

2. Open the ‘Dashboard’ tab in the project manager

3. From the ACTION list on top of the App, select  ‘Activate bought chips’

5. Within the FileDialog, navigate to the folder where you have stored the XML-File containing your chips, select this file and click ‘> Load File’

… specify or change primary storage of chip(s):

1. Open the ‘Project Manager’ tab

2. From the ACTION list, select ‘Chip batch editor’

3. Within the Chip batch editor, scan/enter the barcode(s) on the chip(s) [‘Scan/Type Barcode’]

4. Select ‘Chips –> Biorepository’ from the ‘Select Action’

5. On the popup-menu, select the primary container type (e.g. ‘Box’) and click ‘> Select from database’

6. The Advanced Search window opens. Search for the right container, select by clicking within the search results (left-bottom) and click ‘-> Select’

7. Within the FileDialog, navigate to the folder where you have stored the XML-File containing your chips, select this file and click ‘> Load File’

… get a marker stained and scanned on a chip:

This general workflow and additional requirements are necessary to get a chip stained and imaged:

1. A sample has to be assigned to the chip in the ZellExplorer – Project Manager (see ‘…specify a sample on a chip‘)

2. The antibody stocks have been added to the stock/storage (see ‘…add a new stock antibody to the store‘)

3. The antibodies have to be ordered for the chip (see ‘…order a stain‘)

4. The working solutions has to be produced for this antibody and chip (see ‘…execute a working solution‘)

5. The stain has to be executed (see ‘…execute a stain ‘)

6. By execution of the stain, you will be asked to acknowledge the automatic generation of a scan job. Doing this, the stain will be changed from ‘ordered’ to ‘planned’ and is available in the ZellScan App for scanning.

… open a chip in the project manager:

1. Open the ‘Project Manager’ tab

2. Enter/Scan the chip barcode. The chip will be displayed in the Dashboard tab

3. Doubleclick the chip in the search result list in the Dashboard tab

4. A new tab containing the chip will open

TROUBLESHOOTING:

Problem: The chip is not displayed in the Dashboard tab

Solution: Activate the chip (see ‘…activate bought chips‘)

… specify the sample on a chip:

If no sample is specified for a given chip, no stains or scans can be ordered. Therefore, the sample on the chip must be specified before analyzing biomarkers:

1. Within the respective chip tab, click ‘add sample’ (left-top)

2. You will be asked whether you would like to use an already existing sample [NO] (e.g. prepared PBMC) or specify a new sample [YES].

3. The type of sample (e.g., tissue section, cell solution has to be specified)

4. If you chose to generate a new sample, the sample specification dialog will appear. Here, you can currently only specify the sample source (the individual the sample is derived from). You can specify specific properties like patient ID, age, weight.

5. After specifying the sample source, click ‘> Generate Sample’ (right-bottom)

6. In the popup-dialogs, specify the specific information about fixation of the sample on the chip (Executor, Time, Duration)

7. Select the background scans and additional options (like scanning with/without gaps between positions)

… order a stain

Before you can order any marker/stain, please make sure that you specified the sample on chip. Without specified sample source, no markers can be ordered.

Method 1 (for a single chip or a chipgroup)

1. Within the respective chip or chipgroup tab, from the ACTION list, select  ‘Order Markers’

3. In the Order Stains Dialog window, search and select the markers to be stained. You can speed up search by typing the epitope information into the search box and/or narrowing down the search results by filter for different dyes.

ALTERNATIVELY, you can press ‘Load from old chip’ and type/scan the ID of an already analyzed chip. The ordered markers from this chip will be put in the shopping card.

4. Click ‘Add to card’ to add your selected markers to the shopping card. You will be asked to specify staining parameters (temperature, time, compartment) and priority for the selected stains.

NOTE: For each ‘Add to card’ you can only select one set of staining parameters, so you can first select all surface markers and press ‘Add to card’ followed by a new selection of the intracellular markers – again followed by ‘Add to card’.

You can remove ordered markers any time by selecting one or more markers and click ‘Remove from card’ or press DEL.

5. Click ‘>> Order Stains’ to generate planned stain jobs for the selected chips.

Method 2 (for multiple chips not in the same chipgroup)

1. Open the ‘Project Manager’ tab

2. From the ACTION list, select  ‘Chip batch editor’

3. Within the Chip batch editor, scan/enter the barcode(s) on the chip(s) [‘Scan/Type Barcode’]

4. Select ‘Stains’ from the ‘Select Action’

5. Select ‘Order’ from ‘> Action’ (left-middle)

3. In the Order Stains Dialog window, search and select the markers to be stained. You can speed up search by typing the epitope information into the search box and/or narrowing down the search results by filter for different dyes.

ALTERNATIVELY, you can press ‘Load from old chip’ and type/scan the ID of an already analyzed chip. The ordered markers from this chip will be put in the shopping card.

4. Click ‘Add to card’ to add your selected markers to the shopping card. You will be asked to specify staining parameters (temperature, time, compartment) and priority for the selected stains.

NOTE: For each ‘Add to card’ you can only select one set of staining parameters, so you can first select all surface markers and press ‘Add to card’ followed by a new selection of the intracellular markers – again followed by ‘Add to card’.

You can remove ordered markers any time by selecting one or more markers and click ‘Remove from card’ or press DEL.

5. Click ‘>> Order Stains’ to generate planned stain jobs for the selected chips.

… execute a working solution

1. Within the respective chip tab, click on the test tube icon next to the ordered stain

2. Enter the name of the antibody as it should be displayed later (e.g., ‘anti-human CD4 PE’)

… execute a stain:

1. Within the respective chip tab, click on the gearwheel icon next to the ordered stain

2. Specify the requested information (Workingsolution, Executor)

3. The App will automatically select and display the necessary resulting scan job(s). You might manually modify this in dialog.

TROUBLESHOOTING:

Message: ‘No working solution available’

Solution:execute working solution(s)

… cancel a stain:

1. Within the respective chip tab, click on the trash can icon next to the ordered stain and acknowledge to delete the ordered stain.

___________________________

Start a new projekt

Add subproject

Remove subproject

Add chipgroup to project

Remove chipgroup from project

Add chips to chipgroup

Print ToDo list of ordered markers

(requires Microsoft Word)

Cancel ordered scan

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ZellScanner ONE system – Overview

Zellscanner2

 

ZellScanner ONE consists of selected hardware und software components that are seamless integrated for an easy-to-use Chipcytometry workflow. The user interfaces follow the principles of minimalistic design – which allows the scientist to focus on the important facts: projects, samples, and data visualisation. The modular hardware concept allows the extension of the system for additional scanners, liquid- and chiphandling robots or data storage.

ZellScanner ONE system setup:

ZSO-OV

ZellScanner ONE Hardware

ZellScanner ONE User Interface

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Digital Pathology

Using ZellSafe Tissue chips, we use Chipcytometry to stain cell-bound biomarkers directly in-situ on cryosections and biopsies. Zellkraftwerk partners with drug developers to set-up and execute 30-plex biomarker panels that support oncology drug development programs worldwide. Our assay services are highly-customizable and are designed to support a range of development activities – ranging from early biomarker identification to late stage clinical trials to companion diagnostic development. In tissue cytometry our core expertise is whole slide scanning using 30-plex assays and generation of quantitative cytometric data using tissue-type segmentation as well as segmentation of single cells.

These are the features we are offering in tissue cytometry:

  • Tumor microenvironments: Chipcytometry enables assessing immune response in tumor microenvironments through 30-plexing biomarkers.
  • Accuracy: Highly accurate detection of tumor regions, cells and cellular structures.
  • Quantitative: Multi-dimensional, quantitative readout for Biomarker expression on per cell basis.
  • Pathologist experience: Tissue Cytometry delivers images familiar to pathologists for supporting them in making biomarker-based decisions.

To learn more about Zellkraftwerk’s digital pathology services, just schedule a personal webinar with Zellkraftwerk’ clinical service team.

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