EuroFlow - a guideline for Leukemia & Lymphoma flow cytometry panel

This document serves as an internal guide to prepare for customer visits 1. Situation
In 2009, the Euroflow Consortium (www.euroflow.org) issued a guideline for the flow cytometric immunophenotyping of normal, reactive and malignant leukocytes1). The purpose of the guideline was to move the flow cytometry community closer to standardization. The work will allegedly be published in 2010 in a peer-reviewed journal but that journal has not yet been disclosed. Unlike other previous publications aimed at standardization, the EuroFlow guideline is the first to recommend defined fluorochrome combinations in context of specific clones and vendors. The Euroflow Consortium is a non-profit organization2), actively participating in numerous flow cytometry events and congresses worldwide. Participation at these venues includes lectures and presentations aimed at building awareness and enrollment with the EuroFlow guideline. The guideline is put forward as a cookbook no-questions asked guide-line. The Consortium consists of a handful of the most prestigious scientists within flow cytometry. In total, the panel consists of 124 different flow conjugates, with a split in terms of listed reference products including vendor being: BD: 63 conjugates plus 15 custom conjugates or 63% BC: 12 conjugates or 10% Dako: 7 conjugates or 6% Other: 27 conjugates or 21%

In figure 1, the overall panel from the guideline is depicted. For every disease category highlighted by a red circle, the EuroFlow Consortium has put together an 8-color panel comprising one or more tubes. When each panel is analyzed for Dako portfolio 3 categories emerges: Dakos product is the reference product in the recommended panel Dakos portfolio includes the recommended fluorochrome/antibody/clone but Dako is not recommended Dakos portfolio includes the recommended fluorochrome/antibody combination but with a different clone

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1) EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes, J.J.M Van Dongen et al. http://www.euroflow.org/imagenes/news/euroflow_handout_on_antibody_panels.pdf 2) www.euroflow.org

Table 9

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Below table summarizes the result:

Dako is listed as reference product Panel 1 Acute leukemias screening panel (ALOT) Panel 2 Mature lymphoid cells (LST) MPO/FITC (F0714) CD79a/RPE (R7169) Dako has required clone/fluorochrome but is not listed as reference product CD3/CY (PB982) Dako has the recommended antibody conjugate with different clone

CD56/RPE (R7251*)

Panel 3 Mature lymphoid cells (SST) Panel 4 Plasma cell dycrasias (PCD) Panel 5 B-cell precursor ALL (BCPALL) CD45/PB (PB986) Cylg/APC (C0222) TdT/FITC (F7139)

CD56/RPE (R7251)*

CD56/RPE (R7251)* CD117/APC (C7244) CD117/APC (C7244)

CD4/PB (PB983) CD8/FITC (F0765) CD3/APC (C7225) smlg/FITC (F0434) smlg/RPE (R0436) CD3/APC (C7225) CD8/FITC (F0765) smlg/FITC (F0434) smlg/RPE (R0436) CD38/FITC (F7101)* CD28/RPE (R7163) CD15/FITC (F0830) CD33/RPE (R0745) CD13/RPE (R0715) CD22/APC (C7281) cylg/FITC (F0058) CD2/FITC (F0767)

Panel 6 T-cell ALL (T-ALL) Panel 7 Acute myeloid leukemis and myelodysplastic syndromes (AML & MDS)

Tdt/FITC (F7139)

TdT/FITC (F7139)

CD3/PB (PB982) CD117/RPE (R7145) CD13/RPE (R0715) CD64/RPE (R7219) CD56/RPE (R7251)

Panel 8 B-cell chronic lymphoproliferative diseases (B-CLPD) Panel 9 T-cell chronic lymphoproliferfative diseases (T-CLPD)

CD23/FITC (F0765)

CD56/RPE (R7251)* CD95/RPE (R7154) CD103/FITC (F7138)

Panel 10 NK-cell chronic lymphoprolifeartive diseases (NK-CLPD)

CD16/FITC (F7011) CD13/RPE (R0715) CD15/FITC (F0830) CD22/APC (C7281) CD41/FITC (F7088) CD25/RPE (R0811) CD61/FITC (F0803) CD8/FITC (F0765) smlg/FITC (F0434) smlg/FITC (R0436) CD3/APC (C7225) CD4/PB (PB983) CD5/FITC (F0795)* CD7/FITC (F7276)* CD25/RPE (R0811) CD27/FITC (F7178) CD57/FITC (F7270) CD57/FITC (F7270) CD25/RPE (R8011) CD5/APC (C7242)*

*Please consult Appendix B for results from Dakos internal competitor comparison studies.

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2. Which are the EuroFlow issues that Dako needs to deal with?
Many customers consider the EuroFlow recommendations as the future for leukemia/lymphoma immunophenotyping. The perception is that the fluorochrome combinations selected by EuroFlow including clones and vendors were selected as a result of best-in-class performance. Here are the facts: The Euroflow guideline does not list the number of vendors tested for each marker The Euroflow guideline does not list the number of clones tested for each marker The Euroflow guideline does not reveal the criteria a product must meet to be referenced Some customers believe that they do not need to validate their panels as long as the combination complies with the Euroflow recommendations this is not the case Apparently, BD has bought the name and logo EuroFlow

From the ESCCA Euroflow meeting 2010, it was announced that: Euroflow may potentially only have tested one or two vendors for each antibody, thus it would be impossible to know it the recommended products are indeed best-in-class Dako was never contacted during the process BD was chosen because they offered high discounts

3. Market reaction & Objection handling scenarios

Objection 1: I believe that the references recommended by the EuroFlow panel are of the best quality Reply: The Euroflow panel has not tested all clones from all vendors and nowhere in the guideline do they state to have done so. Apart from the reagents where EuroFlow list Dako as vendor, we have 9 products with the recommended clone/fluorochrome combination and 22 reagents of requested antibody/fluorochrome combination but with a different clone (please see section 1 and Appendix for more details). Further to that, EuroFlow has not defined explicit specifications reflecting the required quality and it is unlikely that Dakos products should not be of at least equal quality, both in terms of signal-to-noise ratio and the explicit degree of fluorescence intensity. Objection 2: When I use the EuroFlow panel I dont need to validate the setup

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Reply: It is correct that you dont need to validate the exact composition of antibody/fluorochrome combination. But when it comes to validating the individual components of the panel there are no exceptions to the Clinical & Laboratory Standard Institute recommendations reflected in guidelines H42a and H43a. In fact EuroFlow gives no indication of what concentration each of their recommended products should be used at. Optimal antibody concentration must be determined during the laboratory validation process.

4. Questions to ask to open up for a dialogue regarding EuroFlow

Does the customer intend to comply with the EuroFlow guideline? If so this means that: o The customer uses all 124 conjugates including custom conjugates? o The customer has or will purchase Infinicyt software? Would the customer be interested in trying/using products that have not been validated by EuroFlow but where Dako can show similar performance as the reference product? Does the customer follow the EuroFlow guideline 100% (if not, this opens for a dialogue regarding the remaining panels) Will the customer use all 124 conjugates? How will the customer decide on the titrations of the antibodies? How does the customer intend to validate the panels? How will the customer get access to custom conjugated antibodies? What will the cost be for running the whole Euroflow system? Has the customer conducted a health benefit to cost analysis for adoption of the EuroFlow guidelines?

5. Key take-home-message
In the most important panel in terms of volume: the acute leukemia panel, Dako has 25% marker coverage. Therefore, if the laboratory has adapted the EuroFlow recommendation Dako should capture 25% of that panels sale. Apart from the recommended markers, Dako has additional 9 reagents of exact clone/fluorochrome combination Further to that, Dako has 22 reagents of recommended antibody/fluorochrome combination but with a different clone

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Where Dakos reagents are not recommended, the reagent has to our knowledge - not been tested by EuroFlow therefore they should not be automatically dis-qualified. It is not correct that customers dont have to validate the individual markers. It is the responsibility of anyone wishing to follow EuroFlow to ensure that individual components work as required if they do not, the concept will not work. This includes control over both signal strength influenced by instrument setup as well as back-ground influenced by unspecific binding but also spill-over from other channels caused by e.g. changes in signal strength or natural decay of tandem fluorochromes.

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6. Appendix A
Analysis of the EuroFlow recommendations and Dako compliance In the following, each panel is listed with information covering: Dakos antibody flourochrome is recommended in the panel Dakos portfolio includes the recommended fluorochrome/antibody/clone Dakos portfolio includes the recommended fluorochrome/antibody but with a different clone

Table 1: The ALOT panel is the initial screening panel for acute leukemia orientation type. It is recommended to screen all types of leukemia wherefore the impact for a vendor being recommended here may have a significant business impact. Dako is recommended to 25% of the total panel (2 reagents out of 8).

Table 2: The lymphoid screening tube is designed for evaluation of several suspected clinical conditions, such as lymphocytosis, lymph node enlargement, splenomegaly, monoclonal serum components etc.

Table 3: Screening tubes designed for evaluation of suspect small samples and samples with very low cell counts, such as fine needle aspirates (FNA), cerebrospinal fluid (CSF) vitreous humor etc.

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Table 4: The 2 tubes aim at identification and enumeration of plasma cells as well as at the discrimination between normal polyclonal plasma cells such as in reactive plasmacytosis versus aberrant monoclonal plasma cells such as in MGUS, MM and PCL.

Table 5: This panel aims at the recognition and classification of all classically defined BCP-ALL or alternative BCP-ALL classifications including immunophenotypic classifications associated with well-defined molecular aberrations, such as specific fusion genes.

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Table 6: This panel consists of 4 tubes aiming at recognizing and classifying all classically defined T-ALL.

Table 7: This is the panel for acute myeloid leukemias and myelodysplastic syndromes.

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Table 8: This panel is designed to diagnose mature B-cell malignancies according to WHO entities using flow cytometric data only.

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Table 9: The EuroFlow T-CLPD panel aims for diagnosis and classification of mature T-cell malignancies.

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Table 10: This panel aims at the discrimination between aberrant and normal/reactive NK-cells.

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Appendix B

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