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FLEX Ready-To-Use antibodies

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FLEX Ready-To-Use antibodies Powered By Docstoc
					FLEX Ready-To-Use antibodies
Part of a system solution supporting
      consistency and quality


                Majken Nielsen, M.Sc., Ph.D.
                   Department Manager

                Research and Development
                   Dako Denmark A/S




                                               1
Agenda

  Ready to Use antibodies - why needed

  The Dako FLEX Ready to Use antibody series

  Ready to Use – before and now

  Flexible protocols – how

  Flexible protocols – risks

  Quality indicators and control tissue

  Validation and Quality Control

  Summary

                                               2
Worlds of …….




                3
Worlds of Immunohistochemistry




     CD23 in B-CLL from 2 different worlds……
                                               4
Choices to take
- standardization and simplification is needed !

   Grossing
                Fixation
                            Processing                                     Preparation
                                           Sectioning                      Preparation
                                                             Drying
                                                                              phase
                                                                              phase

   Deparaffination
                     Pre-treatment
                                     Antibody
                                                Detection
                                                            Counterstain   IHC Staining
                                                                           IHC Staining


   Control
               Cut-off value
                               Tumor entity
                                                 Reporting
                                                                           Interpretation
                                                                           Interpretation
                                                                               phase
                                                                                phase

              314 = 4.8 million procedures (3 choices in 14 steps)
                                                                                            5
Nordic immunohistochemical Quality Control

  Run 8 – 16: 1073 out of 3426 stains (31 %) insufficient
   False negative:
   1.   Too diluted primary ab. conc.                              414 (39
        %)
   2.   Inappropriate primary ab.                       173 (16 %)
   3.   Insufficient HIER                               165 (15 %)
   4.   Inappropriate epitope retrieval                 148 (14 %)
   5.   Unexplained                                         67 ( 6 %)


   False positive:
   1.   Too high primary ab. conc.                          59 ( 6 %)
   2.   Inappropriate primary ab.                           16 (<2 %)
   3.   Excessive retrieval                                 16 (<2 %)
   4.   Unspecific reaction of the detection system         21 (<2 %)
                                                                        6
   5.   Unexplained                                          6 (<1 %)
Our mission

To provide IHC products and solutions that

   Deliver robust and high quality results

   Help standardization in the pathology laboratories

   Streamline the IHC solution

   Improve workflow

   Improve turn around time (TAT)




                                                        7
The basic fundamentals for a technically
optimal IHC performance

  Appropriate tissue fixation and processing

  Good quality sectioning

  Appropriate and efficient epitope retrieval

  Appropriate choice of antibody/clone

  Appropiate antibody dilution

  Robust and sensitive detection system

  Appropriate choice of control material
                                                8
The basic fundamentals for a technically
optimal IHC performance

  Appropriate tissue fixation and processing

  Good quality sectioning

  Appropriate and efficient epitope retrieval

  Appropriate choice of antibody/clone           Ready-to-Use
                                                     (RTU)
  Appropiate antibody dilution                  system solution

  Robust and sensitive detection system

  Appropriate choice of control material
                                                                  9
The Dako FLEX RTU antibody product line

    110 antibodies for routine clinical diagnostics

        Including negative control reagents

    For instrument use

        Autostainer Link instruments

        Dako Autostainer/Autostainer Plus

    One streamed-lined protocol

    Involvement of Key Opinion Leaders in setting the diagnostic quality

    All CE-IVD, IVD class I (minimum)


                                                                     10
The Dako FLEX RTU antibody product line

   110 antibodies for accurate clinical diagnostic results

   Routine use for general- and hematopathology

   85% exisiting Dako antibodies

   6% exisiting Dako antibodies – new clones

   9% against new targets

   Bottle type and number of tests per vial
      Autostainer Link instruments: Automation bottle, 40-60 tests per vial

      Dako Autostainer/Autostainer Plus: Sarstedt bottle, 20-30 tests per vial

   Shelf life minimum 1 year
                                                                              11
RTU antibodies tried without succes

    Inferior sensitivity

       Numerous recommended protocols for a RTU ab series

       Developed over a long time period

       No maintenance activities/protocol optimization

       No standardized protocols

       No involvement of experts to determine the desired diagnostic
       performance

    Poor Workflow solution

    Turn Around Time (TAT) out of focus
                                                                   12
RTU – industry standard before and now

Before                                     Now*
                                               Part of a IHC System Solution
  Both for manual and instrument use           Only for instrument use
  For each RTU Ab multiple protocols           One protocol to fit all RTU abs


     Visualization system A
                                                   EnVisionTM FLEX
     Visualization system B
                                                   or
     Visualization system C
                                                   EnVisionTM FLEX+
     Visualization system D

  TAT out of focus                             TAT in focus
  Quality set by individual manufacturer       Key Opinion Leader involvement
  Control tissue                               Control tissue and defined HE/LE
                                                                                 13
                                           *Dako FLEX RTU, data on file
One standardized protocol
Dako EnVision™ FLEX and FLEX+ system

            K8000/K8010 EnVision™ FLEX, High pH                  K8002/K8012 EnVision™ FLEX+, Mouse, High pH
  EnVision™ FLEX Peroxidase-Blocking Reagent                EnVision™ FLEX Peroxidase-Blocking Reagent
  EnVision™ FLEX /HRP                                       EnVision™ FLEX /HRP polymer
  EnVision™ FLEX DAB+ Chromogen                             EnVision™ FLEX DAB+ Chromogen
  EnVision™ FLEX Substrate Buffer                           EnVision™ FLEX Substrate Buffer
  EnVision™ FLEX Wash Buffer                                EnVision™ FLEX Wash Buffer
  EnVision™ FLEX Target Retrieval Solution, High pH         EnVision™ FLEX Target Retrieval Solution, High pH
                                                            EnVision™ FLEX+ Mouse (LINKER)


                                                      Options
                         K8008/K8018                                               K8009/K8019
  EnVision™ FLEX Hematoxylin                                EnVision™ FLEX+ Rabbit (LINKER)

                             K8004                                                 K8021/K8022
  EnVision™ FLEX Target Retrieval Solution, High pH         EnVision™ FLEX+ Mouse (LINKER)

                             K8005                                                     K8007
  EnVision™ FLEX Target Retrieval Solution, Low pH          EnVision™ FLEX Wash Buffer

                             K8006                                                     K8020
  EnVision™ FLEX Antibody Diluent                           FLEX IHC Microscope Slides


                                                                                                                14
Compatible with 3-in-1 specimen preparation
Standardized protocol
– Simplification
One core protocol
   Pre-treatment in Dako PT Link: one of two Target Retrival Solution options
      1. EnVision™ FLEX, TRS High pH (94%)
      2. EnVision™ FLEX, TRS Low pH (6%)

   Visualization system
      1. EnVision™ FLEX (83%)
      2. EnVision™ FLEX+ (17%)

   Recommended incubation times: Fixed for similar steps
      RTU Ab: 20 min
      EnVision™ FLEX+ (LINKER): 15 min (FLEX+)

      EnVision™ FLEX /HRP polymer: 20 min
      EnVision™ DAB+: 2x5 min                                                   15
Standardized protocol
– Workflow and TAT
For instrument use

  Dako PT Link
      3-in-1 specimen preparation (paraffin-embedded sections )
      HIER (deparaffinized sections )

  Dako Autostainer Platform
      Automated Link Instruments
      Autostainer/Autostainer Plus

  TAT in focus
     Two machine cycles per working day and one overnight run
                                                             16
TAT – the power of parallel processing


                                Up to three runs a day with a
                                minimum of manual work



                               A Dako beta-site study performed in
                               Denver, USA
                               Glasgow, UK
                               Ålborg, Denmark


                               Showed time savings in manual
                               procedures of 15-60 min/day for
                               titrations only


                                                                 17
Standardized protocol
– Key Opinion Leader involvement

Why
Alignment with the desired staining performance
       e.g. Clinical relevance, Specificity, Sensitivity


How:
Eight Key Opinion Leaders from USA, Europe and Japan
       Literary input on individual antibody performance
           e.g. control tissue and definition of HE/LE
       Provided slides with optimal stains of each antibody
       Face-to-face assessment

                                                              18
Key Opinion Leader involvement

                                 Top left towards right:
                                 Dr.Clive Taylor, USA - CAP
                                 Kim McManus, USA
                                 Andrew Dodson, UK - UK
                                 NEQAS
                                 Dr. Robert Osamura, Japan
                                 Dr. Miguel Piris. Spain
                                 Dr. Jahn Nesland, Norway -
                                 NordiQC
                                 Dr. Kengo Takeuchi, Japan

                                 Bottom left towards right:
                                 Sheron Lear, USA
                                 Dr. Assia Bassarova, Norway
                                 Dr. David Dabbs, USA – CAP
                                 Prof. Bharat Jasani, UK - UK
                                 NEQAS
                                 Dr. Alvin Martin, USA - CAP
                                 Lydia Sanchez
                                                      19
Key Opinion Leader involvement
Face-to-face assessment




                                 20
Independent assessment by NordiQC
  AMACR (P504S), Clone 13H4: Optimal           CK7, Clone OV-TL12/30: Optimal
  ASMA, Clone 1A4: Optimal                     CK18, Clone DC 10: Optimal
  BCL2, Clone 124: Optimal                     CK20, Clone Ks20.8: Optimal
  BCL6, Clone PG-B6p: Optimal                  D2-40, Clone D2-40: Optimal
  BSAP, Clone DAK-Pax5: Optimal                Desmin, Clone D33: Optimal
  Calretinin, Clone DAK-Calret 1: Optimal      EMA, Clone E29: Optimal
  Chromogranin A, Polyclonal: Optimal          Ep-CAM, Clone Ber-EP4: Optimal
  CD3, Polyclonal: Optimal                     ER, Clone SP1: Optimal
  CD5, Rabbit Clone SP19: Optimal              ER, Clone 1D5: Optimal
  CD10, Clone 56C6: Optimal                    Ki67, Clone MIB-1: Optimal
  CD15, Clone Carb-3: Optimal                  Mammaglobulin, Clone 304-1A5: Optimal
  CD20, Clone L26: Optimal                     Melan A, Clone A103: Optimal
  CD23, Rabbit Clone SP23: Optimal             MSA (HMB45), Clone HMB45: Optimal
  CD30, Ber-H2: Good                           p53 Protein, Clone DO-7: Optimal
  CD79α, Clone JCB117: Optimal                 PLAP, Clone 8A9: Good
  CD99, 12E7: Good                             PSA, Polyclonal: Optimal
  CD138, Clone MI15: Optimal                   S100, Polyclonal: Optimal
  CDX2, Clone DAK-CDX-2: Optimal               Synaptophysin, Clone SY38: Borderline
  CEA, Polyclonal: Optimal                     TTF1, Clone 8G7G3/1: Borderline
  CK-PAN, Clone AE1/AE3: Optimal               WT1, Clone 6F-H2: Good
                                                                                       21
                              38/40 assessed Sufficient
NordiQC homepage
CD10




Source: http://www.nordiqc.org/Run-27-B8-C2/Assessment/assessment-27-CD10.htm
                                                                                22
1)   Proportion of sufficient stains (optimal or good),
2)   Proportion of sufficient stains with optimal protocol settings only.
Standardized
– and flexible protocols !

Adjustment of the intensity (sensitivity)

Why
IHC evaluation is subjective

Aberrant technique (e.g. fixation, processing etc.)

Aberrant protein expression – biological variation




                                                      23
Flexible protocols

FLEX protocol:

    RTU Ab: 20 min
    EnVision™ FLEX /HRP polymer: 20 min           20-20-2x5
    DAB+: 2x5 min


How adjustment ?

    Lower intensity/sensitivity 10-10-2x5
    Higher intensity/sensitivity 30-30-2x5
    Higher intensity/sensitivity 20-15-20-2x5 (FLEX+)


                                                              24
Flexible protocols

FLEX+ protocol:
   RTU Ab: 20 min
   EnVision™ FLEX+ (LINKER): 15 min
                                                   20-15-20-2x5
   EnVision™ FLEX /HRP polymer: 20 min
   DAB+: 2x5 min


How adjustment ?

    Lower intensity/sensitivity 20-20-2x5 (FLEX)
    Lower intensity/sensitivity 10-15-10-2x5
    Higher intensity/sensitivity 30-15-30-2x5



                                                                  25
Flexible protocols

Protocol modifications may affect the diagnostic precision

   Use of low intensity protocol:
  Dubious reaction or even false negative if choosing the low intensity protocol


   Use of high intensity protocol:
  Neoplasms with non-detectable antigen expression by recommended protocol
  can show positive reaction choosing the high intensity protocol




                                                                                   26
Flexible protocols
Risk - diagnostic precision

           Optimal protocol                    Too low sensitivity




                                                                     27
           Epithelial Antigen, clone Ber-EP4. Lung adenocarcinoma
 Flexible protocols
 Risk - diagnostic precision

             Optimal protocol                   Too high sensitivity




Nuclei neg                                                      Nuclei dubious

             MLH-1. Colon adenocarcinoma-MLH1 gene deficiency

                                                                          28
Flexible protocols
- Verification of performance

Use of Quality indicators

  Defined control tissue

  Cells and/or cellular structures

  Divided into high expression (HE) and low expression (LE) structures

  HE structures are typically moderately to strongly stained

  LE structures are typically weakly to moderately stained

  HE/LE structures are expressed relatively stable

  Typically identified in normal tissue
                                                                  29
Flexible protocols - use of quality indicators
Polyclonal Rabbit Anti-Human Chromogranin A
Control tissue: Colon

                 HE                    LE     Medullary thyroid carcinoma
 FLEX
protocol


  RTU
20-20-2x5




Adjusted
10-10-2x5




                                                                  30
 Flexible protocols - use of quality indicators
 Monoclonal Mouse Anti-Human BCL6, Clone PG-B6p
 Control tissue: Tonsil

                    HE                LE          Follicular lymphoma
  FLEX+
 protocol


    RTU
20-15-20-2x5




  Adjusted
10-15-10-2x5




                                                                  31
Quality indicators - typical performance
Low expression structures (LE)
 Typically weakly to moderately stained




                                …. or moderate to strong but small / discrete



  BCL6, Tonsil




                                                                            32
 Chromogranin A, Colon
Quality indicators - typical performance
Low expression structures (LE)

   Vary in variable protocol settings

        ….even when appearing moderate to strong in the optimal protocol


     Ab dilution 1:1              Ab dilution 1:2        Ab dilution 1:4




    Tonsil stained with monoclonal Rabbit Anti-Human CD23, Clone SP23
                                                                           33
Quality indicators
– Easily accessible control tissue*
                                         100%
                                                    Clinical tissue (11%)
                                         89%
     110 RTU antibodies                           HE in normal tissue,
                                                impossible to identify LE
     Normal vs clinical control tissue                   (22%)
                                         67%
     HE/LE in same tissue type
                                                  HE and LE in different
     HE/LE in different tissue types            normal tissue types (23%)

     No LE                               44%


                                                 HE and LE in the same
                                                normal tissue type (44%)


                                          0%
  *Dako FLEX RTU, data on file                                          34
Quality indicators
– Easily accessible control tissue
Normal tissue the commonly used control tissue
         Tissue                           %HE                  %LE             %HE and LE in the same tissue

         Tonsil                           34.9%                23.9%                23.9%

         Colon/Appendix                   22.0%                16.5%                11,9%

         Liver                            8.3%                 9.2%                 2,8%

         Skin                             3.7%                 2.8%                 0.9%

         Cervix                           2.8%                 1.8%                 1.8%

         Pancreas                         2.8%                 2.8%                 0.9%

         Placenta                         2.8%                 0.9%                 0,9%

         Thyroid                          2.8%                 0.9%                 0.9%

         Brain                            0.9%                 2.8%                 0%

         Other normal tissue1             8.0 %                7.5%                 N.a2
         Clinical tissue                  11%                  N.a2                 N.a2


     1                                                                                                         35
         Other normal tissue: Breast, Fallopian tube, Kidney, Prostate and Thymus
     2   N.a: not applicable
Control tissue

Parameters that should be considered


    Easily accessible

    Stable antigen expression between samples of the same tissue type

    Changes in protocol performance are reflected by changes in
    staining intensity in defined cellular elements/structures within the
    tissue




                          Normal tissue
                                                                            36
The power of quality indicators


    Development has shown that antigen expression

    in majority of clinical samples lies within the

    expression range defined by the HE and LE structures




                                                           37
 One product
 – applied on a broad range of clinical samples
                     FLEX RTU CD79α, Clone JCB117



LE




HE




       Control: Tonsil     Precursor B-lymphoblastic   Plasmacytoma
                              leukemia/lymphoma
                             disseminated to testis                   38
 One product
 – applied on a broad range of clinical samples

               FLEX RTU Calretenin, Clone DAK-Calret 1



LE




HE




       Control: Colon      Granulosa cell tumor   Epithelial mesothelioma
                                                                     39
Dako FLEX RTU antibodies
– Recommended quality indicators

Package inserts ”Performance characteristics”
      Antibody specific control tissue(s)
      Specified HE and LE structures
      HE: ”strong” or ”strong to moderate”
      LE: ”weak” or ”weak to moderate”
      Some antibodies do not have LE structures
      Some antibodies do not have normal control tissue (no HE/LE)

Atlas of Stains
      Clinical application
      Recommended control
      Differential diagnosis
      Images                                                         40
Validation
– Part of development phase before launch
   Does the production lot fulfill design specifications and Intended Use
           Sensitivity
           Specificity
           Repeatability
           Reproducibility
           Robustness
   Extent depends on Intended Use
   Conducted on production lots (filled in sales vials)
   Test on validated instruments
   Test according to Package Insert
       recommended staining protocol (steps and incubation times)
       recommended visualization system (EnVision FLEX / FLEX+)
       recommended instrument platform (PT Link and Autostainer)
                                                                            41
Quality Control
– For each production lot

   Does the production lot fulfill Intended Use
   Consistent performance
   Well-defined Quality Control (QC) test procedure
       According to Package Insert
       Critical parameters evaluated
       Must fulfill acceptance criteria
   Test on validated instruments




                                                      42
Summary
 FLEX RTU antibodies
   110 antibodies for routine clinical diagnostics
   Part of a standardized IHC solution
   For instrument use
   Workflow improved solution - up to three runs a day
   Key Opinion Leader indicated required staining performance
   Standardized and flexible protocols
   Dako recommended Quality indicators
   One product applicable to a broad range of clinical samples
   Atlas of Stains – including stainings on normal tissues
   Intended Use is validated for the recommended protocol
   All productions are quality controlled before shipment
                                                                 43

				
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