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Immunohistochemistry (IHC)

Commercially available antibodies and protocols

Ventana (D5F3)

Ventana protocols using BENCHMARK XT with OptiView

Meta-analysis data using D5F3

Novocastra (5A4)

ETOP ALK Lungscape Protocol

There are two ALK IHC staining procedures, one for laboratories using BOND-MAX, and the other for laboratories using the free-style bench technique – two versions with different Ag retrieval

Staining procedures using free-style bench technique

Staining procedures using BOND-MAX

  1. Retrieval: boiling epitope retrieval solution 2 (ER2) for 30 minutes. It may be that in your lab ER2 solution for 20 minutes is sufficient. (Bond Epitope Retrieval Solution 2 is ready to use and should not be diluted)
  2. Peroxidase block, 5 minutes
  3. Dilution necessary with Leica/Novocastra: 1:10; 15 minutes
  4. Post primary washing, 8 minutes
  5. Polymer 8 minutes
  6. DAB 10 minutes
  7. Hematox 5 minutes

Pressure cooker

ALK IHC staining procedures

  1. Retrieval: Satisfactory staining was achieved with a pressure cooker for 8 minutes citrate pH6, pressure cookers may vary therefore use your standard citrate pH6 pressure cooker retrieval protocol
  2. Peroxidase block, 5 minutes
  3. Dilution necessary with Leica/Novocastra 5A4 antibody: 1:20 and incubate for 30 minutes. Local minor modification (1:25) may be necessary if other Ag retrieval is to be used
  4. Post primary washing, 8 minutes
  5. Use the Novolink detection kit as described in the kit manual

 Microwave

ALK IHC staining procedures

  1. Retrieval: Antigen retrieval was achieved with a microwave (800W) for 20 minutes using citrate buffer pH6
  2. Peroxidase block, 5 minutes
  3. Dilution necessary with Leica/Novocastra 5A4 antibody: 1:10 and incubate for 60 minutes
  4. Post primary washing, 8 minutes
  5. Use the Novolink detection kit as described in the kit manual
  6. From water put the slides in a solution of copper sulphate for 2 minutes. This will darken the pre-existing tan coloured DAB staining to a dark brown colour. (Solution is made as follows: 10 g copper sulphate, 17 g sodium chloride in 2 litres of deionised water)
  7. Continue with protocol with counterstain of haematoxylin for 20 seconds

Other published protocols

Meta-analysis data using 5A4

Origine (1A4)

Comparison of antibody clones used with IHC

Study No. of
Specimens
(% of ALK+)
Antibody
Clone
Antigen
Retrieval
Detection/
Amplification
System
Scoring Standard IHC
Sensitivity
(vs. Standard)
IHC
Specificity
(vs. Standard)
Takeuchi
et al., 2009
21 (52%)

5A4

Target Retrieval
Solution (pH 9.0)
(for all)
iAEP 0 vs. +
(for all)
RT-PCR 100% 100%
5A4   EnVision+     27% 100%
ALK1   iAEP     100% 100%
ALK1   EnVision+     9% 100%
SP8   iAEP     20% 100%
SP8   EnVision+     100% 18%
Rodig
et al., 2009
239 (4%) ALK1 EDTA in pressure
cooker (for both)
EnVision+ 0 vs. + (for
both)
FISH 40% 100%
ALK1   Tyramide amplification     80% 100%
Mino-
Kenudson
et al., 2010
37 (59%) ALK1 EDTA in pressure
cooker (for both)
EnVision+
(for both)
Score >2.7
on image
analysis is
positive
FISH 67% 97%
D5F3         100% 99%
Murakami
et al., 2012
361a (5%) ALK1 Target Retrieval
Solution (pH 9.0)
(for all)
ABC (no
enhancement)
0 vs. +
(for all)
RT-PCR/
FISH
81% 100%
5A4   EnVision FLEX+     100% 100%
    EnVision FLEX+     100% 99.7%
Conklin
et al., 2013
377 (3%) 5A4 According to
manufacturer’s
instructions
(for all)
iAEP 0, 1+, 2+,
or 3+
(for all)
FISH 100% 62.5%
ALK1   EnVision FLEX+     66% 100%
ALK1   ADVANCE     66% 87.5%
5A4   ADVANCE     100% 87.5%
D5F3   ADVANCE     100% 75%
Selinger
et al., 2013
594 (11%) ALK1 Buffer (pH 9.0) in
pressure cooker
EnVision FLEX+ 0, 1+, 2+,
or 3+
(for all)
FISH 100% 99%
5A4 According to
manufacturer’s
instructions
ultraView and
ultraView
Amplification
    100% 98%
D5F3 According to
manufacturer’s
instructions
OptiView and
OptiView
Amplification
    100% 99%
Cutz et al.,
2014
28 (79%) 5A4 Not reported ultraview H-score Pearson
correlation
between
antibodies

5A4 vs D5F3 : 0.89 - 0.97

5A4 vs ALK1 : 0.84

D5F3 Not reported

ultraview

EnVision FLEX+

ALK 1 Not reported EnVision FLEX+
Zwaenepoel
et al., 2014
53 5A4 High pH buffer
(dilution 1:50)
EnVision FLEX+ 0, 1+, 2+,
3+
1+, 2+, 3+ 96% 87-96%
D5F3 High pH buffer
(EnV)
En Vision FLEX+
(D5F3 EnV)
OptiView
Amplification
0, 1+, 2+,
3+
1+, 2+,
3+ (D5F3
EnV)
3+
(D5F3 Ov)
96%
(D5F3 EnV)
93%
(D5F3 Ov)
87-96%
(D5F3 EnV)
100%
(D5F3 Ov)
Condé et al.
2014
79 5A4   OptiView and
OptiView
Amplification
0, 1+, 2+,
3+
2+, 3+ 98% 100%
D5F3 According to
manufacturer’s
instructions
OptiView and
OptiView
Amplification
    98% 100%
Gruber et
al., 2015
218 1A4 pH 6.0 (30 min) OptiView and
OptiView
Amplification
0, 1+, 2+,
3+
1+, 2+, 3+ 100% 95%
D5F3 According to
manufacturer’s
instructions
OptiView and
OptiView
Amplification
0, 1+, 2+,
3+
3+ 99% 95%
Lantuéjoul
et al., 2015
547 5A4   OptiView or
ultraView
Amplification
% of + cells
and
intensity
(1+, 2+, 3+)
10% 1+ 87% 89%
D5F3 According to
manufacturer’s
instructions
OptiView and
OptiView
Amplification
% of + cells
and
intensity
(1+, 2+, 3+)
10% 1+ 92% 76%
Savic et al.,
2015
72 5A4   OptiView
Amplification
Bond Polymer
Refine
Detection
0, 1+, 2+,
3+
1+, 2+, 3+ 96% 100%
D5F3 According to
manufacturer’s
instructions
OptiView and
OptiView
Amplification
0, 1+, 2+,
3+
3+

SP8 is a product of Abcam. Target Retrieval Solution, buffer, EnVision, EnVision FLEX+, and ADVANCE are products of Dako. ultraView (Universal DAB Detection Kit), OptiView (DAB IHC Detection Kit), and ultraView and OptiView Amplification kits are products of Ventana Medical Systems, Inc. Bond Polymer Refine Detection is a product of Leica Biosystems. ABC = avidin biotin complex. 

IASLC ATLAS of ALK and ROS Testing in Lung Cancer, IASLC, Colorado, USA

Positive controls for ALK IHC

  • It is recommended that laboratories include controls alongside their clinical cases being tested for ALK, to ensure that the sensitivity and specificity of their test is satisfactory
  • The appendix has been reported to be a valuable positive tissue control
  • Ganglion cells of the myenteric plexus must show at least a weak to moderate cytoplasmic staining reaction
  • If these cells are negative, a too weak or false negative staining reaction is seen in lung adenocarcinoma with EML4‑ALK translocation

NordiQC. Lung Anaplastic Lymphoma Kinase (lu-ALK) 2015. Available at: http://www.nordiqc.org/downloads/assessments/45_14.pdf
Ibrahim M et al. J Thorac Oncol. 2016;11:2241-47.