SOP FOR PROCEDURE OF ALK D5F3 IMMUNOHISTOCHEMISTRY STAINING OF TISSUE SECTIONS

Standard Operating Procedure (SOP) for ALK (D5F3) Immunohistochemistry Staining of Tissue Sections

1.0 Purpose:

The purpose of this SOP is to establish a standardized procedure for staining sections for ALK (D5F3) Immunohistochemistry study.

2.0 Scope:

This SOP applies to all ALK (D5F3) Immunohistochemistry staining procedures conducted in the laboratory. It serves to control the procedures and guide technicians throughout the process.

3.0 Introduction:

Non-small cell carcinoma (NSCLC) comprises the most common type of lung cancer, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Immunohistochemistry (IHC), particularly in combination with antibodies detecting crucial antigens, is a valuable tool for pathologists in diagnosing NSCLC. Ventana's ALK (D5F3) Rabbit Monoclonal Primary Antibody has demonstrated concordance with ALK FISH Probe, making it sensitive and specific for determining ALK status in NSCLC specimens.

Principle:

The VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody is used for detecting ALK protein in formalin-fixed, paraffin-embedded NSCLC tissue on VENTANA Bench Mark XT or Bench Mark GX IHC automated slide stainer. It aids in identifying patients eligible for XALKORI (crizotinib) treatment.

5.0 Analytical Measuring Range and Limit of Detection:

Not Applicable

6.0 Responsibility:

Technical staff and pathologists

7.0 Abbreviations:

HIER: Heat-induced epitope retrieval

Ag: Antigen

Ab: Antibody

H202: Hydrogen peroxide

D.W.: Distilled water

HRP: Horse radish peroxidase

DAB: DiaMinobenzidine

DPX: Distrene dibutylphthalate xylol

RT: Room temperature

MQ water: Milli-Q water

8.0 Requirements:

Kit reagents

Instrumentation and software

Disposables

Specimen collection and handling materials

9.0 Precautions:

Ensure the use of poly-lysine/APES coated slides for IHC, correct pH of buffers, proper washing between steps, careful handling and disposal of DAB reagent, and use only formalin-fixed, paraffin-embedded tissues.

10.0 Limitations & Interferences:

Avoid exposing FNAC material to alcohol as it can interfere with ALK IHC staining.

11.0 Documentation:

Maintain records in IHC entry, control, and reagent registers, as well as an antibody data file.

12.0 Instructions:

12.1 Pre-Analytical Steps:

Enter specimens in the Immunohistochemistry entry register and assign a department ID.

Prepare reaction buffer and EZ prep solution.

12.2 Analytical Steps:

Follow a series of steps for patient tissue preparation, including deparaffinization, cell conditioning, and primary antibody application, among others.

12.3 Quality Control and Assurance:

Run known positive controls with each batch of samples.

Prepare control blocks from tissues known to be positive for the IHC marker.

Utilize Roche Control for positive and negative controls on slides.

12.4 Calibration and Calibration Verification:

Not Applicable

12.5 Storage of Sample:

Retain paraffin blocks and slides for 10 years at room temperature, filing control slides separately for easy retrieval.

13.0 Reference Range:

Not Applicable

14.0 Critical Value:

Report critical values according to SOP.

15.0 Reporting of Results:

Follow previous SOP for reporting results.

16.0 Contingency Plan:

Have multiple trained staff and backups available in case of reagent shortages, manpower issues, or instrument breakdowns.

17.0 Reference:

Protocol 200 ALX DSF3 (03/25/2014), Procedure: X1 OptiView DAS IHC, Bench Mark XTIHC/staining module.

END OF THE DOCUMENT

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