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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