STANDARD OPERATING PROCEDURE (SOP) FOR IMMUNOHISTOCHEMICAL TESTING OF TISSUE SECTIONS

Standard Operating Procedure (SOP) for Immunohistochemical Testing of Tissue Sections

1.0 Purpose

To establish a standard procedure for staining tissue sections for immunohistochemistry studies.

2.0 Scope

Applicable to all immunohistochemical staining procedures in the laboratory, guiding the technician throughout the process.

3.0 Introduction

Immunohistochemistry is essential for the reporting of surgical pathology cases, aiding in determining the presence and type of tumors.

4.0 Principle

Tissue sections are treated with heat or enzymes to retrieve antigen epitopes. Antibodies and detection systems are then applied under controlled conditions. Staining or its absence is evaluated in the context of clinical history.

5.0 Analytical Measuring Range and Limit of Detection

Not Applicable

6.0 Responsibility

- Technical staff

- Pathologist

7.0 Abbreviations

- IHC: Immunohistochemistry

- HIER: Heat Induced Epitope Retrieval

- Ag: Antigen

- Ab: Antibody

- H₂O₂: Hydrogen Peroxide

- RT: Room Temperature

- DW: Distilled Water

- APES: 3-Aminopropyltriethoxysilane

- HRP: Horseradish Peroxidase

- DAB: Diaminobenzidine

- DPX: Distrene Dibutylphthalate Xylol

- NaOH: Sodium Hydroxide

- MQ Water: Milli-Q Water

8.0 Requirements

Kit Reagents

- Sodium Citrate

- TRIS Buffer

- Antibodies

- Visualization Reagents

- Methanol

- H₂O₂

- Mountant

- Counterstain

- Trisodium citrate (3.47g)

- Citric acid (2.1g)

- pH meter

- NaOH pellets

- 2-liter conical flask

- MQ water

Instrumentation and Software

- Pressure cooker

- Glassware

- Dako open system

- Calibrated pipettes

Disposables

Not Applicable

Other Materials Required but not Provided with the Kit (Reagents and Consumables)

Not Applicable

Specimen Collection and Handling

- Specimen Collection: Room Temperature. Tissues in 10% neutral buffered formalin, immersed within 1 hour of excision, and fixed for 6-72 hours (ER/PR), 6-48 hours (Her2neu).

- Specimen Transport: Room Temperature

- Specimen Storage: Room Temperature

- Specimen and Control Preparation: Room Temperature

9.0 Precautions

1. Use only sections drawn on polylysine/APES coated slides for immunohistochemical staining.

2. Ensure correct pH of buffers.

3. Proper washing between steps to avoid cross-reactions.

4. Handle DAB reagent and its washings with care; ensure proper disposal.

5. Use only formalin-fixed, paraffin-embedded tissues for IHC.

10.0 Limitations & Interferences

Not Applicable

11.0 Documentation

All documents are maintained in:

1. IHC entry register

2. IHC control register

3. IHC reagent register

4. Antibody data file

12.0 Instructions

12.1 Pre-Analytical Steps

12.1.1 Specimen Entry

All specimens are entered into the Immunohistochemistry Entry register and given a department ID (IHC number) e.g., 1/xxxx/Year. This number is noted on the TRF, paraffin blocks, coated slides, and during all steps of processing, staining, examination, and report finalization.

12.1.2 Preparation of Sodium Citrate Buffer (pH 6.0)

1. Weigh trisodium citrate and citric acid as indicated.

2. Add 10 liters of MQ water and place on a magnetic stirrer.

3. Once the solute dissolves, add 5-6 pellets of NaOH to adjust the pH using a pH meter.

4. When pH reaches 6.0, keep the pH probe in the solution for 5-10 minutes for stabilization.

5. Make the volume up to 2 liters by adding MQ water.

12.1.3 Preparation of TRIS Buffer (pH 7.5)

1. Weigh TRIS powder (6.05g) and sodium chloride (8.7g) into a beaker.

2. Add 3.7 ml conc. HCl.

3. Add 1 liter of MQ water.

4. Once the solute dissolves, adjust the pH to 7.5 by adding 8% HCl or 4% NaOH dropwise.

5. This solution is ready for use.

12.2 Analytical Steps

1. Sections are brought down to water with two changes of xylene followed by two changes in alcohol.

2. Antigen Retrieval:

- Pressure Cooker Antigen Retrieval:

- Place slides in a metal slide holder and then in a domestic pressure cooker with pre-warmed sodium citrate (pH 6.0).

- Heat on a hot plate until full pressure develops and there is one whistle.

- Remove cooker from the hot plate and allow to cool until the pressure equalizes.

- Remove the lid and allow the buffer to cool to RT.

3. Wash slides in TRIS buffer (pH 7.5) for three changes. Wipe sections dry.

4. Blocking Endogenous Enzymes:

- Place slides in a freshly prepared solution of methanol and H₂O₂ (47 ml methanol and 3 ml H₂O₂) for 30-45 minutes at RT.

5. Repeat step 3.

6. Incubation with Primary Ab:

- Bring all antibodies to RT before use.

- Mark sections around with a Dako pen to prevent outside spread of Abs.

- Apply enough primary Ab to cover the section.

- Incubate in a moist chamber for 60 minutes at RT.

- Drain the excess Ab.

- Reviewed by the section in charge.

7. Repeat step 3.

8. Incubation with Dako Real Envision/HRP Rabbit Mouse (ENV) Detection Reagent:

- Apply Dako Real Envision detection reagent to cover the sections.

- Incubate in a moist chamber for 30 minutes at RT.

- Drain excess reagent.

9. Repeat step 3.

10. DAB Substrate Chromogen Solution:

- Apply freshly prepared DAB-substrate chromogen solution to the sections to cover them (To 1 ml of Dako Real Substrate Buffer (Bottle B) add 20 μl of Dako Real DAB Chromogen (Bottle C) and mix thoroughly).

- Incubate in a moist chamber for 8-10 minutes at RT.

- Check under a microscope for the development of brown color.

- Drain the excess DAB solution into a separate container.

11. Rinse slides with DW from a wash bottle, avoiding focusing the water jet directly onto the sections. Collect washings for proper disposal.

12. Counterstain:

- Stain slides in Harris's Hematoxylin for 2 minutes.

- Rinse in distilled water.

13. Dehydrate, clear, and mount in DPX.

14. Submit slides with requisition forms and the IHC register to the pathologist for reporting.

12.3 Quality Control and Assurance

1. Known positive controls are run with each batch of samples and evaluated. Patient reporting is done only when controls are acceptable.

2. Control Tissue Selection:

- Control blocks are prepared from tissues known to be positive for the IHC marker.

- First option: Tumor positive for the IHC marker.

- Second option: Normal tissue positive for the marker, considering the manufacturer's recommendations.

- Control tissue is tested along with previously established control and put into use if acceptable reactivity is obtained.

- For new IHC markers, validated tissues are used as positive controls. Control selected shows a range of reactivity, including low positive (i.e., 1+ to 3+).

3. The laboratory uses a polymer-based detection system (biotin-free); therefore, separate negative controls are not run.

12.4 Calibration and Calibration Verification

Not Applicable

12.5 Storage of Samples

- Paraffin blocks and slides are retained for 10 years at room temperature.

- Control slides are filed separately in a slide filing cabinet sequentially for easy retrieval.

13.0 Reference Range

Not Applicable

14.0 Critical Value

Reporting of critical values is done as per guidelines.

15.0 Reporting of Results

15.1 Reporting of ER/PR/Her2 Predictive and Prognostic Markers

Follows guidelines set by CAP/ASCO:

ER and PgR Testing in Breast Cancer

- Positive for ER or PgR: If ≥1% of tumor cell nuclei are immunoreactive. Both average intensity and extent of staining are reported.

- Negative for ER or PgR: If <1% of tumor cell nuclei are immunoreactive in the presence of positive intrinsic controls.

- Uninterpretable for ER or PgR: If no tumor nuclei are immunoreactive and internal epithelial elements present in the sample lack any nuclear staining.

Her-2/neu Testing in Breast Cancer

- Negative (0): No staining in tumor cells.

- Negative (1+): Faint/barely perceptible partial membrane staining in any proportion

15.2 Comments on Decalcified Tissues

"This assay has not been validated on decalcified tissues. Results should be interpreted with caution given the likelihood of false negativity on decalcified specimens.”

16.0 Contingency Plan

In case of reagent shortage, manpower shortage, or instrument breakdown, multiple staff are trained and backups are available.

17.0 References

Theory and Practice of Histological Techniques, John D. Bancroft, Fourth Edition

Handbook of Immunohistochemical Staining Methods, Thomas Boenisch, DAKO Cytomation

Diagnostic Immunohistochemistry, Theranostic and Genomic Applications, David J. Dabbs, 3rd Edition, 2010

Addendum 1: Clinical Utility of IHC Markers

Breast Cancer Diagnostic/Prognostic Markers

Estrogen Receptor (ER): Predictive factor for response to hormonal therapy.

Progesterone Receptor (PR): Predictive factor for response to hormonal therapy.

c-erb B2/Her2-neu: Identifies patients benefiting from Herceptin.

p53: Correlates with tumor grade.

Cathepsin D: Predicts metastatic potential of node-positive tumors.

Ki-67 (MIB-1): Studies proliferative fraction of neoplasms and tumor grade.

Lymphoma Diagnostic/Prognostic Markers

CD3: Diagnosis of T-cell Non-Hodgkin Lymphoma.

CD5: Diagnosis of B-cell CLL/SLL and Mantle Cell Lymphoma.

CD10: Diagnosis of B-Lymphoblastic lymphomas, Burkitt, and Follicular Lymphomas.

CD15: Diagnosis of Hodgkin Lymphoma.

CD20: Detects response to Rituximab therapy.

CD23: Marker for B-cell Lymphoma (SLL/CLL).

CD30: Marker for Hodgkin Lymphoma.

CD45: Leucocyte Common Antigen.

CD45 RO: Marker for Pan T-cell Lymphoma.

BCL-2: Diagnosis of Follicular Lymphoma.

Epithelial Membrane Antigen (EMA): Marker for Hodgkin Lymphoma.

CD68: Marker for Mesenchymal Tumors.

Undifferentiated Tumors Diagnostic Markers

Cytokeratin (Pan Cytokeratin): Marker for Carcinoma.

S100: Marker for Neuroendocrine Tumors.

Vimentin: Marker for Sarcomas and Mesenchymal Neoplasms.

Desmin: Marker for Rhabdomyosarcoma.

Chromogranin A: Marker for Neuroendocrine Tumors.

HMB-45: Marker for Melanoma.

Synaptophysin: Important marker for Neuroendocrine differentiation.

NSE (Neuron Specific Enolase): Marker for Neuroblastic and Neuroendocrine Tumors.

Mesothelioma Diagnostic Markers

Ber-EP4: Differentiates Adenocarcinoma from Mesothelioma.

HBME-1: Marker for Mesothelioma.

Calretinin: Marker for Mesothelioma.

Miscellaneous Diagnostic Markers

CD99 (MIC-2): Diagnosis of Ewing's Sarcoma and T-Cell Lymphoblastic Lymphoma.

CD117 (C-Kit): Marker for Gastrointestinal Stromal Tumor (GIST).

CEA (Carcinoembryonic Antigen): Marker for Gastrointestinal Tumors.

PSA (Prostate Specific Antigen): Diagnosis of Prostate Cancer.

CD34: Marker for Prostate Cancer.

CK7 (Cytokeratin 7): Differential diagnosis of unknown metastases.

CK20 (Cytokeratin 20): Differential diagnosis of unknown metastases.

CD79a: Marker for Pan B-cell Lymphoma.

CD138: Marker for Multiple Myeloma.

SMA (Smooth Muscle Actin): Marker for Mesenchymal Tumors of smooth muscle origin.

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