1 Overview

1.1 Purpose and Scope of Application

This procedure is intended to establish a routine workflow for p16/Ki-67 immunocytochemical dual staining in cervical cytology. It is applicable to the detection of cells with cell-cycle dysregulation in liquid-based cytology samples and can be used as a supplementary method for cervical cytology examination and high-risk human papillomavirus testing, supporting risk assessment and auxiliary interpretation of high-grade cervical epithelial lesions. This procedure is intended only for scientific research, method establishment, and laboratory technical reference, and should not be used as a direct basis for clinical diagnosis, treatment, or medical decision-making.

1.2 Significance of the Biomarkers

(1) p16 is a tumor suppressor protein related to cell-cycle regulation. During persistent infection with high-risk human papillomavirus, the E7 protein can mediate functional inactivation of pRb, leading to p16 overexpression in abnormally proliferating cells. Therefore, strong p16 positivity can suggest abnormal proliferation-related changes in cervical epithelial cells, but dual-stain interpretation should still be based on co-positivity for p16 and Ki-67 within the same cell.

(2) Ki-67 is a nuclear antigen associated with cell proliferation. It reflects the active degree to which cells enter the proliferative cycle and is a commonly used marker for evaluating cell proliferation activity.

(3) Under normal physiological conditions, p16 and Ki-67 are usually not co-expressed in the same cell. If both p16 and Ki-67 are positive within the same cell, this suggests cell-cycle dysregulation and abnormal proliferation, and can serve as an objective marker indicating the risk of high-grade cervical lesions.

1.3 Method Principle

(1) This method is based on liquid-based thin-layer cytology preparation and immunocytochemical dual-staining technology, allowing the expression status of p16 and Ki-67 to be visualized separately within the same cell.

(2) This procedure uses an interpretation system in which p16 is visualized as a brown signal and Ki-67 as a red signal. Dual-stain positivity is not judged solely based on cytomorphological abnormalities, but on the co-expression of p16 and Ki-67 within the same cell.

(3) Dual-staining detection should use validated and compatible p16 primary antibody, Ki-67 primary antibody, and matched detection systems to ensure that both signals can be stably displayed on the same slide and clearly distinguished.

2 Materials and Reagents

2.1 Sample Types

(1) Cervical liquid-based cytology samples.

(2) Unstained slides prepared by membrane-based or sedimentation-based liquid-based thin-layer cytology.

2.2 Main Reagents

(1) p16 primary antibody.

(2) Ki-67 primary antibody.

(3) Tris-EDTA antigen retrieval solution.

(4) TBS or TBS-T washing buffer.

(5) 3% hydrogen peroxide blocking solution.

(6) Protein blocking solution, such as goat serum or BSA blocking solution.

(7) p16 detection system and DAB chromogenic solution.

(8) Ki-67 detection system and Fast Red chromogenic solution.

(9) Hematoxylin counterstain.

(10) Scott’s bluing solution or equivalent bluing reagent.

(11) Aqueous mounting medium.

2.3 Quality Control Materials

(1) Positive control: cervical liquid-based cytology slide or cell block section known to be positive for p16/Ki-67 dual staining.

(2) Internal negative control: mature squamous epithelial cells or other negative background cells on the same slide known to be negative for both p16 and Ki-67.

(3) External negative control: parallel slides processed in the same batch as the test samples, using antibody diluent instead of the primary antibody, or omitting the p16 primary antibody or Ki-67 primary antibody separately.

2.4 Consumables and Equipment

(1) Liquid-based cytology preparation consumables.

(2) Adhesive microscope slides and coverslips.

(3) Humidity chamber.

(4) Constant-temperature antigen retrieval device, water bath, or pressure antigen retrieval device.

(5) Microscope.

3 Specimen Preparation and Pretreatment

3.1 Specimen Requirements

(1) Dedicated unstained slides prepared from residual liquid-based cytology samples are preferred.

(2) The specimen should have relatively intact cell morphology and adequate fixation to support antigen preservation and subsequent interpretation.

(3) Specimen adequacy should be assessed before testing.

3.2 Slide Preparation

(1) Membrane-based or sedimentation-based liquid-based thin-layer cytology preparation should be used.

(2) Cell distribution should be moderate, avoiding severe cell overlap, aggregation, or detachment.

(3) Adhesive microscope slides are recommended to reduce cell detachment during subsequent antigen retrieval and washing steps.

3.3 Fixation

(1) After slide preparation, fixation should be performed according to the requirements of the liquid-based cytology platform.

(2) After fixation, slides should be fully dried and properly stored to avoid repeated exposure to moisture.

4 Experimental Procedure

4.1 Pretreatment After Slide Preparation

(1) Confirm that cells are well attached to the slide and that cell distribution meets interpretation requirements.

(2) If the slide has been stored for a long time, it should be brought to room temperature before staining.

4.2 Antigen Retrieval

(1) Place the slide in Tris-EDTA antigen retrieval solution.

(2) Heat-induced antigen retrieval at pH 9.0 can be used routinely.

(3) Allow the slide to cool naturally to room temperature after retrieval.

(4) Wash with TBS or TBS-T 3 times, 3 min each.

(5) During antigen retrieval, particular attention should be paid to preventing cell detachment.

4.3 Endogenous Peroxidase Blocking

(1) Add 3% hydrogen peroxide blocking solution and incubate at room temperature for 10 min.

(2) Wash with TBS or TBS-T 3 times, 3 min each.

4.4 Protein Blocking

(1) Add goat serum or BSA blocking solution to cover the entire cell area.

(2) Incubate at room temperature for 10 min.

(3) Decant the blocking solution. Vigorous washing is not required.

4.5 p16 Primary Antibody Incubation

(1) Add p16 primary antibody to cover the entire cell area.

(2) Incubate at room temperature for 30–60 min. The specific incubation time should be determined based on antibody validation results.

(3) Wash with TBS or TBS-T 3 times, 3 min each.

4.6 p16 Detection and DAB Color Development

(1) Add the detection system matched to the p16 primary antibody and incubate at room temperature for 20–30 min.

(2) Wash with TBS or TBS-T 3 times, 3 min each.

(3) Add DAB chromogenic solution and develop color for 3–5 min.

(4) Stop the reaction with distilled water.

4.7 Ki-67 Primary Antibody Incubation

(1) Add Ki-67 primary antibody to cover the entire cell area.

(2) Incubate at room temperature for 30–60 min. The specific incubation time should be determined based on antibody validation results.

(3) Wash with TBS or TBS-T 3 times, 3 min each.

4.8 Ki-67 Detection and Fast Red Color Development

(1) Add the detection system matched to the Ki-67 primary antibody and incubate at room temperature for 20–30 min.

(2) Wash with TBS or TBS-T 3 times, 3 min each.

(3) Add Fast Red chromogenic solution and develop color for 8–12 min.

(4) Gently rinse with distilled water to stop the reaction.

4.9 Counterstaining, Bluing, and Mounting

(1) Counterstain with hematoxylin for 20–30 s.

(2) Rinse with tap water, then blue with Scott’s bluing solution for 10–20 s.

(3) Gently rinse with distilled water.

(4) Mount with an aqueous mounting medium.

(5) When a Fast Red chromogenic system is used, ethanol dehydration and xylene clearing are not recommended, as these steps may weaken or eliminate the red signal.

5 Quality Control

5.1 General Requirements

(1) Positive and negative controls should be included in each batch of testing.

(2) The detection platform, reagents, consumables, and equipment should be validated for compatibility before routine use.

(3) Operators and interpreters should receive training and pass competency assessment.

5.2 Positive Control

(1) Known dual-stain-positive cases are recommended as positive controls.

(2) The positive control should display clear and identifiable dual-stain-positive cells.

5.3 Negative Control

(1) The internal negative control is used to confirm that background cells show no abnormal dual-staining signal.

(2) The external negative control is used to determine whether nonspecific staining or detection system contamination is present.

5.4 Quality Control of Antigen Retrieval

(1) If obvious cell detachment occurs after antigen retrieval, retrieval conditions and slide adhesion performance should be reassessed.

(2) For samples with poor cell adhesion, reducing retrieval intensity should be prioritized rather than extending retrieval time.

6 Specimen Adequacy Assessment

6.1 Basic Principles

(1) Specimen adequacy should be assessed according to routine adequacy assessment principles for cervical cytology.

(2) Adequacy assessment should be completed before formal interpretation of dual-staining results.

6.2 Interpretation Conditions

(1) If the specimen is assessed as unsatisfactory and no dual-stain-positive cells are observed, it should be reported as not meeting the conditions for interpretation.

(2) Regardless of whether the specimen is satisfactory or unsatisfactory, if at least one dual-stain-positive cell is present, the result can be interpreted as dual-stain positive.

7 Interpretation Criteria for Staining Results

7.1 Criteria for Dual-Stain Positivity

(1) Both p16 and Ki-67 positive signals are present in the same cell.

(2) In the chromogenic system recommended in this procedure, p16 appears as brown cytoplasmic and/or nuclear staining, while Ki-67 appears as red nuclear staining.

(3) Both signals must be located within the same cell and in the same focal plane.

(4) Interpretation of dual-stain positivity does not depend solely on cytomorphological abnormalities.

7.2 Criteria for Dual-Stain Negativity

(1) Only blue cervical epithelial cells are observed.

(2) Only p16-positive cervical epithelial cells are observed.

(3) Only Ki-67-positive cervical epithelial cells are observed.

(4) If p16 and Ki-67 appear in the same cell cluster area but are not located in the same cell or are not in the same focal plane, the result should be interpreted as negative.

7.3 Handling of Deeply Stained Cells

(1) When p16 staining is too intense and affects assessment of the Ki-67 nuclear signal, the microscope illumination intensity may be appropriately increased.

(2) A final interpretation should be made only after confirming whether a true red signal is present in the nucleus.

8 Dual-Stain Interpretation Workflow

8.1 Low-Power Screening

(1) First scan the entire slide under 10× or 20× magnification.

(2) Prioritize the search for single, scattered suspected dual-stain-positive cells.

8.2 Interpretation of Scattered Cells

(1) If a single, scattered cell meeting dual-stain-positive criteria is identified, the result can be directly interpreted as positive.

(2) If no such cells are found, continue to evaluate cell cluster areas.

8.3 Interpretation of Cell Clusters

(1) First search for single, scattered dual-stain-positive cells at the edge of cell clusters.

① If present, interpret the result as positive.

② If absent, continue to the next step.

(2) Evaluate the p16 staining pattern in the cell cluster.

① If p16 staining is focal, interpret the result as negative.

② If p16 staining is diffuse, continue to the next step.

(3) Evaluate whether the Ki-67-positive nucleus and p16-positive cytoplasm are located in the same focal plane.

① If they are not in the same focal plane, interpret the result as negative.

② If the Ki-67-positive nucleus is embedded within p16-positive cytoplasm and is in the same focal plane, interpret the result as positive.

9 Common Problems and Cause Analysis

9.1 Obvious Cell Detachment

(1) Antigen retrieval conditions are too strong.

(2) Slide adhesion performance is insufficient.

(3) Pretreatment and slide preparation quality are unstable.

9.2 Excessive Background Staining

(1) Nonspecific background staining caused by the chromogenic system.

(2) Insufficient washing or improper control of color development time.

(3) Primary antibody concentration is too high or blocking is insufficient.

9.3 Difficulty in Dual-Stain Interpretation

(1) Excessive color development causes the second color to be masked.

(2) Severe cell overlap makes focal-plane assessment difficult.

(3) Diffuse p16 staining is present in cell clusters, but Ki-67 nuclear localization is unclear.

9.4 Sample Does Not Meet Interpretation Conditions

(1) Insufficient cell quantity.

(2) Excessive nonspecific background staining.

(3) If obvious nonspecific background staining is present in more than 75% of the slide area, the specimen should be reported as not meeting the conditions for interpretation.

10 Reporting Recommendations

10.1 Positive Report

(1) Report as p16/Ki-67 dual-stain positive.

(2) Indicate the presence of cells with cell-cycle dysregulation, supporting the possibility of increased risk of high-grade cervical lesions.

10.2 Negative Report

(1) Report as p16/Ki-67 dual-stain negative.

(2) Indicate that no dual-stain-positive cells meeting interpretation criteria were observed in this test.

10.3 Report for Specimen Not Meeting Interpretation Conditions

(1) Report as specimen not meeting the conditions for interpretation.

(2) If necessary, recommend deciding whether to recollect the sample based on clinical context and other test results.

11 Safety and Operating Standards

11.1 Personal Protection

(1) Laboratory coats and disposable gloves should be worn during the experiment.

(2) DAB, Fast Red, hydrogen peroxide, and other chemical reagents should be handled under standardized conditions.

11.2 Quality and Training Requirements

(1) The detection platform should be validated before implementation.

(2) Operators and interpreters should undergo training, competency assessment, and continuous quality control.

11.3 Waste Disposal

(1) DAB-containing waste, chromogenic substrates, washing solutions, and contaminated consumables should be collected separately.

(2) All waste should be disposed of according to laboratory biological and chemical waste disposal regulations.

12 Related Reagent and Material Selection

Table 1 Reagent selection for p16/Ki-67 immunocytochemical dual staining in cervical cytology

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