Papanicolaou Staining Procedure
Papanicolaou Staining Procedure
1 Overview
1.1 Purpose and Scope
This procedure is used to establish a routine Papanicolaou staining workflow. It is applicable to routine staining and morphological observation of cervical smears, vaginal cytology specimens, non-gynecological exfoliative cytology specimens, fine-needle aspiration cytology specimens, and some body-fluid cytology specimens.
1.2 Principle
(1) Papanicolaou staining is based on wet fixation. Cells are rapidly fixed with 95% ethanol to preserve intracellular proteins, lipids, and nuclear chromatin structures as much as possible, reducing artificial degeneration caused by air drying.
(2) Hematoxylin is mainly used for nuclear staining, making the nuclear membrane, chromatin, and nucleoli clearer.
(3) Orange G (OG) is used for primary staining of keratinized cytoplasm and eosinophilic structures, giving keratinized cells an orange-yellow color.
(4) EA stain is a compound cytoplasmic stain. EA50 or EA36 is commonly used. It can stain the cytoplasm of different cell types green, blue-green, or pink, thereby improving cell typing and abnormal cell recognition.
1.3 Staining Quality Requirements
(1) Nuclear structures should be clear, and chromatin distribution should be distinguishable.
(2) Cell transparency should be high, so that cell outlines and layers can still be observed even when cells overlap.
(3) Cytoplasmic differentiation should be appropriate, with good distinction between keratinized and non-keratinized components.
2 Materials and Reagents
2.1 Sample Types
(1) Cervical or vaginal exfoliated cell smears.
(2) Non-gynecological exfoliative cytology smears.
(3) Fine-needle aspiration cytology smears.
(4) Cytology preparations from cerebrospinal fluid, pleural effusion, ascites, and other body fluids.
2.2 Main Reagents
(1) 95% ethanol fixative.
(2) Distilled water or deionized water.
(3) Hematoxylin staining solution.
(4) Differentiation solution, such as an acid alcohol system.
(5) Bluing solution, such as saturated lithium carbonate or 3% ammonia water.
(6) Orange G staining solution (OG or OG6).
(7) EA staining solution, such as EA50, EA36, or a modified EA stain suitable for the specimen type.
(8) 95% ethanol.
(9) Absolute ethanol.
(10) Xylene.
(11) Neutral balsam.
2.3 Consumables and Equipment
(1) Glass slides and coverslips.
(2) Fixation jars and staining jars.
(3) Forceps and absorbent paper.
(4) Microscope.
(5) Sealed sample box or mailing box.
3 Preparation of Staining Solutions and Samples
3.1 Fixative Preparation
(1) 95% ethanol is recommended as the fixative.
(2) Used fixative should be filtered before reuse.
(3) After prolonged use, the ethanol concentration should be checked. If the concentration falls below 90%, it should be replaced promptly.
3.2 Staining Solution Preparation
(1) Hematoxylin staining solution should be filtered before use to avoid crystals or precipitates affecting staining quality.
(2) The appropriate EA staining formula should be selected according to specimen type. EA36 or EA50 is usually used for gynecological specimens, while EA65 or modified EA may be used for non-gynecological specimens based on laboratory experience.
(3) If EA staining shows abnormal color differentiation, a small amount of phosphotungstic acid may be added when the overall staining is too red; a small amount of saturated lithium carbonate may be added when the overall staining is too blue or green.
(4) A small amount of glacial acetic acid may be added to modified EA staining solution as needed to improve staining performance and enhance stain stability.
3.3 Sample Preparation Requirements
(1) After slide preparation, wet fixation must be performed immediately. Air drying is not allowed.
(2) If specimens need to be transported, they may be removed after 15 min of fixation, a small amount of glycerol can be added to the smear surface, and the slides can be sealed for storage. After receipt by the laboratory, the slides should first be placed in 95% ethanol to remove glycerol before entering the staining workflow.
(3) Drying before or after fixation affects nuclear staining and cytoplasmic color differentiation.
4 Experimental Procedure
4.1 Fixation
(1) After cytology smear preparation, immediately place the slide into 95% ethanol fixative while the specimen is still wet.
(2) The fixation time is generally no less than 15–30 min.
(3) For routine staining, fixation time usually does not exceed 1 week.
(4) Insufficient fixative concentration or delayed fixation may cause artificial cellular degeneration and affect subsequent morphological interpretation.
4.2 Nuclear Staining
(1) Remove the fixed slide and rinse several times with water to remove surface fixative.
(2) Stain in hematoxylin staining solution for 3–5 min. The specific time should be adjusted according to season, age of the staining solution, and desired staining intensity.
(3) Hematoxylin stains faster in summer or when the solution has been stored for a long time, so staining time should be shortened appropriately. In winter or when using freshly prepared staining solution, staining is slower and the time may be extended appropriately.
(4) Nuclear staining can use two strategies:
① Overstaining method: deliberately deepen nuclear staining first, then adjust to the appropriate intensity through differentiation. This is suitable for specimens with abundant mucus or complex background.
② Light staining method: strictly control hematoxylin staining time to achieve appropriate nuclear staining without obvious differentiation. This is suitable for specimens with less mucus and cells that are prone to detachment.
4.3 Differentiation and Bluing
(1) If the overstaining method is used, the slide can be briefly placed in differentiation solution after hematoxylin staining to reduce nuclear staining to the appropriate level and remove excess hematoxylin residue from the cytoplasm.
(2) Rinse immediately with water after differentiation.
(3) Place the slide in bluing solution for several seconds to change the nuclei from reddish-purple to clear blue.
(4) After bluing, rinse thoroughly with running water to avoid alkaline residue affecting subsequent cytoplasmic staining and long-term preservation.
(5) Differentiation solution and bluing solution should be replaced at least once daily.
4.4 Cytoplasmic Staining
(1) Rinse the slide in 95% ethanol.
(2) Stain in Orange G staining solution. The routine staining time is generally short, usually from several seconds to 1–2 min, and should not be too long.
(3) Rinse once or twice in 95% ethanol to remove excess Orange G.
(4) If necessary, briefly transition through 100% ethanol.
(5) Stain in EA staining solution for 3–5 min.
(6) After EA staining, rinse twice in 95% ethanol to remove floating color and stabilize cytoplasmic color differentiation.
4.5 Dehydration, Clearing, and Mounting
(1) Rinse or dehydrate in absolute ethanol.
(2) Continue dehydration in absolute ethanol for about 2 min.
(3) Transfer to xylene for clearing for about 2 min. If xylene becomes cloudy or stained, it should be replaced promptly.
(4) Add neutral balsam and cover with a coverslip to complete mounting.
(5) After mounting, avoid air bubbles and keep the mounting medium evenly distributed.
5 Reference Operation Sequence
95% ethanol fixation (15 min) → water rinse → hematoxylin staining solution (3–5 min) → water rinse → differentiation if needed → water rinse → bluing → water rinse → 95% ethanol rinse → Orange G staining solution (several seconds to 1–2 min) → 95% ethanol rinse → EA50 or EA36 (3–5 min) → 95% ethanol rinse → absolute ethanol dehydration → xylene clearing → neutral balsam mounting
6 Key Points for Result Interpretation
6.1 Cell Nuclei
(1) Nuclear borders should be clear, chromatin distribution should be distinguishable, and nuclear membrane and nucleolar morphology should be easy to identify.
(2) If nuclear staining is too light, details of atypical nuclei may be difficult to show. If nuclear staining is too dark, chromatin granule structure may be obscured.
6.2 Cytoplasm
(1) Keratinized cell cytoplasm is usually orange-yellow.
(2) Non-keratinized squamous epithelium or other cell cytoplasm may appear green, blue-green, or pink.
(3) When color differentiation is ideal, different cell types should show good tonal distinction.
6.3 Transparency
(1) When slide transparency is high, the outlines and layers of overlapping cells can still be recognized.
(2) If dehydration or clearing is insufficient, the slide may appear gray or hazy, affecting microscopic interpretation.
7 Quality Control Points
7.1 Fixation Quality Control
(1) Wet fixation is the key step for successful Papanicolaou staining.
(2) Insufficient fixation, decreased fixative concentration, or smear drying can all lead to nuclear structural distortion, abnormal cytoplasmic color differentiation, and even reduced diagnostic accuracy.
7.2 Staining Solution Quality Control
(1) Hematoxylin should be filtered daily.
(2) The color, clarity, and pH status of EA staining solution should be checked regularly.
(3) Xylene and ethanol should be replaced promptly if contamination, coloration, or turbidity occurs.
7.3 Operational Consistency Control
(1) Specimens in the same batch should use consistent staining times and replacement rhythms as much as possible.
(2) For different seasons, different batches of staining solutions, and different specimen types, staining time should be fine-tuned through preliminary testing or experience.
8 Common Problems and Cause Analysis
8.1 Nuclear Staining Too Light
(1) Hematoxylin staining time is insufficient.
(2) Differentiation time is too long.
(3) The smear dried before fixation.
(4) Bluing is insufficient or running-water rinsing is inadequate.
8.2 Nuclear Staining Too Dark
(1) Differentiation is insufficient.
(2) Differentiation solution concentration is too low.
(3) Fixation with alcohol above 95% may cause staining to appear too dark.
8.3 Cytoplasm Weakly Stained
(1) OG or EA staining time is insufficient.
(2) The staining solution is aged or the concentration has decreased.
(3) Ethanol rinsing is too strong, causing floating color and effective staining components to be excessively removed.
8.4 Poor Cytoplasmic Color Differentiation or Gray-Purple Cytoplasm
(1) The smear dried before fixation.
(2) Hematoxylin staining is too heavy or differentiation is poor, and residual hematoxylin interferes with cytoplasmic staining.
(3) The specimen contains abundant mucus or bacteria, affecting stain penetration and color differentiation quality.
8.5 EA Staining Color Deviation
(1) If the overall staining is too red, a small amount of phosphotungstic acid may be added for correction.
(2) If the overall staining is too blue or green, a small amount of saturated lithium carbonate may be added for correction.
(3) Adding an appropriate amount of glacial acetic acid to modified EA staining solution helps improve staining performance and extend stability during use.
9 Applications
9.1 Routine Cytology Screening
(1) Cervical and vaginal cytology screening.
(2) Exfoliative cytology examination.
9.2 Non-Gynecological Cytology Specimens
(1) Thyroid fine-needle aspiration cytology.
(2) Ovarian tumor fine-needle aspiration cytology.
(3) Cytology examination of cerebrospinal fluid and other body fluids.
9.3 Extended and Automated Applications
(1) Papanicolaou staining can be combined with liquid-based cytology preparation.
(2) In automated cytology platforms, fixation, staining, mounting, and other steps can be standardized.
10 Safety and Operating Standards
10.1 Personal Protection
(1) Wear a lab coat and disposable gloves during the experiment.
(2) Ethanol, xylene, and staining solutions should be handled in a well-ventilated environment.
10.2 Waste Disposal
(1) Waste liquids containing alcohol, xylene, and staining residues should be collected separately by category.
(2) All waste liquids and contaminated consumables should be disposed of according to laboratory chemical and biological sample handling regulations.
11 Selection of Key Reagents and Materials for Papanicolaou Staining
Cat. No. | Product Name | Grade and Purity | Corresponding Step | Use |
Water | for biotechnology nuclease-free, sterile | Rinsing / solution preparation | Used as standardized experimental water | |
Mayer hematoxylin staining solution | BioReagent, Biological Stain, for microscopy | Nuclear staining | Used for cell nuclear staining | |
Improved Harris Hematoxylin Staining Solution | BioReagent,Suitable for microbiology,for microscopy | Nuclear staining | Optional nuclear stain for Papanicolaou staining | |
Hematoxylin Staining Solution (Gill No.2) | BioReagent, Biological Stain, for microscopy | Nuclear staining | Suitable for routine cytological nuclear staining | |
Hematoxylin Staining Solution (Gill No.3) | BioReagent, Biological Stain, for microscopy | Nuclear staining | Suitable for systems requiring stronger nuclear staining | |
Lithium Carbonate Saturated Aqueous Solution | BioReagent,for microscopy,Biological Stain | Bluing | Used for hematoxylin bluing | |
Lithium Carbonate Aqueous Solution (0.05%) | BioReagent,Biological Stain,for microscopy,0.05% | Bluing | Used for mild bluing treatment | |
Ammonia Solution (0.1%) | BioReagent,Biological Stain,for microscopy,0.1% | Bluing | Used in ammonia-water bluing systems | |
Orange G6 Staining Solution | BioReagent,Biological Stain,for microscopy | Cytoplasmic primary staining | Used for staining keratinized cytoplasm and eosinophilic components | |
EA36 Staining Solution | BioReagent,Biological Stain,for microscopy | Cytoplasmic counterstaining | Used for cytoplasmic color differentiation in the EA36 system | |
Papanicolaou Stain Solution (Papanicolaou EA36) | BioReagent,Biological Stain,for microscopy | Cytoplasmic counterstaining | Used in the Papanicolaou EA36 system | |
Papanicolaou Stain Solution (Papanicolaou EA50) | BioReagent,Biological Stain,for microscopy | Cytoplasmic counterstaining | Used in the Papanicolaou EA50 system | |
Papanicolaou staining solution (EA65) | BioReagent,Biological Stain,for microscopy | Cytoplasmic counterstaining | More suitable for color differentiation in some non-gynecological specimens | |
Xylene | Premium-Grade Reagents, ≥99%, xylene isomer and ethyl benzene | Clearing | Used for clearing after dehydration | |
Xylene | Anhydrous Grade, ≥98%, mixture of isomers | Clearing | Used for routine clearing | |
Xylene | ACS, ≥98.5%, isomers plus ethylbenzene | Clearing | Used for clearing in standardized experimental systems | |
Neutral gum | FMP | Mounting | Used for coverslip mounting of prepared slides |