The core of Papanicolaou staining is not coloration by a single dye, but the formation of layered differences among nuclei, keratinized components, cytoplasm of mature squamous epithelial cells, and cytoplasm of non-keratinized cells through nuclear staining, keratinized cytoplasmic staining, and polychromatic cytoplasmic counterstaining. Orange G-6, EA36, and EA50 all belong to the cytoplasmic staining system, but their functional emphases differ: Orange G-6 mainly highlights keratinization-related structures, whereas EA36 and EA50 are responsible for polychromatic cytoplasmic differentiation and cell-type discrimination.

Keywords: Papanicolaou staining; Orange G-6; EA36; EA50; cytological staining; keratinized cells; cytoplasmic counterstaining; squamous epithelial cells; cytopathology

1 Layered Staining Logic in the Papanicolaou Staining System

1.1 Division of Functions between Nuclear Staining and Cytoplasmic Staining

(1) Nuclear staining layer

Papanicolaou staining usually first uses hematoxylin-based staining solutions to stain the nuclei, allowing clear visualization of the nuclear membrane, chromatin granules, nucleoli, and nuclear atypia. The quality of nuclear staining directly affects the interpretation of cytological diagnostic indicators such as nuclear enlargement, hyperchromasia, increased nuclear-cytoplasmic ratio, and irregular chromatin texture.

(2) Keratinized cytoplasmic staining layer

Orange G-6 mainly acts on keratinized or highly keratinization-related cytoplasmic components. Its function is not to generally intensify cytoplasmic staining, but to preferentially highlight keratinized cells, dyskeratotic cells, and keratin-rich areas, thereby clearly distinguishing these structures from non-keratinized cells.

(3) Polychromatic cytoplasmic counterstaining layer

EA36 and EA50 mainly perform cytoplasmic counterstaining and cell-type differentiation. Through differential binding among eosin, light green, and toning components, the EA system enables mature squamous cells, intermediate cells, parabasal cells, glandular epithelial cells, and some reactive cells to show different color tones.

1.2 Chemical Basis of Cytoplasmic Staining

(1) Competition among acidic dyes

Orange G, Eosin Y, Light Green SF Yellowish, and Bismarck Brown are mostly acidic dyes or cytoplasmic staining components. They bind to cytoplasmic proteins, keratin, mucin, and other cytoplasmic structures to different degrees. Differences in dye molecular size, diffusion rate, and tissue affinity form the basis of polychromatic differentiation in Papanicolaou staining.

(2) Regulation by phosphotungstic acid

OG and EA systems often contain regulatory components such as phosphotungstic acid. Phosphotungstic acid can influence competitive binding among acidic dyes, so that staining is not a simple superimposition but a selective cytoplasmic differentiation process. Its concentration and formulation ratio affect the balance among orange-yellow, pink, and blue-green tones.

(3) Influence of the ethanol system

OG and EA staining solutions are usually ethanol-based. Ethanol concentration affects cellular dehydration, dye diffusion, and cytoplasmic transparency. Water carryover, insufficient dehydration, or dilution of staining solutions may cause grayish cytoplasm, weakened layering, or turbid background.

2 Functional Positioning of Orange G-6

2.1 Main Functions

(1) Demonstration of keratinized cytoplasm

Orange G-6 is mainly used to demonstrate keratinized cytoplasm, dyskeratotic cells, and keratin-rich structures. Mature keratinized squamous cells, keratinized debris, and some keratinizing abnormal cells may appear orange-yellow, orange-red, or orange-brown in OG-6.

(2) Indication of keratinization status

The value of OG-6 lies in highlighting keratinization-related cells rather than universally intensifying all cytoplasmic staining. In cervical cytology and other squamous epithelial cytology specimens, orange-yellow cytoplasm usually indicates a higher degree of keratinization or higher keratin content; however, lesion assessment still requires integration with nuclear morphology and cellular arrangement.

(3) Connection with EA counterstaining

OG-6 is applied before EA counterstaining and serves to pre-label keratinized structures. Ideally, the orange-yellow signal produced by OG-6 should remain clear after subsequent EA staining and should not be completely masked by EA counterstaining.

2.2 Typical Staining Features

(1) Keratinized cells

The cytoplasm of keratinized cells is usually orange-yellow to orange-red and may be thin, transparent, or dense. The nuclei may be pyknotic, deeply stained, or absent. This staining pattern helps identify keratinized backgrounds and keratinizing squamous epithelial components.

(2) Dyskeratotic cells

Dyskeratotic cells often show dense orange-stained cytoplasm, small cell size, hyperchromatic nuclei, or marked nuclear atypia. OG-6 can enhance the visibility of these cells, but their diagnostic significance still depends on nuclear atypia, nuclear-cytoplasmic ratio, and abnormal cellular maturation.

(3) Non-keratinized cells

Non-keratinized intermediate cells, parabasal cells, and glandular epithelial cells should not be extensively or strongly orange-stained by OG-6. If a large number of non-keratinized cells show orange staining, prolonged staining time, insufficient differentiation, or abnormal balance in subsequent EA counterstaining should be considered.

2.3 Methodological Characteristics

(1) High selectivity

OG-6 has relatively high selectivity for keratinization-related components and is an important step in distinguishing keratinized from non-keratinized cytoplasm in Papanicolaou staining. Its staining quality directly affects recognition of clues related to keratinizing lesions.

(2) Time sensitivity

Insufficient OG-6 staining may result in inadequate orange staining of keratinized cells and weakened keratinization clues. Excessive staining may cause generalized yellowish background or mild orange staining of non-keratinized cells, reducing cytoplasmic differentiation quality.

(3) Risk of counterstain masking

When EA counterstaining is too strong, the orange-yellow signal produced by OG-6 may be weakened by a green or gray-green background. This balance is particularly important when using EA50.

3 Functional Positioning of EA36

3.1 Main Functions

(1) Mild cytoplasmic counterstaining

EA36 is used after OG-6 to complete polychromatic cytoplasmic counterstaining, allowing squamous epithelial cells at different stages of maturation to show pink, light green, blue-green, or gray-green differences. Its tone is relatively soft and emphasizes cellular transparency and layered differentiation.

(2) Suitability for gynecological cytology

EA36 is commonly used in gynecological cytology staining systems, especially for differentiating superficial cells, intermediate cells, parabasal cells, and inflammatory backgrounds in cervical exfoliative cytology. Its staining profile is more favorable for preserving the transparent cytoplasmic effect typical of conventional Papanicolaou staining.

(3) Coordination between nucleus and cytoplasm

The counterstaining intensity of EA36 should be coordinated with hematoxylin nuclear staining and OG-6 orange staining. Nuclear staining that is too pale, overly strong EA staining, or insufficient OG signal can all affect the diagnostic layering between nuclei and cytoplasm in Papanicolaou staining.

3.2 Functions of Staining Components

(1) Eosin Y

Eosin Y mainly gives mature squamous cell cytoplasm a pink to orange-pink tone, especially in superficial cells and some mature cells. When the eosin effect is too strong, the overall specimen may appear excessively pink.

(2) Light Green SF Yellowish

Light Green SF Yellowish mainly stains less mature cells, metabolically active cells, glandular epithelial cells, and some reactive cells blue-green, gray-green, or pale green. This component is an important basis for distinguishing non-keratinized cells from mature squamous cells in the EA system.

(3) Bismarck Brown

Bismarck Brown can regulate the overall tone and background layering of EA staining solutions. Its proportion varies among different formulations; therefore, different EA36 products may show differences in overall color tone.

3.3 Typical Staining Features

(1) Superficial squamous cells

The cytoplasm of superficial squamous cells is usually pale pink, orange-pink, or light orange-pink, with relatively high transparency. The nuclei are usually small, deeply stained, or pyknotic.

(2) Intermediate squamous cells

The cytoplasm of intermediate squamous cells often appears blue-green, gray-green, or pale green, with clear cytoplasmic borders. This feature helps evaluate the maturation level of squamous epithelium.

(3) Parabasal cells and repair cells

Parabasal cells, repair cells, and some reactive cells in inflammatory backgrounds may show more prominent green or blue-green staining. If the green tone is too strong and the cytoplasm appears overly dark, excessive EA staining or insufficient differentiation should be considered.

4 Functional Positioning of EA50

4.1 Main Functions

(1) Enhanced cytoplasmic counterstaining

EA50 also belongs to the EA series of cytoplasmic counterstains, but it usually produces stronger cytoplasmic contrast and a more prominent green tone. Its functional emphasis is to enhance cytoplasmic recognition, making non-keratinized cells, glandular-like cells, and cell outlines in complex backgrounds clearer.

(2) Suitability for non-gynecological cytology

EA50 is often used for specimens requiring stronger cytoplasmic contrast, such as serous cavity effusions, respiratory specimens, urine, brushing specimens, and fine-needle aspiration cytology specimens. These specimens contain complex cellular sources, background proteins, inflammatory components, and degenerative cells, requiring stronger counterstaining to assist cell-type recognition.

(3) Difference from EA36

EA36 is more suitable for soft, transparent, and finely layered gynecological cytological staining, whereas EA50 is more suitable for enhancing cytoplasmic contrast and cellular border visualization in complex specimens. The difference between them is not simply one of staining strength, but also of specimen suitability and interpretive requirements.

4.2 Typical Staining Features

(1) Non-keratinized epithelial cells

EA50 can make the cytoplasm of non-keratinized epithelial cells appear distinctly blue-green, gray-green, or green, facilitating observation of cytoplasmic thickness, cellular borders, and cell-group architecture.

(2) Glandular epithelial cells

The cytoplasm of glandular epithelial cells often appears blue-green, pale green, or gray-green. In specimens of glandular cell origin, EA50 can enhance the borders of cell clusters, cytoplasmic texture, and background contrast.

(3) Complex background components

In effusion, sputum, or fine-needle aspiration specimens, EA50 can enhance the contrast between cells and the background. However, if staining is too strong, proteinaceous background, mucus, or inflammatory debris may also become more intensely stained, reducing overall transparency.

4.3 Methodological Characteristics

(1) Stronger counterstaining capacity

EA50 has relatively strong cytoplasmic counterstaining capacity and is suitable for specimens requiring enhanced cytoplasmic contrast. In cervical cytology, however, inadequate control may produce excessive green cytoplasmic staining and disrupt the soft layering of conventional Papanicolaou staining.

(2) Sensitivity to differentiation conditions

After EA50 staining, dehydration, differentiation, and clearing steps have a considerable influence on the final color tone. Insufficient dehydration may lead to grayish cytoplasm, whereas excessive differentiation may result in pale coloration.

(3) Higher requirement for balance with OG-6

Because EA50 provides stronger counterstaining, it is more likely to weaken the orange-yellow keratinization signal of OG-6. Therefore, when EA50 is used, the staining time of OG-6, the staining time of EA50, and subsequent dehydration steps should be carefully balanced.

5 Functional Distinction among Orange G-6, EA36, and EA50

Table 1. Core Functional Differences among Orange G-6, EA36, and EA50

5.1 Differences in Functional Level

(1) Orange G-6 for keratinization recognition

The core value of Orange G-6 lies in recognizing keratinized or keratin-rich structures. It is a key cytoplasmic staining step in identifying keratinizing squamous epithelial components and dyskeratotic cells.

(2) EA36 for mild differentiation

EA36 is mainly used for cytoplasmic stratification of squamous cells at different maturation stages in gynecological cytology. Its staining style emphasizes transparency, softness, and stable layering.

(3) EA50 for enhanced contrast

EA50 is more suitable for specimens with complex cellular origins or those requiring strengthened visualization of cytoplasmic borders. Its stronger counterstaining facilitates cell-type recognition in non-gynecological specimens.

5.2 Differences in Color Layers

(1) Orange-yellow layer

This layer is dominated by OG-6 and mainly indicates keratinized or keratin-rich structures. It should be clear without excessive diffusion and should not be completely masked by EA counterstaining.

(2) Pink layer

This layer is mainly contributed by eosin in the EA system and is commonly seen in the cytoplasm of mature squamous cells. In EA36, this layer is usually relatively soft.

(3) Green to blue-green layer

This layer is mainly contributed by Light Green SF Yellowish and is commonly seen in intermediate cells, parabasal cells, glandular epithelial cells, and some reactive cells. EA50 usually makes this layer more prominent.

6 Selection Strategies for Different Specimen Types

6.1 Cervical Cytology Specimens

(1) Routine screening

Cervical cytology emphasizes clear nuclear structures, transparent cytoplasm, and distinct maturation layering of squamous cells. The combination of OG-6 and EA36 is well suited to forming the soft orange-yellow, pink, and blue-green layering characteristic of conventional Papanicolaou staining.

(2) Observation of keratinizing lesions

When dyskeratotic cells, keratinized debris, or keratinizing squamous epithelial lesions are present, the staining quality of OG-6 is particularly important. The key observation is whether the orange-yellow cytoplasm is clear and localized to keratinized cells rather than representing generalized yellowish background staining.

(3) Specimens with inflammatory background

In specimens with heavy inflammatory background, overly strong EA36 staining may increase staining of inflammatory cells, mucus, or background debris. Background clarity should be controlled through staining time and dehydration steps.

6.2 Non-Gynecological Cytology Specimens

(1) Serous cavity effusions

Effusion specimens often contain mesothelial cells, inflammatory cells, degenerative cells, and tumor cells. The stronger cytoplasmic counterstaining capacity of EA50 helps visualize cell-group architecture and cytoplasmic borders, but excessive staining of background proteins should be avoided.

(2) Respiratory specimens

Sputum, brushing, or lavage specimens often contain mucus, inflammatory cells, squamous epithelial cells, and glandular-like cells. OG-6 can demonstrate keratinized or squamous metaplastic components, while EA50 can enhance cytoplasmic recognition of non-keratinized cells and glandular-like cells.

(3) Urine and fine-needle aspiration cytology

Urine and fine-needle aspiration specimens vary considerably in cellularity, degeneration, and background components. EA50 can improve cytoplasmic contrast, but overly deep staining may affect observation of perinuclear structures and cytoplasmic details.

Table 2. Suitability of OG-6, EA36, and EA50 in Different Specimens

7 Common Staining Deviations and Cause Analysis

7.1 OG-6-Related Deviations

(1) Insufficient orange staining of keratinized cells

Common causes include insufficient OG-6 staining time, aged staining solution, poor dehydration conditions, or overly strong EA counterstaining. The result is weakened color distinction between keratinized and non-keratinized cells, making keratinization clues less prominent.

(2) Generalized yellowish background

Excessive OG-6 staining, insufficient differentiation, or excessive carryover between steps may cause a generalized yellowish background. This reduces cellular transparency and interferes with identification of keratinized cells.

(3) Abnormal orange staining of non-keratinized cells

If intermediate cells, glandular cells, or background cells show extensive orange staining, reduced OG-6 selectivity, prolonged staining time, or abnormal cellular fixation and dehydration status should be considered.

7.2 EA36-Related Deviations

(1) Cytoplasm too pale

Insufficient EA36 staining time, decreased effective dye concentration, or excessive dehydration may result in pale cytoplasmic staining and indistinct cellular maturation layering.

(2) Insufficient green tone

When the effect of the light green component is insufficient, intermediate cells and parabasal cells may lack blue-green layering, and the overall specimen may appear overly pink. This is unfavorable for distinguishing squamous cells at different maturation stages.

(3) Overall grayish tone

Water carryover into the EA staining solution, incomplete dehydration, or abnormal clearing steps may cause the cytoplasm to appear grayish and reduce cellular transparency.

7.3 EA50-Related Deviations

(1) Excessive green staining

Prolonged EA50 staining or insufficient differentiation may cause excessive green cytoplasmic staining, making cellular layering appear rigid and even masking intracellular details.

(2) Enhanced background staining

Non-gynecological specimens often contain abundant proteins, mucus, and cellular debris. EA50 may intensify staining of background components. If the background is too dark, staining time and dehydration conditions should be adjusted.

(3) Weakening of OG-6 signal

When EA50 counterstaining is too strong, the orange-yellow color of keratinized cells may be visually weakened by a green or gray-green background. In this case, the staining times of OG-6 and EA50 should be rebalanced.

Table 3. Common Staining Deviations and Adjustment Strategies

8 Reagent Selection for Papanicolaou Staining

Table 4. Basic Reagents in the Papanicolaou Staining System

Table 5. Ready-to-Use Papanicolaou Staining Solutions and Supporting Products

Orange G-6, EA36, and EA50 perform different levels of cytoplasmic differentiation in Papanicolaou staining. OG-6 highlights keratinized structures, EA36 provides mild cytoplasmic stratification suitable for gynecological cytology, and EA50 enhances cytoplasmic contrast in complex specimens. Together with nuclear staining, dehydration, and clearing steps, these components determine the diagnostic readability of Papanicolaou staining.

For more related articles, please see below:

[1] Papanicolaou's Stain