Nuances of the Papanicolaou stain

1 Department of Pathology, Deenanath Mangeshkar Hospital and Research Centre, Pune, India.

Meherbano M. Kamal

2 Department of Pathology, Government Medical College, Nagpur, Maharashtra, India.

Pramodini R. Deotale

2 Department of Pathology, Government Medical College, Nagpur, Maharashtra, India.

Hirkini Mankar

2 Department of Pathology, Government Medical College, Nagpur, Maharashtra, India.

* Corresponding author: Dr. Meherbano M. Kamal, Professor, Department of Pathology, Government Medical College, Nagpur, Maharashtra, India. moc.liamg@lamakmm.rd, moc.liamg@02enawahtas.atkajarp

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Abstract

The impressive list of achievements of Dr. G. N. Papanicolaou and his tedious journey from normal to abnormal human cell includes the importance of wet fixation of cells and the development of the unique polychromatic Pap stain. The 5-dye Pap stain method evolved through 2 salient phases. The first being the development of wet fixation using alcohol-ether to enhance cellular transparency and the second phase saw the introduction of various cytoplasmic counterstaining methods using orange G and EA (light green, Bismarck brown, eosin) and phosphotungstic acid, facilitating the distinction of cell types. The specific characteristics of the staining method is, the cellular transparency combined with crisp nuclear staining, achieved through tailored cellular fixation and cytoplasmic staining using variable dye and pH combinations. With little modifications if any the Pap stain continues to be applied uniformly globally. However, institutional supply of dyes and chemicals from different companies make minor modifications, that remain consistent, an essential part of the staining protocol. This chapter describes the preparation and principles of various components of the stain that are being currently used in our department.

Keywords: Papanicolaou stain, Pap stain, Wet fixation, Fixatives, Gill’s modified OG-6, Gill’s modified EA, Rehydration of Pap smears

THE PAPANICOLAOU (PAP) STAIN – STAIN PREPARATION AND PROCEDURE IN DETAILS

Among the major achievements in the history of cytopathology, the eponymous Pap stain method formalized by Dr. George N. Papanicolaou in 1942 was foundational. [1, 2,3] The PAP stain has been used all over the world since half a century for the staining of cervicovaginal smear. Many modifications have been published after George Papanicolaou described the original staining technique in 1942. The staining procedure varies with various staining protocols and methodologies used in different laboratories. The PAP stain is a polychromatic counterstaining method consisting of stains such as Orange G 6 (OG6) and modified eosin azure (EA). The strength of the Pap stain is such that it results in:

To achieve this, proper fixation of the smear is one of the most important prerequisite factors.

FIXATION AND FIXATIVES

The purpose of cytological fixatives is to maintain the cytomorphologic characteristics and diagnostically essential elements of the cell. Ethyl alcohol is the fixative specifically recommended for cytological preparations. Fixation coarsens the cell structures and sharpens nuclear chromatin pattern and its details. Commonly used cytological fixatives include wet fixatives and dry fixatives.

WET FIXATIVES

95% ethyl alcohol and ether in equal volume. Nowadays, it is not used as it is inflammable in nature and has a pungent odor

The cytology laboratory of G.M.C, Nagpur, uses 95% ethanol as the fixative for routine cytological smears for both cervicovaginal and fine-needle aspiration cytology smears. The slides should be fixed immediately in the fixative solution, as even a slightest air drying of the smear can alter cytomorphological features causing diagnostic problems. [ 1] The smear should be fixed at least for 20–30 min to assure adequate fixation. However, prolonged fixation for several days or even weeks will not alter cellular features. Fixative solution along with the smear may be refrigerated in such situations to minimize the evaporation.

DRY OR COATING FIXATIVES

Dry or coating fixatives are major substitutes for wet fixatives in special situations like cancer detection camps or when transportation of smears from distant collection centers is required. These fixatives are available commercially and are either aerosol (applied by spraying) or liquid based (dropped over a smear). They are composed of an alcohol base, which fixes the cells and a wax-like substance that forms a thin protective coating over the smear. Any ordinary hair spray can be substituted as coating fixative.

Method

The Pap smear is prepared in the usual way and immediately fixed with “dry fix” spray; or a few drops of liquid based fixative solution are put on the smear

While applying the spray, the bottle must be held at least 10–12 inches away from glass slide, which will prevent layering and hole formation

The coating fixatives must be removed from the smear before staining. For this, slides are kept in 80% ethyl alcohol for 1–2 h. Sometimes, two changes of 80% alcohol may be required to remove the coating fixative completely.

If the coating fixative is not removed entirely before staining, it gives a “bubbling” effect to the smear which interferes with the diagnosis. [ 2]

STAIN PREPARATION

Nuclear stain hematoxylin

Hematoxylin is the most widely used nuclear stain. It is commercially available as an amorphous brown-colored powder form, which is extracted from heartwood of Central America Logwood – “Haematoxylum campechianum.”

Hematoxylin itself is not a dye, it has to be oxidized to “hematin” which is actual staining component

The natural oxidation process or ripening of hematoxylin in aqueous solution takes place gradually over a period of time by keeping the stain bottle in the sunlight area at least for a month

The instant ripening can be achieved chemically by the addition of oxidizing agents such as sodium iodate or mercuric oxide

This stain can be used either progressively (Box 1) (without the use of acid alcohol differentiation) or regressively (Box 2)