Papanicolaou stain
Encyclopedia
Papanicolaou stain is a multichromatic staining histological
technique developed by George Papanikolaou, the father of cytopathology
.
Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smear
s), sputum
, brushings, washings, urine
, cerebrospinal fluid
, abdominal fluid, pleural fluid
, synovial fluid
, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells.
Pap staining is a very reliable technique. As such, it is used for cervical cancer
screening
in gynecology. The entire procedure is known as Pap smear
.
The classic form of Pap stain involves five dye
s in three solutions:
When performed properly, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue, and violet. The chromatin
patterns are well visible, the cells from borderline lesion
s are easier to interpret and the photomicrographs are better. The staining results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted.
On a well prepared specimen, the cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink at once.
Pap stain is not fully standardized; it comes in several versions, subtly differing in the exact dyes used, their ratios, and timing of the process.
The EA stain contains two mutually incompatible chemicals, Bismarck brown and phosphotungstic acid, which precipitate each other, impairing the useful life of the mixture and compromising the differential staining of eosin and light green. The descriptions of the compositions of the staining solutions vary by source and differ even in Papanicolaou's own publications. Mixtures of the same name from different vendors therefore can differ in composition, occasionally producing different or poor results.
in 65% ethanol
fixative
, and use of Richard-Allan Hematoxylin-2 and Cyto-Stain
, resulting in a 90-second process yielding transparent polychromatic stains.
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...
technique developed by George Papanikolaou, the father of cytopathology
Cytopathology
Cytopathology is a branch of pathology that studies and diagnoses diseases on the cellular level. The discipline was founded by Rudolf Virchow in 1858. A common application of cytopathology is the Pap smear, used as a screening tool, to detect precancerous cervical lesions and prevent cervical...
.
Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smear
Pap smear
The Papanicolaou test is a screening test used in to detect pre-cancerous and cancerous processes in the endocervical canal of the female reproductive system. Changes can be treated, thus preventing cervical cancer...
s), sputum
Sputum
Sputum is mucus that is coughed up from the lower airways. It is usually used for microbiological investigations of respiratory infections....
, brushings, washings, urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
, cerebrospinal fluid
Cerebrospinal fluid
Cerebrospinal fluid , Liquor cerebrospinalis, is a clear, colorless, bodily fluid, that occupies the subarachnoid space and the ventricular system around and inside the brain and spinal cord...
, abdominal fluid, pleural fluid
Pleural cavity
In human anatomy, the pleural cavity is the potential space between the two pleura of the lungs. The pleura is a serous membrane which folds back onto itself to form a two-layered, membrane structure. The thin space between the two pleural layers is known as the pleural cavity; it normally...
, synovial fluid
Synovial fluid
Synovial fluid is a viscous, non-Newtonian fluid found in the cavities of synovial joints. With its yolk-like consistency , the principal role of synovial fluid is to reduce friction between the articular cartilage of synovial joints during movement.-Overview:The inner membrane of synovial joints...
, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells.
Pap staining is a very reliable technique. As such, it is used for cervical cancer
Cervical cancer
Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. One of the most common symptoms is abnormal vaginal bleeding, but in some cases there may be no obvious symptoms until the cancer is in its advanced stages...
screening
Screening (medicine)
Screening, in medicine, is a strategy used in a population to detect a disease in individuals without signs or symptoms of that disease. Unlike what generally happens in medicine, screening tests are performed on persons without any clinical sign of disease....
in gynecology. The entire procedure is known as Pap smear
Pap smear
The Papanicolaou test is a screening test used in to detect pre-cancerous and cancerous processes in the endocervical canal of the female reproductive system. Changes can be treated, thus preventing cervical cancer...
.
The classic form of Pap stain involves five dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
s in three solutions:
- A nuclear stain, haematoxylinHaematoxylinHaematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the heartwood of the logwood tree. When oxidized it forms haematein, a compound that forms strongly coloured complexes with certain metal ions, the most notable ones being Fe and Al salts. Metal-haematein complexes are used...
, is used to stain cell nucleiCell nucleusIn cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
. The unmordantedMordantA mordant is a substance used to set dyes on fabrics or tissue sections by forming a coordination complex with the dye which then attaches to the fabric or tissue. It may be used for dyeing fabrics, or for intensifying stains in cell or tissue preparations. The term mordant comes from the Latin...
haematein may be responsible for the yellow color imparted to glycogenGlycogenGlycogen is a molecule that serves as the secondary long-term energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue...
. - First OG-6 counterstainCounterstainA counterstain is a stain with color contrasting to the principal stain, making the stained structure more easily visible.An example is the malachite green counterstain to the fuchsine stain in the Gimenez staining technique....
(-6 denotes the used concentration of phosphotungstic acidPhosphotungstic acidPhosphotungstic acid , tungstophosphoric acid , is a heteropoly acid with the chemical formula 31240. It normally present as a hydrate. EPTA is the name of ethanolic phosphotungstic acid, its alcohol solution used in biology. It has the appearance of small, colorless-grayish or slightly...
; other variants are OG-5 and OG-8). Orange GOrange GOrange G or orange gelb is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder.-Staining:...
is used. It stains keratinKeratinKeratin refers to a family of fibrous structural proteins. Keratin is the key of structural material making up the outer layer of human skin. It is also the key structural component of hair and nails...
. Its original role was to stain the small cells of keratinizing squamous cell carcinomaSquamous cell carcinomaSquamous cell carcinoma , occasionally rendered as "squamous-cell carcinoma", is a histologically distinct form of cancer. It arises from the uncontrolled multiplication of malignant cells deriving from epithelium, or showing particular cytological or tissue architectural characteristics of...
present in sputumSputumSputum is mucus that is coughed up from the lower airways. It is usually used for microbiological investigations of respiratory infections....
. - Second EA (Eosin Azure) counterstain, comprising three dyes; the number denotes the proportion of the dyes, eg. EA-36, EA-50, EA-65.
- Eosin YEosin YEosin Y is a form of eosin....
stains the superficial epithelial squamous cells, nucleoliNucleolusThe nucleolus is a non-membrane bound structure composed of proteins and nucleic acids found within the nucleus. Ribosomal RNA is transcribed and assembled within the nucleolus...
, ciliaCiliumA cilium is an organelle found in eukaryotic cells. Cilia are slender protuberances that project from the much larger cell body....
, and red blood cellRed blood cellRed blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...
s. - Light Green SF yellowishLight Green SF yellowishLight Green SF yellowish, or Light Green, Acid Green, Lissamine green SF, Acid Green 5, Food Green 2, FD&C Green no. 2, Green No. 205, Acid Brilliant Green 5, Pencil Green SF, or C.I. 42095, is a green triarylmethane dye. It is used in histology for staining collagen; for that purpose it is a...
stains the cytoplasmCytoplasmThe cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
of other cells, including non-keratinized squamous cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCFFast Green FCFFast Green FCF, also called Food green 3, FD&C Green No. 3, Green 1724, Solid Green FCF, and C.I. 42053, is a sea green triarylmethane food dye. Its E number is E143....
, however it produces visually different results and is not considered satisfactory by some. - Bismarck brown YBismarck brown YBismarck brown Y is a diazo dye. It is used in histology for staining tissues. It stains acid mucins to yellow color. It can be used with live cells...
stains nothing and in contemporary formulations it is often omitted.
- Eosin Y
When performed properly, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue, and violet. The chromatin
Chromatin
Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
patterns are well visible, the cells from borderline lesion
Lesion
A lesion is any abnormality in the tissue of an organism , usually caused by disease or trauma. Lesion is derived from the Latin word laesio which means injury.- Types :...
s are easier to interpret and the photomicrographs are better. The staining results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted.
On a well prepared specimen, the cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink at once.
Pap stain is not fully standardized; it comes in several versions, subtly differing in the exact dyes used, their ratios, and timing of the process.
The EA stain contains two mutually incompatible chemicals, Bismarck brown and phosphotungstic acid, which precipitate each other, impairing the useful life of the mixture and compromising the differential staining of eosin and light green. The descriptions of the compositions of the staining solutions vary by source and differ even in Papanicolaou's own publications. Mixtures of the same name from different vendors therefore can differ in composition, occasionally producing different or poor results.
Ultrafast Papanicolaou stain
Ultrafast Papanicolaou stain is an alternative for the fine needle aspiration samples, developed to achieve comparable visual clarity in significantly shorter time. The process differs in rehydration of the air-dried smear with saline, use 4% formaldehydeFormaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...
in 65% ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
fixative
Fixative
A fixative is a stabilizing or preservative agent:*Fixative , a liquid usually sprayed over a finished piece of artwork to better preserve it and prevent smudging...
, and use of Richard-Allan Hematoxylin-2 and Cyto-Stain
Cyto-Stain
Cyto-Stain, or CytoStain, is commercially available mix of staining dyes for polychromatic staining in histology. It provides results comparable to Papanicolaou staining, but in less number of operations and in shorter time...
, resulting in a 90-second process yielding transparent polychromatic stains.
See also
- H&E stainH&E stainH&E stain, HE stain or hematoxylin and eosin stain is a popular staining method in histology. It is the most widely used stain in medical diagnosis; for example when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E and termed H&E...
, other popular staining technique - Wright stain, used for cerebrospinal fluid and suspected lymphomaLymphomaLymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. Treatment might involve chemotherapy and in some cases radiotherapy and/or bone marrow transplantation, and can be curable depending on the histology, type, and stage...
s - Staining (biology)Staining (biology)Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...