- 1 Pornoisenicolaou stain
- 2 Ultrafast Papanicolaou stain
- 3 Pap test
- 4 Contents
- 5 • 1 Technical aspects
- 6 • 2 Practical aspects
- 7 • 3 References
- 8 1 Technical aspects
- 9 Studies of the accuracy of conventional cytology report:
- 10 1.3 - Studies of the accuracy of liquid based monolayer cytology report:
- 11 3. References
Nicolaou stain (also Pap stain) is a multichromatic staining histological technique developed by George Papanikolaou, the first one to develop the cytopathology. Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smears), 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 dyes in three solutions: ==
• A nuclear stain, haematoxylin, is used to stain cell nuclei. The unmordanted haematein may be responsible for the yellow color imparted to glycogen.
• First OG-6 counterstain (-6 denotes the used concentration of phosphotungstic acid; other variants are OG-5 and OG-8). Orange G is used. It stains keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.
• Second EA (Eosin Azure) counterstain, comprising of three dyes; the number denotes the proportion of the dyes, eg. EA-36, EA-50, EA-65.
• Eosin Y stains the superficial [[epithelial squamous cells, nucleoli, cilia, and red blood cells.
• Light Green SF yellowish stains the cytoplasm of all other cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCF, however it produces visually different results and is not considered satisfactory by some.
• Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.
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 lesions are easier to interpret, the photomicrographs are better, and the stained cells are pretty. 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 and once. Pap stain is not fully standardized; it comes in several versions, subtly differing in the exact dyesused, 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% formaldehyde in 65% ethanol fixative, and use of Richard- Allan Hematoxylin-2 and Cyto-Stain, resulting in a 90-second process yielding transparent polychromatic stains.  See also
H&E stain, other popular staining technique Wright stain, used for cerebrospinal fluid and suspected lymphomas
For the similarly named guitar player and actor see Pat Smear In gynecology, the Papanikolaou test or Papanicolaou test (also called Pap smear, Pap test, cervical smear, or smear test) is a medical screening method, invented by Georgios Papanikolaou, primarily designed to detect premalignant and malignant processes in the ectocervix. It may also detect infections and abnormalities in the endocervix and endometrium.
The endocervix may be partially sampled with the device used to obtain the ectocervical sample, but due to the anatomy of this area, consistent and reliable sampling cannot be guaranteed. As abnormal endocervical cells may be sampled, those examining them are taught to recognize them.
The endometrium is not directly sampled with the device used to sample the ectocervix. Cells may exfoliate onto the cervix and be collected from there, so as with endocervical cells, abnormal cells can be recognised if present but the Pap Test should not be used as a screening tool for endometrial malignancy.The pre-cancerous changes (called dysplasias or cervical or endocervical intraepithelial neoplasia) are usually caused by sexually transmitted human papillomaviruses (HPVs). The test aims to detect and prevent the progression of HPV-induced cervical cancer and other abnormalities in the female genital tract by sampling cells from the outer opening of the cervix (Latin for "neck") of the uterus and the endocervix.
The sampling technique changed very little since its invention by Georgios Papanikolaou (1883–1962) to detect cyclic hormonal changes in vaginal cells in the early 20thcentury until the development of liquid based cell thinlayer technology. The test remains an effective, widely used method for early detection of cervical cancer and pre-cancer. The UK's call and recall system is among the best; estimates of its effectiveness vary widely but it may prevent about 700 deaths per year in the UK. It is not a perfect test. "A nurse performing 200 tests each year would prevent a death once in 38 years. During this time she or he would care for over 152 women with abnormal results, over 79 women would be referred for investigation, over 53 would have abnormal biopsy results, and over 17 would have persisting abnormalities for more than two years. At least one woman during the 38 years would die from cervical cancer despite being screened." HPV vaccine may offer better prospects in the long term. It is generally recommended that sexually active females seek Pap smear testing annually, although guidelines may vary from country to country. If results are abnormal, and depending on the nature of the abnormality, the test may need to be repeated in three to twelve months. If the abnormality requires closer scrutiny, the patient may be referred for detailed inspection of the cervix by colposcopy. The patient may also be referred for HPV DNA testing, which can serve as an adjunct (or even as an alternative) to Pap testing. About 5% to 7% of pap smears produce abnormal results, such as dysplasia, possibly indicating a pre-cancerous condition. Although many low grade cervical dysplasias spontaneously regress without ever leading to cervical cancer, dysplasia can serve as an indication that increased vigilance is needed. Endocervical and endometrial abnormalities can also be detected, as can a number of infectious processes, including yeast and Trichomonas vaginalis. A small proportion of abnormalities are reported as of "uncertain significance".
• 1 Technical aspects
• 1.1 Liquid based monolayer cytology • 1.2 Human papillomavirus testing • 1.3 Automated analysis
• 2 Practical aspects
• 3 References
1 Technical aspects
Samples are collected from the outer opening or os of the cervix using an Aylesbury spatula or (more frequently with the advent of liquid-based cytology) a plastic-fronded broom. The cells are placed on a glass slide and checked for abnormalities in the laboratory. The sample is stained using the Papanicolaou technique, in which tinctorial dyes and acids are selectively retained by cells. Unstained cells can not be visualized with light microscopy. The stainschosen by Papanicolau were selected to highlight cytoplasmic keratinization, which actually has almost nothing to do with the nuclear features used to make diagnoses now. The sample is then screened by a specially trained and qualified cytotechnologist using a light microscope. The terminology for who screens the sample varies according the country; in the UK, the personnel are known as Cytoscreeners, Biomedical scientists (BMS), Advanced Practitioners and Pathologists. The latter two take responsibility for reporting the abnormal sample which may require further investigation.
Studies of the accuracy of conventional cytology report:
• sensitivity 72% • specificity 94%
In the United States, physicians who fail to diagnose cervical cancer from a pap smear have been convicted of negligent homicide. In 1988 and 1989, Karen Smith had received pap smears which were argued to have "unequivocally" shown that she had cancer; yet the lab had not made the diagnosis. She died on March 8 1995. Later, a physician and a laboratory technician were convicted of negligent homicide. These events have led to even more rigorous quality assurance programs, and to emphasizing that this is a screening, not a diagnostic, test, associated with a small irreducible error rate.
== 1.2 - Liquid based monolayer cytology ==
Since the mid-1990s, techniques based around placing the sample into a vial containing a liquid medium which preserves the cells have been increasingly used. The media are primarily ethanol based. Two of the types are Sure-Path (TriPath Imaging) and Thin-Prep (Cytyc Corp). Once placed into the vial, the sample is processed at the laboratory into a cell thin-layer, stained, and examined by light microscopy. The liquid sample has the advantage of being suitable for low and high risk HPV testing and reduced unsatisfactory specimens from 4.1% to 2.6%. Proper sample acquisition is crucial to the accuracy of the test; clearly, a cell that is not in the sample cannot be evaluated.
1.3 - Studies of the accuracy of liquid based monolayer cytology report:
• sensitivity 61% to 66% • specificity 82% to 91% Some, but not all studies , report increased sensitivity from the liquid based smears. Human papillomavirus testing
The presence of HPV indicates that the person has been infected, the majority of women who get infected will successfully clear the infection within 18 months. It is those who have an infection of prolonged duration with high risk types (e.g. types 16,18,31,45) that are more likely to develop
""==Cervical Intraepithelial Neoplasia== ""
Italic text due to the effects that HPV has on DNA. Studies of the accuracy of HPV testing report:• sensitivity 88% to 91% (for detecting CIN 3 or higher) to 97% (for detecting CIN2+) • specificity 73% to 79% (for detecting CIN 3 or higher) to 93% (for detecting CIN 3 or higher) By adding the more sensitive HPV Test, the specificity may decline. However, the drop in specificity is not definite.  If the specificity does decline, this results in increased numbers of false positive tests and many women who did not have disease having colposcopy and treatment. A worthwhile screening test requires a balance between the sensitivity and specificity to ensure that those having a disease are correctly identified as having it and equally importantly those not identifying those without the disease as having it. Due to the liquid based pap smears having a false negative rate of 15-35%, the American College of Obstetricians and Gynecologists and American Society for Colposcopy and Cervical Pathology have recommended the use of HPV testing in addition to the pap smear in all women over the age of 30.
Regarding the role of HPV testing, randomized controlled trials have compared HPV to colposcopy. HPV testing appears as sensitive as immediate colposcopy while reducing the number of colposcopies needed. Randomized controlled trial have suggested that HPV testing could follow abnormal cytology or could precede cervical cytology examination. A study published in April 2007 suggested the act of performing a Pap smear produces an inflammatory cytokine response, which may initiate immunologic clearance of HPV, therefore reducing the risk of cervical cancer. Women who had even a single Pap smear in their history had a lower incidence of cancer. "A statistically significant decline in the HPV positivity rate correlated with the lifetime number of Pap smears received."
1.3 - Automated analysis In the last decade there have been successful attempts to develop automated, computer image analysis systems for screening. Automation may improve sensitivity and reduce unsatisfactory specimens. One of these has been FDA approved and functions in high volume reference laboratories, with human oversight.
2. Practical aspects The physician or operator collecting a sample for the test inserts a speculum into the patient's vagina, to obtain a cell sample from the cervix. A pap smear appointment is normally not scheduled during menstruation. The procedure is usually just slightly painful, because of the neuroanatomy of the cervix. However, this can depend on the patient's anatomy, the skill of the practitioner, psychological factors, and other conditions. Results usually take about 3 weeks. Slight bleeding, cramps, and other discomfort can occur afterwards.
Other tests, including the TruTest, an endometrial biopsy used for early detection of uterine cancer, can be performed during the same visit.
1. ^ Raffle AE, Alden B, Quinn M, Babb PJ, Brett MT (2003). "Outcomes of screening toprevent cancer: analysis of cumulative incidence of cervical abnormality and modelling of cases and deaths prevented". BMJ 326 (7395): 901. doi:10.1136/bmj.326.7395.901. PMID 12714468. 2. ^ a b c d e Coste J, Cochand-Priollet B, de Cremoux P, et al (2003). "Cross sectional study of conventional cervical smear, monolayer cytology, and human papillomavirus DNA testing for cervical cancer screening". BMJ 326 (7392): 733. doi:10.1136/bmj.326.7392.733. PMID 12676841. ACP Journal Club 3. ^ a b Ronco G, Cuzick J, Pierotti P, et al (2007). "Accuracy of liquid based versus conventional cytology: overall results of new technologies for cervical cancer screening randomised controlled trial". doi:10.1136/bmj.39196.740995.BE. PMID 17517761. 4. ^ a b c d e f Kulasingam SL, Hughes JP, Kiviat NB, et al (2002). "Evaluation of human papillomavirus testing in primary screening for cervical abnormalities: comparison of sensitivity, specificity, and frequency of referral". JAMA 288 (14): 1749-57. PMID 12365959. 5. ^ Cuschieri KS, Cubie HA, Whitley MW, et al (2005). "Persistent high risk HPV infection associated with development of cervical neoplasia in a prospective population study". J. Clin. Pathol. 58 (9): 946-50. doi:10.1136/jcp.2004.022863. PMID 16126875. 6. ^ a b c Cuzick J, Szarewski A, Cubie H, et al (2003). "Management of women who test positive for high-risk types of human papillomavirus: the HART study". Lancet 362 (9399): 1871-6. PMID 14667741. 7. ^ Arbyn M, Buntinx F, Van Ranst M, Paraskevaidis E, Martin-Hirsch P, Dillner J (2004). "Virologic versus cytologic triage of women with equivocal Pap smears: a meta-analysis of the accuracy to detect high-grade intraepithelial neoplasia". J. Natl. Cancer Inst. 96 (4): 280- 93. PMID 14970277. 8. ^ http://screening.iarc.fr/colpochap.php?lang=1&chap=4 9. ^ Wright TC, Cox JT, Massad LS, Twiggs LB, Wilkinson EJ (2002). "2001 Consensus Guidelines for the management of women with cervical cytological abnormalities". JAMA 287 (16): 2120-9. PMID 11966387. 10.^ ASCUS-LSIL Traige Study (ALTS) Group. (2003). "Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance". Am. J. Obstet. Gynecol. 188 (6): 1383-92. PMID 12824967. 11.^ , J Inflamm 2007;4. 12.^ Biscotti CV, Dawson AE, Dziura B, et al (2005). "Assisted primary screening using the automated ThinPrep Imaging System". Am. J. Clin. Pathol. 123 (2): 281-7. PMID 15842055. 13.^ Davey E, d'Assuncao J, Irwig L, et al (2007). "Accuracy of reading liquid based cytology slides using the ThinPrep Imager compared with conventional cytology: prospective study". doi:10.1136/bmj.39219.645475.55. PMID 17604301. Categories: Oncology | Gynecology