Cytology

Cytology is the branch of diagnosis medicine, which deals with the study of individual cells, and/or tissue fragments spread on laboratory slide and stained appropriately. In cytology, the Medical Laboratory technologists learned how to identify the normal cells of the female genital tract, how to detect abnormalities from infections and inflammation as well as the basics of the Pap test. As a continuation to that, Medical Laboratory Technologists study on how to differentiate between benign lesions and potentially malignant lesions on the cytology smears. More will be explained on different types of carcinoma that occur in cervical cancer. There will also be an emphasis on the proper running of a cytological laboratory.

Medical Laboratory Technologists should be able to tell if a sample shows an abnormality and also what is the nature of that lesion. They should also be well-versed in the laboratory management aspects of cytology, such as quality control, how to effectively run a lab and how to handle samples from the moment they arrive until the report is dispatched back to the physician. Cytology deals with the study of epithelial cells and non-epithelial cells. It gives an understanding of normal cytology of female genital tract through microscopic examination of Pap smear. Medical Laboratory Technologists are also exposed to epithelial changes due to inflammations, infections, hormonal influences as well as regenerative and degenerative processes.

Main Reference Textbook:
Diagnostic Cytology and its Histopathology Basis, Koss,L 4th Edition, Lippincott


Additional Reference Materials:
The Manual of Cytotechnology, American Society of Clinical Pathologist, 1997
A Manual of cytotechnology

Basic Histopathology, A color atlas and text, 4th Edition 2002. Wheather

World Health Organization Terminology

In the 1950s, some cytologists began to promote a more scientifically accurate terminology that would allow cytological diagnoses to translate directly into histological diagnoses. This terminology (Table 15) was later adopted by the World Health Organization (WHO) (Riotton et al., 1973). The WHO terminology allows more precise correlation between cytological and histopathological findings, but is difficult to use since it includes a number of different entities.

These are mild dysplasia, moderate dysplasia, severe dysplasia, epidermoid carcinoma in situ, epidermoid carcinoma in situ with minimal stromal invasion, invasive epidermoid microcarcinoma and invasive epidermoid carcinoma. Studies have shown high rates of intra-observer and inter observer variation with cervical cytology in general (Yobs et al., 1987; Klinkhamer et al., 1988; Selvaggi, 1999; Stoler & Schiffman, 2001).

Classification systems that utilize more diagnostic categories have inherently higher rates of variability than do classification systems with fewer diagnostic categories (Yobs et al., 1987; Selvaggi, 1999; Stoler & Schiffman, 2001; Kundel & Polansky, 2003). Other limitations of the WHO terminology are that it does not adequately deal with non-neoplastic conditions nor with specimen adequacy. Despite its limitations, many cytologists around the world continue to utilize the WHO terminology.

Cervical Cytology and Cytological Terminology

Cervical Cytology

Cytological testing involves collection of exfoliated cells from the cervix and microscopic examination of these cells after staining. The concept of utilizing exfoliative cytology to identify women with invasive cervical cancer was introduced by Papanicolaou and Babes in the 1920s (Papanicolaou, 1928; Papanicolaou & Traut, 1941). Subsequently, Papanicolaou refined the technique and demonstrated that conventional cytology could also be used to identify precancerous lesions of the cervix (Papanicolaou, 1954).

The shift in emphasis from using cytology as a way to identify cases of invasive cervical cancer to using it to identify women with high-grade precursor lesions who are at risk for subsequently developing invasive cervical cancer was highly significant, as it meant that cervical cytology could be used to actually prevent the development of cervical cancer rather than simply identify cases at an early stage. In the 1960s, cervical cytology began to be widely used in many developed countries as a technique for cervical cancer prevention. Although the method was introduced over a half century ago, cytology-based screening programmes continue to be the mainstay of cervical cancer prevention.

Cytological terminology

Papanicolaou classes

The terminology developed by Papanicolaou separated cervical cytological findings into five categories or classes (Table 14) (Papanicolaou, 1954). At the time the classification was developed, there was only limited understanding of the relationship between cervical cancer precursor lesions and invasive cancers. Moreover, invasive cervical cancer was common and cervical cytology was initially viewed as a way of detecting early stage, easily treated cancers.

Therefore, the Papanicolaou classification system focused on how closely the exfoliated cells resembled those from an invasive cancer. Although the Papanicolaou classification was modified many times over the years, the problems inherent in this classification remain. For example, although is clear how Class I and Class V translate into known histological entities, Classes II, III or IV correlate less clearly with standard histopathological lesions. For example, should a carcinoma in situ be classified as Class IV and all grades of dysplasia as Class III, or does mild dysplasia correspond to Class II? There was also no consensus as to what other non-neoplastic conditions were combined in Class II.

Such ambiguity in the Papanicolaou classification resulted in its non-uniform use by different cytologists. Modifications of the Papanicolaou classifications are still used in some countries. In the Netherlands, a modified Papanicolaou system (CISOE-A) is used for classification. This redefined and subdivided the Papanicolaou classes in order to make the terminology correlate with histopathological terminology (Hanselaar, 2002).

Cervical Intraepithelial Neoplasia (CIN) Terminology

As a result of advances in understanding of the pathogenesis of cervical cancer, the cervical intraepithelial neoplasia (CIN) terminology was introduced in the late 1960s (Richart, 1968, 1973). The CIN concept emphasized that dysplasia and carcinoma in situ represent different stages of the same biological process, rather than separate entities. It had a major impact on how precancerous lesions were treated, since all types of cervical cancer precursor were considered to form a biological and clinical continuum.

In the CIN terminology, mild dysplasia is classified as CIN 1, moderate dysplasia as CIN 2 and severe dysplasia and carcinoma in situ are grouped together and classified as CIN 3 (Table 15). The CIN terminology is still widely used in many countries for reporting both histological and cytological diagnoses.

Atypical squamous cells (ASC)

‘Atypical squamous cells’ (ASC) is used when cytological findings are considered suggestive but not diagnostic of a squamous intraepithelial lesion (SIL) (Figure 31). The term ASC was retained in the 2001 Bethesda System because of the wide recognition that these cells imply a significant risk for an underlying high-grade cervical intraepithelial lesion (SIL). In various studies, the prevalence of CIN 2 or 3 in women with ASC has varied between 10% and 20% (Wright et al., 2002). The ASC category roughly correlates with the ‘borderline dyskaryosis’ category used in the United Kingdom. However, neither the WHO terminology nor the CIN terminology incorporates a category similar to ASC.

The 2001 Bethesda System also clearly separate s A S C from reactive/reparative changes and an interpretation of ASC should not be made whenever a cytopathologist identifies minor cytological abnormalities. The term ASC should be used only when the cytological findings are suggestive, but not diagnostic, of SIL. Currently, approximately 4–5% of all cervical cytology specimens are classified as ASC in the USA (Jones & Davey, 2000).

The ‘atypical squamous cell’ category is formally subdivided into two subcategories: ‘atypical squamous cells – of undetermined significance’ (ASCU S or ASC-US) and ‘atypical squamous cells – cannot exclude a high-grade SIL’ (ASC-H). This subdivision was felt to be important because women with ASC-H (Figure 32) are at considerably higher risk for having CIN 2 or 3 and of being high-risk HPV DNA-positive than are women with ASCUS (Genest et al., 1998; Sherman et al., 1999, 2001; Selvaggi, 2003).

Information from the US National Cancer Institute ASCUS–LSIL Triage Study (ALTS) clinical trial indicates that the risk that a woman with AS C-H has CIN 2 or 3 is over twice that of a woman with ASCUS (Sherman et al., 2001). Moreo ver, the prevalence of high-risk HPV DNA- positivity among women with ASC-H is almost as h igh as that of women with a high-grade squamous intraepithelial lesion (HSIL) cytologic a l result. Therefore the recommended management of women with ASCUS and ASCH differs (Wright et al., 2002).

Low-Grade Squamous Intraepithelial Lesion

The LSIL category in the Bethesda System includes both HPV effects and CIN 1 (i.e. mild dysplasia). Most cytologists consider the cytopathic effects of HPV, including multinucleation, perinuclear halos and nuclear atypia with irregular nuclear outlines and hyperchromasia, to overlap the cytological features of CIN 1. These features are referred to as ‘koilocytosis’, a term derived from the Greek koilos, meaning hollow. The classical studies of Reagan and others identified the key cytological features of CIN 1 (Table 18) (Reagan & Hamomic, 1956).

The cells are of the superficial or intermediate cell type. They are classically described as having nuclei 4–6 times the size of a normal intermediate-cell nucleus (Figure 33).

However, nuclei may vary in size and, in many cases of LSIL that are characterized by marked HPV cytopathic effects, are only twice the size of a normal intermediate-cell nucleus. The nuclei are usually hyperchromatic, and multinucleation is common. The chromatin is finely granular and uniformly distributed. The cells typically occur as individual cells or as sheets of cells with well defined cell borders.

High-Grade Squamous Intraepithelial Lesion

Because the Bethesda System combines moderate and severe dysplasia together with carcinoma in situ in the HSIL category, there is wide variation in the cytological appearance of HSIL. When applying the 2001 Bethesda System, many cytopathologists utilize the option of subdividing HSIL into CIN 2 and CIN 3 lesions. As the severity of the lesion increases, the degree of differentiation and the amount of cytoplasm decreases, the nuclear: cytoplasmic ratio increases, and the degree of nuclear atypia also increases. HSIL of the moderate dysplasia type typically contains cells similar to those seen in LSIL, as well as atypical immature cells of the parabasal type (Figure 34).

The nuclei of these cells are more hyperchromatic and irregular than typically seen in LSIL. In severe dysplasia, the overall size of the cells is reduced compared to mild and moderate dysplasia, but because the cells demonstrate minim al differentiation, the nuclear: cytoplasmic ratio is greatly increased. In severe dysplasia, there are usually considerably greater numbers of neoplastic cells that are typically found individually.

Carcinoma in situ can be of the small-cell type, of the large-cell non-keratinizing type or of the large-cell keratinizing (pleomorphic) type. Although separation of carcinoma in situ into these three different cytological types has little clinical significance, all three have quite different cytological appearances. Small-cell lesions consist of small basal-type cells similar to those seen in severe dysplasia but which demonstrate even less cy toplasm and higher nuclear: cytoplasmic ratios (Figure 35).

Because of their small size, these cells can easily be overlooked during routine screening and such cases account for a disproportionate percentage of false negative cytological results. The cells of large-cell non-keratinizing lesions typically form syncytial-like cell sheets in which individual cell membranes are difficult to identify. These cells have enlarged, hyperchromatic nuclei and minimal amounts of cytoplasm. The keratinizing large-cell type of carcinoma in situ is composed of pleomorphic, highly atypical cells, many of which have thick keratinized cytoplasm. These cells are often spindled or tadpole-shaped and have extremely dense nuclear chromatin (Figure 36).

Invasive Squamous Cell Carcinoma

Cytologically, squamous-cell carcinomas of the cervix are subdivided into keratinizing and non-keratinizing types.

Non-keratinizing carcinomas (Figure 37) typically have large numbers of malignant cells that form loose cell sheets and syncytial arrangements. The cells have enlarged nuclei with coarsely clumped chromatin, prominent macronucleoli and focal chromatin clearing. A key cytological feature is the presence of a ‘dirty’ background containing blood and necrotic material. This is often referred to as a tumour diathesis. This characteristic background is usually less prominent in liquid-based cytology specimens.

Cervical smears from women with kera tinizing carcinomas contain malignant cells of a variety of shapes and sizes (Figure 38). Some of the cells are pleomorphic or tadpole-shaped with nuclei that are irregular in shape and quite hyper chromatic. U nlike non-keratinizing squamous-cell carcinoma, keratinizing squamous-cell carcinomas usually do not have a ‘dirty’ background or evidence of tumour diathesis.

Atypical Glandular Cells (AGC)

Glandular Cell Abnormalities

Glandular cell abnormalities are categorized into four categories: atypical glandular cells (AGC), atypical glandular cells – favor neoplasia, adenocarcinoma in situ and adenocarcinoma. Whenever possible, atypical glandular cells are categorized as to whether they are endocervical or endometrial in origin.

Atypical glandular cells (AGC)

Glandular cytological abnormalities are considerably less common than squamous abnormalities and most cytologists tend to be less comfortable recognizing and diagnosing them. In addition, the criteria used to differentiate reactive endocervical changes from neoplasia are less well established than those used for squamous lesions.Cytologists even have difficulty in differentiating atypical endocervical cells from cases of CIN 2 or CIN 3 that have extended into endocervical crypts. This accounts for the high prevalence of squamous abnormalities (approximately 30%) detected in women referred to colposcopy for AGC (Eddy et al., 1997; Veljovich et al., 1998; Ronnett et al., 1999; Jones & Davey, 2000; Krane et al., 2004).


The cytological features of atypical glandular cells vary depending on the degree of the underlying histopathological abnormality and whether or not the cells are endocervical or endometrial in origin. Atypical glandular cells of endocervical origin frequently form dense two- or three-dimensional aggregates that have minor degrees of nuclear overlapping. In some cases, the chromatin is somewhat granular and nuclear feathering can be seen at the periphery of the cellular aggregates (Figure 39).

In cases interpreted as atypical glandular cells – favor neoplasia, there is more marked cytological abnormality and typically a greater number of abnormal cells.

Adenocarcinoma In Situ (AIS)

In cases of adenocarcinoma in situ, there are usually a larger number of atypical glandular cells that form crowded cellular clusters (Figure 40).

The sheets are usually three-dimensional. The cells within these sheets occasionally form rosettes and have extensive feathering of the cells at the periphery. Individual endocervical cells are highly atypical with enlarged round, oval or elongated nuclei that vary in size from cell to cell. In most cases, the chromatin is coarsely clumped and multiple mitoses are seen.

Adenocarcinoma

Invasive adenocarcinomas should be subclassified into the endocervical or endometrial type whenever possible. The cytological diagnosis of invasive adenocarcinoma is relatively straightforward.

Adenocarcinoma cells from either an endocervical or an endometrial primary type have enlarged nuclei, high nuclear: cytoplasmic ratios, coarsely clumped chromatin and prominent nucleoli (Figure 41). They can occur singly or in clusters.

Conventional Cervical Cytology

The importance of proper specimen collection cannot be overemphasized. Although no formal studies have demonstrated that educating clinicians on the optimal technique of obtaining cervical cytology samples improves specimen quality, there is considerable anecdotal evidence that this is important (Krieger et al., 1998). One half to two thirds of false negative cervical cytology results are attributable to either poor patient conditions at the time the cervical specimen is collected or the manner in which it is collected (Morell et al., 1982; Gay et al., 1985; Vooijs et al., 1985; Agency for Health Care Policy and Research, 1999). Therefore it is important that clinicians and nurses obtaining specimens be adequately trained in specimen collection and that they avoid situations that may reduce the performance of the test (McGoogan et al., 1998). This is especially important in low-resource settings, where women may undergo screening only once or twice in their lifetime.

Preparing the woman

Whenever possible, appointments for a cervical cytology examination should be scheduled approximately two weeks after the first day of the last menstrual period. Patients should be instructed to avoid sexual intercourse and douching for 24 to 48 hours before having the cytology specimen collected. In addition, women should not use any intravaginal products or medicine for several days before the smear is taken. Women using an intravaginal estrogen product should discontinue its use several days before the examination. Circumstances that may interfere with the interpretation of a cervical cytology test include active menstruation, significant cervical or vulvovaginal infections and a timing less than eight weeks post-partum. When a woman is actively menstruating, blood and cellular debris from the endometrium tend to obscure the cells on the smear, particularly during the first few days. Similarly, a cytology specimen should not be obtained when an abnormal vaginal or cervical discharge is observed. Women with a discharge should be evaluated for cervicitis and vaginitis using appropriate tests and be treated before the cytology specimen is taken, otherwise the specimen may be compromised by the inflammatory exudates or mildly reactive cells may be misinterpreted as a significant cytological abnormality. There is controversy as to the ideal timing of post-partum smears. Smears obtained less than eight weeks postpartum are often difficult to interpret because of marked inflammation and reparative changes, so a high rate of mild cytological abnormalities may be diagnosed. Another factor that can adversely affect the interpretation of cervical cytology specimens is severe atrophy. Although one should strive to collect specimens under ideal conditions, failure to comply with suggested screening intervals presents a greater risk to women. For previously noncompliant women, particularly those at risk for cervical neoplasia, a smear obtained under less than ideal conditions is preferable to no smear at all.

Equipment

To collect a conventional cervical cytological specimen, the equipment required is a speculum, a light source, a collection device, a glass slide and fixative. Since most cervical cancer precursors and invasive cancers occur in the transformation zone, the use of specially designed devices that sample this area is recommended. The most common is a wooden or plastic spatula that conforms to the curvature of the portio. It is critical that the endocervical canal be sampled in order to obtain reasonable sensitivity (Martin Hirsch et al., 1999) and many spatula-type devices have extended tips designed to collect cells from this area. Either a moistened cotton swab or a brush-type endocervical sampler device (e.g., cytobrush) can be used to collect a second sample directly from the endocervical canal after the portio has been sampled (Koonings et al., 1992; Kohlberger et al., 1999). Recently developed collection devices that sample the endocervix and exocervix simultaneously do not provide a significantly lower false negative rate than the combination of spatula and a conical cervical brush (Szarewski et al., 1993). There is no consensus as to whether a single-slide technique, with both samples of the ectocervix and endocervix placed on the same slide, or a technique in which the two samples are put on two separate slides is preferable. Comparative studies of the two techniques have reported similar results (Saitas et al., 1995; Quackenbush, 1999). The single-slide approach has the advantage of reducing screening time and laboratory workload and it decreases the storage space required for archiving slides. When a single-slide technique is utilized, there also is no consensus on whether the specimens from the ectocervix and endocervix should be mixed together on the slide or kept separate as in the V (vagina) C (ectocervix) E (endocervix) technique.

Collecting the sample

A conventional cytology specimen is typically obtained using a spatula and conical cervical brush. The slide must first be labelled with the woman's name or number. Laboratories should have a written protocol specifying what is considered adequate labelling and should not accept inadequately labelled specimens. The person collecting the specimen should ensure that a test requisition is accurately and legibly filled out before collecting the specimen. The information most commonly requested by laboratories includes:

• Woman's name and indication if there has been a name change in the last five years. Some laboratories also use unique patient identifier numbers

• Date of birth or age

• Menstrual status (date of last menstrual period, whether the woman is pregnant, post partum, on hormone replacement therapy, or has had a hysterectomy)

• Previous history of abnormal cervical cytology, or treatment for CIN or cancer

• Whether the clinician considers the woman to be at high risk for developing CIN or cancer. Possible risk factors include smoking, infection with HIV, lack of previous screening and multiple partners.

• Specimen source – vaginal or cervical

Good visualization of the cervix is important for obtaining an adequate specimen. Cervical cytology specimens are generally collected with the woman in the dorsolithotomy position. A sterilized or single-use bivalve speculum of appropriate size is inserted into the vagina in such a manner as to allow complete visualization of the cervical os and as much of the transformation zone as possible. The cervix should not be contaminated with lubricant or water-soluble gel that may obscure the smear. Therefore the smear must be obtained before any bimanual examination. Gentle removal of excess mucus and discharge from the cervix with a large cotton-tipped applicator can produce a better-quality smear (Kotaska & Matisic, 2003), but vigorous cleansing may remove many of the most easily exfoliated cells. Saline should not be used to help clear debris from the surface of the cervix. It is also preferable not to apply 3–5% acetic acid to the cervix before taking the cytology specimen, as this can reduce the cellularity of the smear and produce poor staining (Griffiths et al., 1989; Cronje et al., 1997). Before the specimen is collected, the cervix should be carefully inspected with the naked eye for grossly visible masses or ulcerations that may indicate an invasive cervical cancer. If a grossly visible lesion isidentified, the woman should be referred for further confirmation. In many cases, the lesion can be directly sampled and the cellular sample obtained can be submitted separately for cytological assessment. The procedure for collecting cells from the cervix varies depending on the type of device used and the number of slides to be prepared. If a spatula and conical cervical brush are utilized, the first step is to place the spatula firmly against the ectocervix with the long projection extending into the endocervical canal. The spatula is then rotated several times 360° around the portio and removed. It is important to ensure that the entire squamocolumnar junction is sampled, since this is the site where most CIN lesions develop. In most women, the spatula will come into contact with the squamocolumnar junction if the pointed end is placed in the os, but in young women with a large ectopy, the spatula may need to be moved laterally to sample a peripherally positioned squamocolumnar junction. When rotating the spatula, it is easy to miss part of the cervix; this can be alleviated by directly visualizing the cervix while sampling. Transfer is best performed by using the spatula to thinly spread the cells onto the glass slide. It is important to ensure that as much cellular material as possible is transferred from both sides of the spatula. The endocervical canal is then sampled, using a conical cervical brush, which is placed in the endocervical canal so that the last few bristles remain visible and then gently rotated 90° to 180° once. One such rotation will adequately sample the endocervical canal and generally does not produce bleeding. Material from both sides of the spatula should be spread onto the slide. If collection devices that simultaneously sample both the endocervix and the ectocervix are used, the manufacturers' directions should be followed for each type of device. Cell fixation must be performed within a few seconds of specimen collection in order to prevent air-drying, which obscures cellular detail and hinders interpretation (Somrak et al., 1990). Immersing the slide in alcohol or spraying it with a specially formulated spray fixative can prevent air-drying. With immersion fixation, the slide is either immersed in alcohol and transferred to the laboratory in the container of alcohol or allowed to fix for 20 to 30 minutes in the alcohol, removed and allowed to air-dry. Various different spray fixatives are available. Only spray fixatives specifically designed for cytological specimens should be used and the manufacturer's instructions for a given product must be followed. The fixative should be liberally applied such that the slide appears moist over its entire surface. In order to prevent disruption of the cellular layer on the slide, the container of spray fixative should generally be held 15–25 cm from the slide during application.

Performance of conventional cytology

Despite the proven effectiveness of cervical cytological screening in reducing the incidence of cervical cancer, over the last decade the accuracy of cervical cytology has been questioned. Two factors need to be considered when assessing the accuracy of any screening or diagnostic test. One is whether the test is specific in detecting a given condition; the other is the sensitivity of the test for detecting the condition. Several large meta-analyses have indicated that both the sensitivity and specificity of cervical cytology are lower than previously thought (Fahey et al., 1995; McCrory et al., 1999; Nanda et al., 2000). [The Working Group considered the estimates of cytology test performance obtained through these meta-analyses to be of concern, given current cytology practices. In particular, it felt that it is very unlikely that specificities as low as 60–70% would be observed in a modern cytological screening practice.]. Even within the confines of research studies, a wide range of performance has been reported.

Liquid-based Cervical Cytology

Liquid-based cytology (LBC) was introduced in the mid-1990s as a way to improve the performance of the test. Rather than having the clinician prepare the cytological specimen at the bedside by spreading the exfoliated cells onto a glass slide, the cells are transferred to a liquid preservative solution that is transported to the laboratory, where the slide is prepared. A number of different LBC techniques are in use worldwide.(Figure 29).

These include ThinPrep®, SurePath™, Cytoscreen™, Cyteasy®, Labonord Easy Prep, Cytoslide, SpinThin and PapSpin. The first two of these are approved for use in the USA by the Food and Drug Administration (FDA) and are the most widely used methods worldwide. They are therefore the best characterized in terms of performance. With the ThinPrep method, clumps of cells and mucus are broken up by mechanical agitation and then the liquid preservative solution is filtered through a membrane filter with a pore size specifically designed to trap epithelial cells while allowing contaminating red blood cells and inflammatory cells to pass through.

The epithelial cells collected on the membrane filter are then transferred onto a glass slide and stained. This produces a relatively thin, monolayer-type preparation. The ThinPrep-2000 processor allows one specimen to be processed at a time, whereas the newer ThinPrep-3000 processor is more fully automated and allows up to 80 samples to be processed at a time. In contrast, with the SurePath method, clumps of cells and mucus are broken up by aspiration through a syringe. The cell suspension is then layered on top of a density gradient and the red blood cells and inflammatory cells are separated from the epithelial cells by density gradient centrifugation. The resulting cell pellet containing predominantly epithelial cells is then inserted into a robotic workstation, where it is resuspended and transferred to a glass microscope slide.

The SurePath method allows up to 48 samples to be processed at a time. LBC is purported to have a number of advantages over conventional cervical cytology. These include a more representative transfer of cells from the collection device to the glass slide, a reduction in the number of unsatisfactory cytology specimens, the availability of residual cellular material for subsequent molecular testing or for making additional glass slides, and possibly increased detection of HSIL.

Performance of liquid-based cytology methods

Numerous studies have evaluated the comparative performance of the two most commonly used LBC methods (ThinPrep and SurePath) and conventional cytology with respect to test positivity, their sensitivity and specificity for identification of CIN, the time required for evaluation of the specimens, and specimen adequacy. Although there is reasonable agreement that LBC improves specimen adequacy and reduces screening time compared to conventional cytology, there is considerable controversy surrounding the relative sensitivity and specificity of the two approaches, largely due to a lack of well designed comparative studies. Most comparative studies have utilized one of two types of study design: split-sample studies and historical control studies. Split-sample studies collect cells from the cervix using a single collection device and a conventional cervical cytology specimen is prepared first. Residual cells remaining on the device are then transferred to a liquid based cytology preservative.

Therefore each woman acts as her own control and detection rates in conventional and LBC specimens are compared. The other widely used study design, known as ‘direct to vial’, compares the performance of LBC collected in the routine manner (direct transfer to the preservative solution) during a given time period with historic control data obtained using conventional cytology. Both study designs have significant limitations. With split-sample studies, it is difficult to ensure that the two cytology specimens are comparable. Since the conventional cytology slide is prepared first and the LBC specimen is prepared second, this design would seem to lead inherently to bias against LBC. Therefore it has been argued that split-sample studies do not demonstrate the full benefit that could be obtained when LBC is utilized in routine clinical practice. Studies utilizing historical controls avoid the need to prepare several cytology specimens from a single woman, but introduce other potential biases, including the comparability of the populations being compared.

Other significant limitations found in many of the studies evaluating LBC include failure to compare test performance with a reference standard of ‘blinded’ colposcopy/biopsy and a study population of women followed up for a prior cytological abnormality rather than women undergoing routine screening. A review of new cervical cytology methods conducted in 2001 for the US Preventive Services Task Force and the Agency for Healthcare Research and Quality found that out of 962 potentially relevant studies, not one met their predefined inclusion criteria (Hartmann et al., 2001). This was commonly due to lack of an adequate reference standard, but most studies were excluded for more than one reason.

Quality Assurance (QA) and Quality Control (QC) in Cervical Cytology

An advantage of cervical cytology over screening methods such as visual screening is that even though quality assurance and quality control programmes can be developed for both, the availability of archival glass slides facilities such programmes. Various definitions for quality control and quality assurance are used by laboratories.

In general, quality control can be thought of in terms of the actual assessments that are done to ensure high quality and quality assurance can be thought of in terms of the entire process of maintaining minimum standards and continually striving for excellence. Quality assurance should be a coordinated effort that is designed to control, detect and prevent the occurrence of errors and hopefully to improve patient care. In general, there are three stages to the process of quality control (Bozzo, 1991):

• Setting standards for what one wishes to control and defining the benchmarks;
• Developing a mechanism for assessing what one wishes to control;
• Defining the response to be taken when deficiencies are identified.

For cervical cytology screening, quality assurance programmes can include a number of types of activity and should take into account country and location-specific needs. What may be considered acceptable or even mandatory in one setting may serve simply to limit the availability of screening in other settings. It is critical, however, that any cervical cytology laboratory or programme has an established quality assurance programme. In general, it is preferable for cytology services to be centralized as much as possible, to facilitate quality assurance. The use of computerized data collection systems that can integrate cytological findings, histological findings and follow-up information is highly desirable (Miller et al., 2000).

Preanalytic Quality Control

Preanalytic quality control measures include the records that laboratories should maintain relating to specimen receipt, preparation of specimens, staining of specimens and upkeep of equipment and microscopes, as well as records of personnel and their training and education.

Various Benign Disorders that occur in Endocervical Region

Basal Cell Hyperplasia

It is rarely seen in cervical scrape smears. In form of flat clusters of small, spherical or polygonal cells. Cells have scanty cytoplasm relatively large, dense but regular nuclei. The nuclei vary in size and shape and may show mitotic activity. The recognition of the basal endocervical cells is easier if well-differentiated columnar cells are attached to the periphery of such cluster.

Squamous Metaplasia

It is a replacement of normal endocervical epithelium by squamous epithelium of varying degrees of maturity.It is very frequent even that may be confined to a small area of the endocervix and involves surface epithelium and the glands. Squamous metaplasia is a normal, physiological event during maturation of the female genital tract.

Atypical Squamous Metaplasia

Squamous metaplasia in tissue and smears show light to severe abnormalities. The slight changes are cell and nuclear enlargement or binucleation confined to a few cells within the cluster. Severe changes include: significance cellular and nuclear enlargement, variability in nuclear sizes, and coarse granulation of chromatin and presence of prominent nucleoli.

Tubal and tubo-endometroid Metaplasia

Endocervical epithelium show features of tubal or endometrial epithelium. Tubal metaplasia is a replacement of the normal epithelium lining the endocervical surface and gland by epithelium of tubal type such as columnar ciliated cells, clear secretory cells. Nuclear abnormalities may be significant and may classified as atypical glandular cells of unknown significance (AGUS)

Changes that occur in Nuclei of Malignant cells

Nuclear abnormalities are the dominant morphologic feature of cancer cells. Key changes observed:

Nuclear enlargement (N/C ratio)

Irregularities of nuclear configuration and contour

• Notches and Protrusions (“nipples”)

Nuclear texture: hyperchromasia and coarse granulation of chromatin

• Hyperchromasia - Changes in nuclear chromatin (dark staining)
• Coarse granulation of chromatin - Thickening of nuclear membrane

Abnormalities of sex chromatin in females

• Sex chromatin body (Barr body) – represent inactive X chromosome in female cells. Presence of two or more Bar bodies may observed in cancer cell

Multinucleation in cancer cells

• Nuclear: Grooves, Creases

Nucleolar abnormalities

• Eosinophilic center, surrounded by border nucleolus-chromatin. Number and size of nucleoli increase. Comma-shaped (cookie-cutter shaped)

Abnormalities of cell cycle and mitoses

• Mitotic rate - Exceed significantly the rate of normal tissue of origin. Abnormal mitoses - Abnormal number and distribution of chromosomes. Excessive number of mitotic spindle

Cytology and Histopathology Lab

Cytology is the science that utilizes the cells that have desquamated freely from epithelium surfaces body cavities or cells that have been forcibly removed from various issues and fluid to arrive at the diagnosis. The specimens that are process from cytology laboratory are gynaecological specimen for pap smear and non-gynaecological specimen such as fluid, sputum and brushings.

The work that been conducted are labeling of cytological specimen, preparation of smear for non-gynae specimen, staining using May Grunwald Giemsa stain and Papanicolaou stain, process of aspiration material from FNAC, quick dip, cell block and destining procedure. The tasks that I was assigned or observed during my practical in Cytology Laboratory are Labeling of Cytological Specimen, Preparation of Smear for Non-Gynae Specimen, May Grunwald Giemsa Stain, Papanicolaou Stain is to demonstrate nuclear details and preserve of keratin and Process of Aspiration Material from FNAC is to ensure that aspirated material are processed correctly and properly as well as to achieve optimal preservation of cells and nuclear shapes which are important in making the diagnosis.

The purpose of Labeling of Cytological Specimen is to establish the anatomical source of cytological specimen. Preparation of Smear for Non-Gynae Specimen is to concentrate cells and smears preparation for staining procedures. May Grunwald Giemsa Stain are too demonstrate cytoplasmic detail and extracellular substances and also to demonstrate cytoplasmic and nuclear size. The role of histopathology laboratory is to check and process tissues (sections of tissues) for specimens that are take from surgery, biopsy or even autopsy. Section quality, interpretation, and diagnosis depend on the right method when taking and sending the specimens to the laboratory. Clinical information must be written clearly on the request from fast interpretation

Histopathological examination is made to examine the tissue which is taken from operation, tissues taken without operation, for example endoscopy (non-surgically removed tissues) and also autopsy tissue. During my time in this histopathology lab, almost all the works have been take over by the Hospital Besar Ipoh. The specimens that been received are packed and delivered to Hospital Besar Ipoh. Therefore, most of my work was to label the specimen and record all the detail in the log book of histopathology. However, the lab technician told me that the work that were done here were:

1) Labeling of Histopathological Specimen.

2) Foucet-Van Gieson (Bile Pigment Stain).

3) Routine Morphological Staining Using Haematoxylin and Eosin.

4) PAS and Neutral Mucopolysaccharide.

5) Gordon and Sweet’s (Reticulin Fibers).

6) Alcian Blue Stain.

7) Grocott Gomori Methenamine Sliver (GGMS) for Fungi.

8) PAS with Diatase.

9) Special Stain: Congo Red for Amyloid.

10) Diff Quick (Modified Giemsa).

11) Ziehl Neelsen Stain for Acid Bacilli.

12) Masson Trichrome for Muscle and Collagen.

13) PAS with Alcian Blue.

14) Special Stain: Perl’s Prussian Blue for Iron Pigment.

15) Special Stain: Verhoeff-Van Gieson for Elastic Fibres.

16) Special Stain: Southotungistic Mucicarmine for Acid Mucopolysaccharide.

17) Special Stain: Phospotungistic Acid Haematoxylin for Striated Muscle.

18) Prepare Slide From Tissue Paraffin Block.

19) Grossing of Histopathological Specimen.