Fig. 1.1
Unsatisfactory due to scant squamous cellularity. Endocervical cells are seen in a honeycomb arrangement (LBP, ThinPrep at 10× magnification)
Fig. 1.2
Unsatisfactory – scant cellularity (LBP, SurePath). Although this image cannot be directly compared to a microscopic field, this SurePath slide had fewer than 8 cells per 40× field. A SurePath specimen with this level of cellularity throughout the preparation would have fewer than 5,000 cells
Fig. 1.3
Satisfactory, but borderline squamous cellularity (LBP, SurePath). At 40×, there were approximately 11 cells per field when ten microscopic fields along a diameter were evaluated for squamous cellularity; this would give an estimated total cell count between 5,000 and 10,000
Fig. 1.4
Satisfactory, but borderline squamous cellularity (LBP, ThinPrep): 10× fields of a ThinPrep specimen should have at least this level of cellularity to be considered satisfactory. At 40× magnification of this ThinPrep specimen, there were approximately four cells per field, which would correspond to slightly over 5,000 cells. Note that this level of cell density would be unsatisfactory in a SurePath LBP (see Fig. 1.2), corresponding to less than 5,000 cells because of the smaller preparation diameter
Fig. 1.5
Squamous cellularity is satisfactory in this LBP from a 70-year-old woman with an atrophic cell pattern (LBP, SurePath). LBPs may show less nuclear enlargement than conventional preparations due to fixation in the suspended state. The transformation zone component(s) may be difficult to assess in atrophy
Fig. 1.6
Unsatisfactory specimen reprocessing. Original preparation (a, left) from a 54-year-old woman was unsatisfactory due to scant squamous cellularity and excessive blood (LBP, ThinPrep). Reprocessing with glacial acetic acid resulted in a satisfactory sample (b, right)
Fig. 1.7
Satisfactory vaginal cytology from a 56-year-old, status post total hysterectomy (with no cervix remaining) for endometrial adenocarcinoma (LBP, ThinPrep). Cellularity was estimated to be <5,000 but it was considered satisfactory since the source was vaginal
Fig. 1.8
(a, b) Low-cellularity but satisfactory specimen in woman with history of radiation (LBP, ThinPrep; contributed by Fang Fan, MD)
Fig. 1.9
Low-cellularity but satisfactory specimen from a woman with history of pelvic radiation (LBP, SurePath)
Fig. 1.10
Atrophy: borderline cellularity in LBP preparations from two different postmenopausal women (LBP, ThinPrep). Parabasal cells can be seen isolated (a, left) or in clusters (b, right). It may be difficult to distinguish parabasal-type cells from squamous metaplastic cells in specimens showing atrophy due to a variety of hormonal changes including menopause, postpartum changes, and progestational agents
Fig. 1.11
Unsatisfactory specimen from a 39-year-old woman (LBP, ThinPrep). Abundant endocervical cells and mucus are seen; however, the squamous component is inadequate
An adequate liquid-based preparation (LBP) from a woman with a cervix should have an estimated minimum of at least 5,000 well-visualized/well-preserved squamous or squamous metaplastic cells. This range applies only to squamous cells. Endocervical cells and completely obscured cells should be excluded from the estimate. Women who have had chemo- or radiation therapy, who are postmenopausal with atrophic changes, or who are post-hysterectomy may have samples with fewer than 5,000 cells, and such specimens may still be considered adequate at the discretion of the laboratory. The patient history must be taken into consideration in such cases. Samples with less than 2,000 cells, however, should be considered unsatisfactory in most circumstances.
Some have advocated that LBPs with 5,000–20,000 cells are of borderline or low squamous cellularity. In specimens with suspected low cellularity, an estimation of total cellularity can be obtained by performing representative field cell counts. A minimum of ten microscopic fields, usually at 40×, should be assessed along a diameter that includes the center of the preparation and the average number of cells per field estimated. When there are holes or empty areas on the preparation, the percentage of the hypocellular areas should be estimated, and the fields counted should reflect this proportion. Although both LBPs have similar numbers of cells overall, SurePath™ (BD Diagnostics, Durham, NC) slides have a higher cell density than do ThinPrep™ (Hologic, Inc., Bedford, MA) slides because of the smaller preparation diameter with SurePath™ (see Table 1.1). Siebers et al. evaluated several different protocols for estimation of low cellularity ThinPrep™ specimens and found that counting five fields along a horizontal diameter and five fields along a vertical diameter (SKML protocol) at 10× had the best correlation with a reference method that utilized image analysis software for counting cells [13]. However, when all of their measurements at different objective powers were merged, the differences between the SKML and the Bethesda protocols (as noted above) were not statistically significant.
Table 1.1
Guidelines for estimating cellularity of liquid-based preparations
FN20 eyepiece/10× objective | FN20 eyepiece/40× objective | FN22 eyepiece/10× objective | FN22 eyepiece/40× objective | ||||||
|---|---|---|---|---|---|---|---|---|---|
Prep. diameter (mm) | Area (mm2) | Number of fields at FN20, 10× | Number of cells/field for 5K total | Number of fields at FN20, 40× | Number of cells/field for 5K total | Number of fields at FN22, 10× | Number of cells/field for 5K total | Number of fields at FN22, 40× | Number of cells/field for 5K total |
13 | 132.7 | 42.3 | 118.3 | 676 | 7.4 | 34.9 | 143.2 | 559 | 9.0 |
20 | 314.2 | 100 | 50.0 | 1,600 | 3.1 | 82.6 | 60.5 | 1,322 | 3.8 |
Table 1.1 provides the average number of cells per field required to achieve a minimum of 5,000 cells on an LBP given the preparation diameter and field number of the eyepiece (ocular). For individuals using eyepieces and preparations not shown, the formula is: number of cells required per field = 5,000/(area of preparation/area of field). The diameters of SurePath and ThinPrep preparations are 13 and 20 millimeters (mm), respectively. The diameter of a microscopic field in millimeters is the field number of the eyepiece divided by the magnification of the objective. The area of the field is then determined by the formula used to calculate the area of a circle [pi × radius squared, πr 2]. The magnification power of the ocular does not affect this calculation [14, 15]. For additional explanation of the pertinent optical principles, see http://www.microscopyu.com/articles/formulas/formulasfieldofview.html.
Figures 1.1, 1.2, 1.3, 1.4, and 1.5 show cell coverage or density in unsatisfactory, borderline satisfactory, and satisfactory liquid-based preparations. These are not reference images, as they do not represent an entire microscopic field; thus, the cell density shown in the images cannot be compared directly to Table 1.1 for estimation of squamous cellularity.
In some instances, the cellularity on the prepared slide may not be representative of the collected sample. Slides with fewer than 5,000 cells should be examined to determine if the reason for the scant cellularity is a technical problem related to slide preparation such as an excessively bloody specimen. If a technical problem is identified and corrected, a repeat preparation may yield adequate cellularity (Fig. 1.6a, b). However, the adequacy of each slide should be determined separately and not cumulatively. Attempts to determine cellularity cumulatively by summing the cellularity of multiple inadequate slides may be confounded by uncertainty regarding the true cellularity of the specimen (not the slide), which might be substantially less than in a specimen with normal slide cellularity. This matter is in need of more research, and hence this guideline may be subject to change in the future. Given the relatively low minimum criterion for adequate cellularity, caution is warranted in borderline cases. The report should clarify whether blood, mucus, lubricant, inflammation, or technical artifact contributed to an unsatisfactory sample or whether the problem was simply low squamous cellularity.
1.2.3 Conventional Preparations (Figs. 1.12–1.16):
Fig. 1.12
Squamous cellularity: this image depicts the appearance of a 4× field of a conventional preparation with approximately 75 cells. The specimen is unsatisfactory if all fields have this level, or less, of cellularity. It is to be used as a guide in assessing the squamous cellularity of a conventional smear (Used with permission, © George Birdsong, 2003)
Fig. 1.13
Squamous cellularity: this image depicts the appearance of a 4× field of a conventional preparation with approximately 150 cells. If all fields have this level of cellularity, the specimen will meet the minimum cellularity criterion, but by only a small margin (Used with permission, © George Birdsong, 2003)
Fig. 1.14
Squamous cellularity: this image depicts the appearance of a 4× field of a conventional preparation with approximately 500 cells. A minimum of 16 fields with similar (or greater) cellularity are needed to call the specimen adequate (Used with permission, © George Birdsong, 2003)
Fig. 1.15
Squamous cellularity: this image depicts the appearance of a 4× field of a conventional preparation with approximately 1,000 cells. A minimum of eight fields with similar (or greater) cellularity are needed to call the specimen adequate (Used with permission, © George Birdsong, 2003)
Fig. 1.16
Squamous cellularity: this image depicts the appearance of a 4× field of a conventional preparation with approximately 1,400 cells. A minimum of six fields with similar (or greater) cellularity are needed to call the specimen adequate (Used with permission, © George Birdsong, 2003)
An adequate conventional cervical specimen should contain an estimated minimum of approximately 8,000–12,000 well-preserved and well-visualized squamous epithelial cells. As was noted above for liquid-based preparations, this minimum cell range should be estimated, and laboratories should not count individual cells in conventionally prepared slides. This cellularity range should not be considered a rigid threshold and comments related to lower cellularity in post-therapy and vaginal specimens also apply to conventional preparations. “Reference images” of known cellularity are illustrated in Figs. 1.12, 1.13, 1.14, 1.15, and 1.16. These reference images have been computer edited to simulate the appearance of 4× fields on conventional preparations. Cytologists should compare these images to specimens in question to determine if there are a sufficient number of fields with approximately equal or greater cellularity than the reference images. For instance, if an image corresponding to a 4× field with 1,000 cells was used as the reference, a specimen would need to have at least eight such 4× fields to be deemed to have adequate cellularity.
1.2.4 Explanatory Notes
Strict objective criteria may not be applicable to every case. Some slides with cell clustering, atrophy, or cytolysis are technically difficult to count, and there may be clinical circumstances in which a lower cell number may be considered adequate. Laboratories should apply professional judgment and employ hierarchical review when evaluating these uncommon borderline adequacy cases. It should also be kept in mind that the minimum cellularity criteria described here were developed for use with cervical cytology specimens.
The recommendation for a minimum cellularity of 5,000 cells for an LBP is based on relatively limited scientific evidence [16, 17]. This threshold is lower than the 8,000–12,000 minimum cellularity for conventional preparations, because LBPs, by virtue of the preparation methodology, present a more random (and presumably more representative) sample of the collected cervical material. Although there are significant differences between ThinPrep and SurePath, there are not sufficient data to justify different minimum cellularities for the LBPs currently on the market.
The relationship of number of cells present on a slide and the detection sensitivity for epithelial lesions has only been rarely investigated. One study reported a higher rate of detection of high-grade lesions when cellularity on LBPs exceeded 20,000 [18]. However, this study did not assess false-negative rates vs. cellularity. Investigators have attempted to perform seeding experiments to determine if there are minimal cellularity requirements for successful identification of abnormal cells in LBPs; however, no conclusions were reached, leading the authors to suggest that a pragmatic approach be maintained with minimum cellularity being set at 5,000–10,000 squamous cells [19]. Kitchener et al, in a recent, very thorough study involving 56 laboratories in the United Kingdom, assessed the relationships between cellularity, abnormal cell counts, and detection of abnormalities in liquid based cervical cytology preparations. They concluded that a minimum acceptable cell count of 15,000 and 5000 for SurePath and ThinPrep, respectively, would probably achieve the best balance between maintaining low levels of inadequate slides and not compromising the chances of detecting abnormalities. Although these suggested cell counts differ for the two preparation types, the proportion of slides which fell under the respective cutoffs were similar for the two preparations, and actually tended to be lower with SurePath [62]. Laboratories may choose to append a quality indicator comment such as “borderline or low squamous cellularity” for specimens that meet minimal criteria for satisfactory cellularity but have only 5,000–20,000 cells.
Cellularity can be quickly and reproducibly estimated in LBPs [16, 20]. Some manufacturers include estimation of LBP cellularity during training. Preliminary studies show that reference image methodology for conventional preparations is quickly learned and has better interobserver reproducibility than the previous Bethesda 10 % slide coverage criterion [21]. Additional studies relating sensitivity to cell number would be useful for all preparation types.
The College of American Pathologists (CAP) survey data shows that the 50th percentile rates for unsatisfactory specimens in US laboratories are 1.0, 1.1, and 0.3 % for conventional, ThinPrep, and SurePath preparations, respectively [22]. Unsatisfactory rates that significantly differ from these thresholds in an individual laboratory should prompt careful evaluation for the possible causes relating to sampling methodology, preparation technique, patient population, or interpretation thresholds.
1.3 Endocervical/Transformation Zone (EC/TZ) Component (Figs. 1.17–1.22)
Fig. 1.17
Endocervical cells (CP). Distinct cytoplasmic borders are seen in the cluster of cells on the left, giving a “honeycomb” appearance. The cell cluster on the right is seen from a side view, giving the “picket fence” appearance
Fig. 1.18
Endocervical cells (LBP, SurePath). Cellular dissociation is more frequent in liquid-based preparations than in conventional smear preparations
Fig. 1.19
Endocervical cells (LBP, SurePath). Routine screening, 27-year-old woman, NILM on follow-up. Normal endocervical cells may appear in large hyperchromatic fragments, often in the center of some LBPs. The thickness of the fragment may give the appearance of architectural disarray; however, note normal appearing cells at the periphery of the fragment. Additionally, focusing up and down through the fragment reveals normal spacing of cells, distinct cytoplasmic borders, and bland nuclear chromatin. Normal endocervical cell groups with this appearance should not be confused with neoplastic clusters that show more crowding (even within a single layer of cells), nuclear enlargement, nuclear membrane irregularity, and abnormal chromatin pattern
Fig. 1.20
Transformation zone component (LBP, SurePath). Normal endocervical cells from the upper region of the endocervical canal can closely mimic squamous metaplastic cells
Fig. 1.21
Normal squamous metaplastic cells (LBP, SurePath). Routine screening. Twenty-eight-year-old woman
Fig. 1.22
Atrophy (CP). Degenerated cells in mucus and parabasal-type cells should not be counted in assessing transformation zone sampling. It may be difficult to distinguish parabasal-type cells from squamous metaplastic cells in specimens showing atrophy due to a variety of hormonal changes including menopause, postpartum changes, and progestational agents. In such cases, the laboratory may elect to make a comment about the difficulty of assessing the transformation zone component
The presence of transformation zone sampling is not necessary for an adequate specimen – only squamous cellularity, as noted above, is necessary. However, laboratories should report the presence or absence of a transformation zone component as it may be a useful quality assurance measure. For both conventional and liquid-based preparations, an adequate transformation zone sample requires at least ten well-preserved endocervical or squamous metaplastic cells, singly or in clusters (Figs. 1.17, 1.18, 1.19, 1.20, 1.21, and 1.22). The presence or absence of a transformation zone component is reported in the Specimen Adequacy section unless the woman has had a total hysterectomy. Degenerated cells in mucus and parabasal-type cells should not be counted in assessing transformation zone sampling. In such cases, the laboratory may elect to make a comment about the difficulty of assessing the transformation zone component. See Fig. 1.22 for discussion regarding difficulties in differentiating metaplastic and parabasal cells.
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