High Risk Pattern Group 1-
Crowded Sheets and Unusual Glandular Cells

Crowded Sheets

High grade abnormality may present as crowded groups or sheets of cells, for example tissue fragments and microbiopsies, with approximately 20 to many thousands of nuclei. These are prominent in the smear and visually easy to detect, but are readily misinterpreted because they may resemble common benign cell types20. Every crowded cell sheet or thick cell group should be considered a high risk pattern.

Figure 13

Whatever benign entity they may resemble, crowded sheets from a high grade squamous abnormality have these features in common:

• Nuclei are crowded and overlap
• The sheets are usually more than three nuclei thick
• There is unpredictable variation in:
  - Nuclear size
  - Nuclear structure
  - Nuclear border
  - Chromatin density
• Some nuclei have abnormal nuclear structures
• Polarity is sometimes disturbed
• Mitosis and apoptosis may be present

Unpredictable variation between nuclei, and the presence of some specific abnormal features, are the keys to diagnosis. Individually, some nuclei may seem quite normal.

Figure 14

Accompanying dysplastic cells may assist diagnosis, 38,39 but they may be of lower grade, and so not helpful in the diagnosis of high grade abnormality. It is important to recognise abnormal crowded sheets on their own, as frequently there are no other abnormal cell types and no single abnormal cells present. 40

The potential diagnostic information available in crowded sheets can be overwhelming. The tendency is to take a global view, rather than attending to the abundance of fine details. However, diagnosis is in the details. To manage the assessment of so many details, it is useful to break the assessment of crowded sheets into a number of smaller steps, attending separately to the search for each particular piece of significant information.

Figure 15

Interpretation of crowded sheets

Examination of thick areas

Interpretation of nuclear features within a thick, overlapping group can be difficult. Diagnostic information may be obtained by examining nuclei at the edges of the group. 36 However, edge nuclei may be distorted, and cytoplasm at the edges of a HSIL sheet may be relatively plentiful or may resemble endocervical cytoplasm. Examining only the cells at the edge of the group could lead to under diagnosis.

Interpretation requires examination of nuclei throughout the whole group. With proper microscope adjustment (Köhler illumination), many sections or planes of focus can be examined by focussing carefully through these thick crowded sheets. Minor adjustment of the substage diaphragm can optimise definition of detail.

Figure 16

Figure 17

Figure 18

Crowding

Nuclear crowding is significant if:

• There is overlapping
• Spacing is not regular
• The distance between coplanar nuclei does not display an obvious ‘so-close-no-closer’ minimum gap between most adjacent nuclei as seen in normal cell sheets

Crowded nuclei scramble over each other, ignoring the personal space of others. Nuclei may be crowded in this disordered way even if they are not closely packed.

Figure 17	Figure 18	Figure 19
Figure 20	Figure 21

Overlapping
Overlapping is significant if near coplanar nuclei overlap by more than one third of a nuclear diameter.

Figure 20

Polarity
Polarity within high grade crowded sheets may be very disordered, jumbled or chaotic38 but not always. Polarity may be normal, with nuclei trending along the group. When nuclei are round, a sheet can seem ordered because there are no long nuclear axes to be misaligned. Polarity may show swirling or whorling patterns. It may also appear well defined with nuclei running at right angles to the length of the sheet, reflecting the ordered, vertical polarity often seen in histopathology of Cervical Intraepithelial Neoplasia Grade 3 (CIN 3). Furthermore cells at the sheet edge may be flattened. 41 This may reflect a histological appearance in which two or three layers of cells at the surface of a CIN 3 mature slightly and flatten.

Figure 21	Figure 22	Figure 23
Figure 24	Figure 56

	Normal, ordered polarity along the group
	Polarity is not assessable if nuclei are round
	Disordered, chaotic polarity
	Swirling polarity
	Polarity across a sheet, with flattening at one edge

Table 4

Sheet thickness

Sheet thickness of 3 nuclei or more is significant. Most sheets from high grade squamous abnormalities are more than 3 nuclei thick.

Figure 25

Figure 26

Figure 27

Nuclei should actually be counted, not guessed. The mucoid or pale immature cytoplasm of many high grade sheets may be quite transparent, so even quite thick sheets may appear deceptively thin, and the test may surprise.

Figure 19

Figure 28

Figure 29

Figure 30

Table 5

Sheet thickness of three or more nuclei is significant.

Nuclear Variation*
Nuclear size

Nuclear enlargement is more reliable as a feature in low grade lesions. 38 It is an unreliable criterion for high grade abnormality. In high grade sheets nuclei may be smaller than normal nuclei. Variation in relative nuclear size is far more important than absolute size. A range of sizes, smallest to largest, of 2x diameter or more is significant while 3x diameter or more is suspicious. Only compare nuclei in a similar state of preservation, as degeneration may be accompanied by shrinkage or swelling.

Figure 31

Figure 32

Chromasia

Hyperchromasia is not necessary for diagnosis. 11 Nuclei in abnormal crowded sheets may be pale, so the sheet as a whole may not appear dark. Unpredictable variation in chromasia is far more important than absolute hyperchromasia. Examples of unpredictable variation include:

• A larger nucleus darker than a smaller one
• Equal sized nuclei of very different densities

Compare cells of a like state of preservation, as degeneration often increases staining density.

Figure 1

Figure 33

Figure 34

Nuclear structure

Variation in nuclear structure is extremely important, and is far more important than the general nuclear structure within the group. Significant variation includes:

• Coarse, generally large chromocentres vs fine, generally small chromocentres
• Open structure (widely spaced chromocentres) vs close packed structure (little space between chromocentres)
• Clear or pale parachromatin vs dense or dark parachromatin
• Few prominent chromocentres vs many or no chromocentres
• Even chromatin distribution vs irregular chromatin distribution

The diagnosis of abnormality is more certain if the range of nuclear structures includes several specific abnormal structures. Some combinations are particularly significant, for example pale, fine, open nuclei together with coarse, densely hyperchromatic nuclei.

Figure 35

Figure 36

Degeneration and air drying smudge nuclear structure, so key information may be lost. Nevertheless diagnosis, or at least strong suspicion, may still be possible in degenerate cells, relying on other features such as unpredictable variation in nuclear size and chromasia.

Figure 37

Nucleoli

In high grade abnormal sheets, nucleoli may be:

• Absent or present
• Large or small
• Single or multiple
• Rounded or angular

Their presence adds nothing either way to the diagnosis, but if a reactive process is being considered, the absence of nucleoli in some or all cells is an abnormal criterion.

Figure 22

Figure 29

Figure 38

Nuclear border

The chromatinic nuclear border in abnormal sheets may vary from nucleus to nucleus. Some have a distinct but thin, pencil line border. In others the border may be prominent. In some the internal nuclear structure simply stops at the nucleus edge, without a defining line. In benign cells the nuclear borders of all nuclei within the cell population tend to be similar.

Figure 33

Figure 36

Table 6

Specific abnormal nuclear structures

Abnormal chromatin structure in at least some nuclei is a key abnormal criterion 11, 38 but a careful search on high magnification may be needed to find it. (Appendix 4)

Figure 35

Figure 36

Nuclear shape

Nuclei in high grade abnormal sheets are usually round to oval, not irregular or abnormally shaped. 41,42 Nevertheless, if abnormal nuclear shapes are present they are a bonus and strong evidence for abnormality. Degenerative and distorted angular shapes and nuclear nipple protrusions are often present but are not relevant to diagnosis. (Appendix 1)

Figure 39

Figure 40

Figure 41

Figure 42

Mitoses

Mitoses are sometimes present and are very significant, 39 especially if embedded in the cell sheet with epithelial nuclei in focal planes above and below. This indicates that the mitosis is above the basal layer in the epithelium, a feature of HSIL.

Figure 15

Figure 24

Figure 26

Figure 43

Apoptosis

Apoptosis occurs in cervical neoplasia, increasing in incidence with increasing grade of lesion. 43 The presence of apoptosis in an epithelial fragment raises suspicion of high grade abnormality. Suspicion is even greater if mitoses are also present. In normal epithelium other forms of programmed cell death such as maturation and exfoliation predominate, apoptosis is rare.

Figure 44

Figure 45

In apoptosis a damaged or dysfunctional cell breaks down into small inactive fragments, which are then engulfed by surrounding cells. 44 Apoptosis is recognised by the presence of groups of small, black, brown or pale, rounded or irregular fragments of nuclear and cellular debris. Some apoptotic bodies show an internal structure of dark and light areas, the darker material presenting as an eccentric, rounded clump or as a crescent-like band.

Figure 44

Figure 44

Figure 45

Apoptosis needs to be distinguished from normal debris and nuclear fragments arising from degeneration of polymorphonuclear leukocytes in inflammation. Degenerate leukocyte lobes can be recognised by comparison with nearby similar polymorphs in various stages of degeneration.

Apoptosis should also be distinguished from keratohyaline granules. Keratohyaline is usually seen in single cells. Keratohyaline granules are homogeneous, having no internal structure, and their staining is locally predictable. The presence of keratohyaline granules in immature metaplastic cells is an abnormal feature, but is unrelated to apoptosis.

Bare and part-bare nuclei

Part-bare nuclei at the edge of a cell sheet, and bare nuclei in the background suggest abnormality.

Figure 9	Figure 20	Figure 31
Figure 44	Figure 46	Figure 49

Table 7

*Refer to all figures in crowded sheets for examples