Abstract
The Liquid-Based Cytology (LBC) technique was originally developed for gynecologic cervical smears, but progressively gained widespread consensus after having demonstrated its usefulness both in non-gynecologic and fine-needle aspiration cytology samples. The main advantages of this methodology are the reduction in confounding factors, the even distribution of cells on a thin layer, and the possibility to obtain more slides from the same sample. The above described advantages of LBC strongly encourage the replacement of conventional preparations with LBC in the routine cytologic evaluation of endometrial cellular samples.
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Notes
- 1.
In LBC, the brush of the device is immersed in a vial of preservative-fixative solution where it is vigorously rotated several times to ensure the release of the cells collected. The abundant cytologic material obtained is deposited as cell clusters of various sizes on the slide. The device is removed from the vial, then the sample is ready for processing after about 30 min in fixative solution and is stable for several weeks thereafter.
When endometrial cells are fixed, cell clusters maintain their three-dimensional pattern resembling, essentially, microbiopsies; this allows a direct correlation between histological and cytological architectures. Hence, the adoption of cytoarchitectural criteria reflecting the histological growth pattern appears to be more useful for the cytologic assessment of endometrial lesions. Moreover, it should be noted that endometrial cells are subjected to mechanical trauma during specimen preparation to obtain an LBC sample, such as that caused by mixing the vial with a vortex mixer.
According to a recent investigation, it was concluded that the endometrial cell clusters are not yet completely fixed in the interval between 30 min and 1 h of fixation, so the mechanical trauma during the SP-LBC process may destroy at least part of them and the three-dimensional structure of the cell clusters may be partially or totally lost (Fig. 7.11). Instead, numerous single or several endometrial cells were observed in the background of the specimen. Nishikawa et al. [19] reported that as far it concerns the number of endometrial cell clusters with the major axis of 200 μm or more, a fixation time ≺6 h produced an average number of 9.3 (range: 0–22), while a fixation time ≥ 18 h produced a significantly higher average number of 71.3 (range: 3–313).
On the other hand, as for the number of single or small dispersed clusters of endometrial cells, their average number with a fixation time <6 h was 132.2 (range: 29–508), while a fixation time ≥18 h showed a significantly lower average number of 35.7 (range: 5–593). This result suggests that fixation time plays a major role in the protection of endometrial cell clusters from physical impact during the LBC preparation process. A fixation time of 18 h or more is essential for the endometrial SP-LBC preparation (Fig. 7.12).
- 2.
CyL is a 25% methanol buffer solution; it is used for hemolysis, prevention of protein precipitation, dissolution of mucus, and morphologic maintenance in samples. If a cytologic sample from the uterine cervix is inadequate for massive blood contamination in a TP preparation, a mixture of glacial acetic acid (GAA) additive and CyL recommended by Hologic is used as hemolytic treatment, and the TP preparation is remade. However, it has been reported that this treatment adversely affects the morphologic appearance of cervical squamous cells and glandular cells due to the influence of GAA [20, 21].
- 3.
Cell suspensions were prepared by adding some blood to the cellular samples, treated with a hemolytic agent, i.e., CyL, then they were left for up to 1, 6, and 24 h, respectively. After different time intervals of hemolysis, TP preparations were made and stained with the Papanicolaou method. All of the obtained preparations were examined for cellularity, nuclear area, and brightness level of nuclear chromatin.
As a result, the cellularity “without CyL treatment” was 27.8 (Fig. 7.13a), “immediately after” was 195.4 (Fig. 7.13b), “after 1 h” was 266.2 (Fig. 7.13c), “after 6 h” was 148.6 (Fig. 7.13d), and “after 24 h” was 99.4 (Fig. 7.13e). The cellularity values found in samples obtained after “1 h” were higher than those found in samples obtained “immediately after.” The cellularity in samples obtained at “24 h” was significantly lower than that found in samples obtained “immediately after,” “1 h,” and “6 h.” Moreover “24 h” of CyL treatment in samples without adding blood caused reductions in cellularity, nuclear area, and tendency toward the brightness of nuclear chromatin.
If CyL treatment is delayed until the next day in routine practice, this may lead to a general decrease in cellularity and to an increase in degenerative changes in the cell suspension. Hence, it was estimated that a hemolytic treatment of “1 h” represented the optimal choice.
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Norimatsu, Y., Nishikawa, T., Kobayashi, T.K., Fulciniti, F. (2022). Processing Methodology of Endometrial Cytology Samples. In: Hirai, Y., Fulciniti, F. (eds) The Yokohama System for Reporting Endometrial Cytology. Springer, Singapore. https://doi.org/10.1007/978-981-16-5011-6_7
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