The Cellient Automated Cell Block System (Hologic) can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. This study showed that the Cellient system for paraffin sections can be combined with HPV testing thanks to the formalin-free BoonFix. In two additional studies it was shown that such samples can also be used for morphotyping the vaginal microbiome and preparing cytologic ThinPrep slides.
The Cellient Automated Cell Block System (Hologic), producing paraffin blocks could provide an ideal method for cytohistologists as it compares favorably with traditional cell block sectioning [
In a pilot study of 14 HIV-positive women with ASCUS or HSIL, the Cellient system for cytohistology was exploited to analyze p16 positive dyskeratocytes in HPV-positive cervical scrapes [
From each sample six ThinPrep slides (Hologic) were prepared in the T3000. The remaining material in the vial was stored. The cytology ThinPrep slides were scored according to the Bethesda system [
The BoonFixed samples were analyzed for 25 HPV genotypes using a highly sensitive PCR-reverse hybridization Line Probe Assay, the so-called INNO-LiPA HPV genotyping extra system (Innogenetics, Belgium). The SPF10 primer set amplifies a 65-bp region in the L1 open reading frame [
All the remaining material in the vial was used for the Cellient method. This was collected into a cassette and loaded into the instrument. Eosin was applied and vacuum-drawn through the sample. Alcohol was similarly applied and drawn through the sample for dehydration. To clear the alcohol, the procedure was repeated with xylene. The sample was then embedded in paraffin and finally embedded in an additional layer of paraffin during processing in the finishing station.
From the Cellient paraffin block eight serial sections were cut, two for the Papanicolaou staining, one for the hematoxylin eosin (HE) staining, and five for the p16 immunostainings.
Metaplastic cells are defined as cells with a vesicular nucleus and a nuclear-cytoplasm ratio of around 0.5 [
Koilocytotic cells display a clear zone well demarcated around the nucleus [
Squamous pearls are whirls of squamous cells. In the ThinPrep slides these pearls display small pyknotic nuclei. The cytoplasmic staining is either turquoise or red. In the Papanicolaou-stained paraffin Cellient sections, the cytoplasm is either bright red or dark green; nuclei are not always in the section.
In the ThinPrep slides, dyskeratotic cells have a pyknotic nucleus, a nuclear cytoplasmic ration between 2 and 6, and orange or green staining cytoplasm. In the Papanicolaou-stained Cellient paraffin sections, the cytoplasm is green or orange-red. In these sections, the pyknotic nuclei of the dyskeratotic cells can be relatively large. In the HE stain, dyskeratotic cells have bright red cytoplasm. In the p16 Cellient paraffin sections, dyskeratotic cells can display brown cytoplasm and brown-blue nuclei or intensely brown pyknotic nuclei in which the blue (hematoxylin) staining is hidden by the brown color.
The boon fixative BoonFix can be used for cervical scrapes [
In total, 11 HPV genotypes were found, two low risk (lr) (HPV44 and HPV70) and nine high risk (HPV16, HPV26, HPV51, HPV52, HPV53, HPV56, HPV58, HPV66, HPV69). In Table
Low-risk and high-risk HPV genotypes and biomarker p16 staining.
Case | Low-risk HPV | High-risk HPV | p16 positive cells |
---|---|---|---|
1 | 52, 69 | Dyskeratotic cells | |
2 | 70 | 52, 69 | Dyskeratotic cells |
3 | 51 | No p16 staining | |
4 | 70 | 66 | No p16 staining |
5 | 51, 66 | No p16 staining | |
6 | 16, 56 | CIN 1 cells | |
7 | 16, 56 | CIN 1 cells and dyskeratotic cells | |
8 | 16, 51, 53 | Metaplastic cells | |
9 | 16, 53, 66 | Koilocytotic cells | |
10 | 16, 53 | Metaplastic cells | |
11 | 16 | CIN 3 cells and dyskeratotic cells | |
12 | 44 | 26, 52 | CIN 3 cells |
13 | 70 | 52 | CIN 3 cells |
14 | 58 | CIN 3 cells and dyskeratotic cells |
In Table
Cytology of the ThinPrep slide and cytohistology of the Cellient paraffin sections.
Case | Cytology | Cytohistology | Cell types |
---|---|---|---|
1 | ASCUS | Atypia | Dyskeratosis and koilocytosis |
2 | ASCUS | Atypia | Dyskeratosis and koilocytosis |
3 | ASCUS | Atypia | Dyskeratosis and koilocytosis |
4 | ASCUS | Negative | Koilocytosis |
5 | ASCUS | Negative | Squamous pearls |
6 | ASCUS | CIN 1 | CIN 1 fragments and dyskeratosis |
7 | ASCUS | CIN 1 | CIN 1 fragments and metaplasia |
8 | ASCUS | Atypia | Dyskeratosis and metaplasia |
9 | ASCUS | Warty atypia | Koilocytosis |
10 | ASCUS | Negative | Metaplasia |
11 | HSIL | CIN 3 | CIN 3 fragments and dyskeratosis |
12 | HSIL | CIN 3 | CIN 3 fragments and koilocytosis |
13 | HSIL | CIN 3 | CIN 3 fragments and koilocytosis |
14 | HSIL | CIN 3 | CIN 3 fragments and dyskeratosis |
The Cellient Automated Cell Block System was able to provide high quality histology and outstanding images obtained with the biomarkers p16, all this on cervical scrapes on which 22 HPV genotypes were established by PCR.
Nuclear and cytoplasmic p16 positivity was observed in metaplastic cells, koilocytes, and dyskeratotic cells. Of the CIN 1 epithelial fragments, 10–30% of the cells were p16 positive, whilst in CIN 3 epithelial fragments over 50% stained positive. This is in accordance with our experience of p16 in Shandon cytoblock paraffin sections. Note that p16 positivity can also be observed in the cytohistology of adenocarcinoma of the endocervix [
It is well-known that HPV infection can induce a disturbance of the keratinization process leading to dyskeratosis. The Papanicolaou method (in which dye competition between light green and eosin is exploited) is well-suited to detect these dyskeratotic cells by their strikingly orange color, whilst others stain intensely green. It should be noted that the pyknotic nuclei of dyskeratotic cells can be rather large suggesting polyploidy. Such dyskeratotic cells can have relatively large nuclei and accordingly be designated as atypical. The biomarker p16 is used to detect overexpression of p1
The BoonFixed samples can in addition be used for morphotyping the vaginal microbiome. In a study of South African women, the vaginal microbiome was assessed by the Leiden BV scoring system based on morphotyping the stained bacteria. The cases were identified as Leiden bacterial vaginosis (BV) negative (BV−;
The 51 HIV positive women were frequently infected with HPV16 and HPV18. In addition, HPV35, HPV52, HPV33, and HPV66 were often detected in these samples.
In BoonFixed cervical scrapes HPV testing can be combined with cytology on ThinPrep slides. The cross-sectional study of Dols et al. [
Cytology score of the ThinPrep slides versus HPV testing.
Negative cytology (WNL) | ASCUS-LSIL | HSIL | Total | |||||
---|---|---|---|---|---|---|---|---|
|
% |
|
% |
|
% |
|
% | |
hrHPV positive | 58 | 42.0 | 35 | 89.7 | 16 | 94.1 | 109 | 56.2 |
lrHPV positive | 68 | 49.3 | 27 | 69.2 | 11 | 64.6 | 106 | 54.6 |
HPV negative | 43 | 31.2 | 0 | 0 | 43 | 22.2 | ||
| ||||||||
Total | 138 | 71.1 | 39 | 20.1 | 17 | 8.8 | 194 | 100 |
Cytology score versus hrHPV.
Negative cytology (WNL) | ASCUS-LSIL | HSIL | ||||
---|---|---|---|---|---|---|
|
% |
|
% |
|
% | |
hrHPV16 | 8 | 5.8 | 15 | 38.5 | 5 | 29.4 |
hrHPV18 | 6 | 4.3 | 1 | 2.6 | 2 | 11.8 |
hrHPV31 | 5 | 3.6 | 2 | 2.9 | 0 | |
hrHPV33 | 11 | 8.0 | 3 | 7.7 | 1 | 5.9 |
hrHPV35 | 6 | 4.3 | 3 | 7.7 | 4 | 23.5 |
hrHPV39 | 3 | 2.2 | 1 | 2.6 | 2 | 11.8 |
hrHPV45 | 1 | 0.7 | 0 | 0 | ||
hrHPV51 | 10 | 7.2 | 3 | 7.7 | 2 | 11.8 |
hrHPV52 | 17 | 12.3 | 9 | 23.1 | 7 | 41.2 |
hrHPV56 | 3 | 2.2 | 3 | 7.7 | 0 | |
hrHPV58 | 3 | 2.2 | 8 | 20.5 | 0 | |
hrHPV59 | 0 | 1 | 2.6 | 0 | ||
hrHPV66 | 7 | 5.1 | 13 | 33.3 | 2 | 11.8 |
hrHPV68 | 2 | 1.4 | 2 | 2.9 | 2 | 11.8 |
| ||||||
Total | 138 | 39 | 17 |
We conclude that the Cellient system for paraffin histology can be successfully combined with HPV testing and morphotyping the vaginal microbiome thanks to BoonFixing.