The Cellient System for Paraffin Histology Can Be Combined with HPV Testing and Morphotyping the Vaginal Microbiome Thanks to BoonFixing

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.


Introduction
1.1. The Cellient System. 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 [1]. Although such automated techniques have an up front cost, they could prove to be cost-effective by saving preparation time for the cytologist, and thereby allowing more samples to be assessed in the required timeframe of clinical practice. In the paraffin sections, we can analyze p16 staining of dyskeratocytes [2] in cases classified as ASCUS (atypical squamous cells of unknown significance) and as HSIL (high-grade squamous intraepithelial lesion) [3].

First Pilot Study of Combining Cellient
Histology with HPV Testing 2.1.1. Cervical Scrapes. 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 [4].   [5]. Detection after PCR is based on the principle of reverse hybridization. Amplification products are subsequently hybridized using specific oligonucleotide probes in a single typing strip [6]. For the current study, HPV-positive scrapes were included.

The Cellient Paraffin Sections.
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.

Morphologic Criteria in ThinPrep and Cellient Slides.
Metaplastic cells are defined as cells with a vesicular nucleus and a nuclear-cytoplasm ratio of around 0.5 [7]. In the p16 stain, the vesicular nature of the nucleus is still visible.
Koilocytotic cells display a clear zone well demarcated around the nucleus [7]. Particularly in the ThinPrep cytology slides it is easy to identify koilocytes.
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 Papanicolaoustained 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.

BoonFix Is a Boon Fixative.
The boon fixative BoonFix can be used for cervical scrapes [8] and for biopsies processed by microwave technology [9]. Note that the Cellient method is based on vacuum techniques and not on microwave technology as it is used on a large scale in the LCPL since 1987 [10,11]. This fixative does not contain formalin nor methanol, the latter being an important component of Preserve of Hologic. In the Hologic system using Preserve, it is advised not to do PCR within one month of cervical sampling. In contrast, when the cervical scrape is suspended in BoonFix, PCR is possible on archival samples. In the studies presented herewith, Preserve from Hologic was not tested. To the best of our knowledge, no papers on this subject were published.
In Table 2 the cytologic diagnoses of the Papanicolaoustained ThinPrep slide classified as ASCUS and as HSIL and the histology diagnoses of the Papanicolaou-and HEstained Cellient paraffin slides are presented. In three cases, metaplastic cells were observed in the paraffin Cellient sections, in seven cases koilocytosis, in one squamous pearls, and in seven dyskeratosis. As many as five cases (1, 2, 7, 11, and 14) had p16 positive dyskeratocytes. Only in case 3, 4, and 5 no p16 staining cells were observed in the Cellient paraffin sections.

The Cellient Images. 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 [12].
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 p16 INK4a protein in cervical premalignant and malignant lesions [13][14][15]. Galgano et al. [16] conclude that immunocytochemical staining for p16 is a useful and reliable adjunct for the identification of CIN 2+. We can add that in the Cellient paraffin sections of cervical scrapes, the p16 positive staining dyskeratotic cells can be identified and classified according to their nuclear size and nuclear cytoplasmic ratio. 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.

HPV Testing and Cytology Scoring of ThinPrep Slides.
In BoonFixed cervical scrapes HPV testing can be combined with cytology on ThinPrep slides. The cross-sectional study of Dols et al. [17] was performed for 194 HIV-positive women. The cervical samples were genotyped for HPV types, and slides were evaluated for atypical squamous cell changes according to the Bethesda classification system. The cytology score of the ThinPrep slides versus lrHPV and hrHPV testing is presented in Tables 3 and 4. Prevalence of high grade squamous intraepithelial dysplasia (HSIL) was 9%. Overall, more than half (56%) of women were infected with an hrHPV type, 94% of women with HSIL ( = 16), 90% of women with LSIL ( = 35), and 42% of women within normal limits (WNL) ( = 58) tested positive for hrHPV. Overall, the most prevalent hrHPV subtypes were HPV16 (26%) and HPV52 (30%).

Conclusion
We conclude that the Cellient system for paraffin histology can be successfully combined with HPV testing and morphotyping the vaginal microbiome thanks to BoonFixing.