**2. Materials for cytology determination and evaluation and notes on specimen preparation**

Cytodiagnosis is a test that involves the observation of cell images taken from a patient to determine the benign/malignant nature of the cells obtained, among other things.

Cytodiagnosis can be performed by collecting cells in body fluids, such as sputum, urine, pleural fluid, or ascites, or through cell collection with a needle or rubbing with a brush. Depending on the method used to collect the cells, it is classified as exfoliative cytology, puncture aspiration cytology, abrasion cytology, or imprint cytology.

#### **2.1 Exfoliative cytology**

Cells spontaneously detach from tissue, often into liquid, such as sputum, urine, milk, pleural fluid, and ascites. The obtained cytogram is often accompanied by


(*Also specify if "negative for squamous intraepithelial lesion"*) *Epithelial Cell Abnormalities* Squamous Cell • Atypical squamous cells ○ Of undetermined significance (ASC-US) ○ Cannot exclude HSIL (ASC-H) • Low-grade squamous intraepithelial lesion (LSIL) (*Encompassing: HPV/mild dysplasia/CIN-1*) • High-grade squamous intraepithelial lesion (HSIL) (*Encompassing: moderate and severe dysplasia, CIS; CIN-2 and CIN-3*) ○ *<sup>W</sup>*ith features suspicious for invasion *(if invasion is suspected)* • Squamous cell carcinoma Glandular cell • Atypical ○ Endocervical cells (NOS *or specify in comments*) ○ Endometrial cells (NOS *or specify in comments*) ○ Glandular cells (NOS *or specify in comments*) • Atypical ○ Endocervical cells, favor neoplastic ○ Glandular cells, favor neoplastic • Endocervical adenocarcinoma *in situ* • Adenocarcinoma ○ Endocervical ○ Endometrial ○ Extrauterine ○ Not otherwise specified (NOS) *Other Malignant Neoplasms (specify)* **Adjunctive testing** *Provide a brief description of the test method(s) and report the result so that it is easily understood by the clinician* **Computer-assisted interpretation of cervical cytology** *If case examined by an automated device, specify the device and result*

#### **Educational notes and comments appended to cytology reports (optional)**

*Suggestions should be concise and consistent with clinical follow-up guidelines published by professional organizations (references to relevant publications may be included)*

Abbreviation: CIN, cervical intraepithelial neoplasia; CIS, carcinoma *in situ*; HPV, human papillomavirus; NOS, not otherwise specified; Pap, Papanicolaou.

*Source: Reprinted from [5].*

#### **Table 2.**

*The 2014 Bethesda system.*

degeneration. Care must be taken to consider this part of the image when making a decision. However, there is little burden on the patient, such as pain.

#### **2.2 Abrasion cytology**

Cells are collected by rubbing the surface of the tissue in the cervix and bronchi. Cell images should be dried without ethanol fixation as soon as possible after the smear.

#### **2.3 Imprint cytology**

Cells are collected by rubbing tissue taken from biopsies or surgery onto a glass slide. As with abrasion cytology, fixation is required immediately after the smear. Care should also be taken not to spoil lymph nodes or undifferentiated tumors if they are suspected.

#### **2.4 Puncture aspiration cytology**

The needle test is used to collect lesions from areas under the skin, such as the mammary glands, thyroid gland, and lymph nodes. Fresh cells are obtained, and care must be taken to ensure that the smear and preparation of the specimen are performed as close as possible to recapitulate the situation *in vivo*. Introducing artifacts will prevent correct assessment and diagnosis.

Approximately 10–15 years ago, there was a debate in the Japanese domestic cytological society about the smear method for mammary gland puncture aspiration cytology. The author believes that the smear should be blown at an angle and fixed as it is (see **Figure 2**). At that time, there was an opinion within the academic society that lightly fitting two glass slides together was the correct approach. However, when a trace amount of liquid component is placed between the glass slides, it is technically impossible to lightly align them because of the surface tension, ruining the precious sample.

The author had a bitter experience in understanding this, as shown in **Figures 3** and **4**. These are cases in which the smears are made by matching the glass and destroying the cells; thus, a definitive diagnosis cannot be made. At an academic conference in Japan, several researchers stated that a thick smear is not good because it does not allow the staining solution to enter the smear. For example, if a specimen is cancer, the tumor is a manifestation of a fast cell proliferation rate and high cell density. Adding an artifact to force the stain to cover the tumor in its entirety would destroy the structure of the finalized cell population and result in misdiagnosis. Thus, staining cytology specimens should not be mistaken for paintings.

At symposiums and other meetings of academic societies in Japan, the results were not determined, and the tests were subsequently performed using the chosen approach of each institution. As a result, since around 2015, the rate of positive diagnosis has declined, especially for puncture aspiration cytology of the mammary gland. Guidelines for surgical and treatment methods also indicated tissue biopsy using a thick biopsy needle as the mainstream approach, while preoperative testing for hormone receptors, HER2 sensitivity, and Ki-67 index became the standard [16–18].

#### **Figure 2.**

*Standard correct method of fine needle aspiration cytology. (excerpt from an educational video provided by AstraZeneca). (a) this is performed to confirm that the needle is definitely inside the tumor and is done with ultrasound guidance. When the needle tip enters the target site (inside the tumor) aspirate for about 1 minute. Keep the pistol primed (do not scatter cancer cells in the needle). Rotate the needle and gently move it back and forth to push the tumor cells into the needle. Insert, and always return suction when removing the needle. (b) Angle the tip of the needle about 40° and lightly push. Blow onto a glass slide. (c) Point the cut surface of the needle point slightly diagonally instead of straight down. When sprayed, the cells are dispersed almost evenly. (d) Spray the smeared slide glass as it is quickly fixed in alcohol. (e) When remove the alcohol, if it is smeared to the extent shown here, then it is generally okay. (f) Sometimes cells remain at the base of the needle. (g) If this occurs, cap the needle upside down on a slide glass and hit strongly.*

#### **Figure 3.**

*Fibroadenoma. (a) Laminated glass smear method. (b) Spray smear method. (a) Reported as class III because the cells collapsed due to rubbing together and could not be accurately determined. (b) The bilayered structure of myoepithelial and glandular cells is clear because the cells are not broken.*

#### **Figure 4.**

*Ductal carcinoma. (a) Laminated glass smear method. (b) Spray smear method. (c) Smashing smears. (d) Macroscopic image of slide specimen: (d-1) use of the spray smear method; (d-2) use of smashing smears. (a) the individual cells are stretched by the effects of rubbing together. (b, c) the cancer cells have not collapsed. The stacked structure is also preserved.*

We hope that the time will come when puncture aspiration cytology will be actively utilized for the follow-up of benign lesions, such as fibroadenomas.

Pancreatic cancer is among the most lethal cancers worldwide because of the limited availability of techniques for the early detection of signs and symptoms. Ultrasound-guided pancreatic puncture aspiration is now also actively performed in some major hospitals [19, 20]. In some hospital facilities, cytological examinations and tissue biopsies are performed together. As the author noted [21], both tests have their own advantages and disadvantages. Unlike tissue biopsy, cytology allows specimens to be obtained from background findings. In the preparation of the tissue specimen, the

**Figure 5.**

*Adenocarcinoma of the pancreas. (a) Cytology, class V. necrotic material was obtained in the background indicated by the red arrows ( ). (b) Histology, no malignancy. Only red blood cells and histiocytes were observed in the background. Source: Excerpted from ref. [21], partially modified.*

background findings are the parts that are removed by roughing the paraffin block [21]. The presence of cystic or necrotic material in the specimen is a very important diagnostic finding (see **Figure 5**).

#### **2.5 Cell block specimens**

Traditionally, cell block specimens have been used when fibrin precipitation has caused cells to adhere to each other and clump together, making smears on glass slides impossible. Sometimes, useful findings can be obtained when the clumped material is used for specimen preparation in the same way as tissue fragments [22–24]. Therefore, immunostaining has recently become increasingly necessary. The reasons for this are the emergence of cancers of unknown primary origin and the widespread use of antibodies for immunostaining.

More recently, when the only symptom is fluid accumulation in the body cavity, such as pleural or ascites fluid, this has been diagnosed as cancer on cytological examination. However, imaging findings of the surrounding organs are not abnormal. Therefore, cell block specimens are prepared from the remaining smear sediment; then, immunostaining is performed to deduce the primary tumor (see **Figure 6**). Based on this, an increasing number of centers are processing specimens with the aim of preparing cell blocks from the outset. Body cavity fluid, which is collected in large quantities, is useful because it tends to have surplus sediment.

Specifically, this process uses disposable tubes made of soft material, as shown in **Figure 7**. Samples are rotated faster than normal centrifugation (3000 rpm, 5 min); then, the supernatant is removed and fixing solution (15% neutral buffered formalin) is added. The sample is left to fix for approximately one day. The specimen is then cut with scissors as shown in the diagram, placed in an embedding cassette, and processed in an automatic embedding machine. Specimens are then prepared in the same way as normal tissue specimens. Ideally, during paraffin embedding, the sediment is picked up with tweezers from inside this tube and embedded directly into the embedding dish, but thin sections can also be made by embedding the entire Japanese tube. An alternative method is to use collodion. The use of sodium alginate has also been considered but should be avoided because it has been reported that sodium alginate may cause some antibodies to become unstained in rare cases.

#### **Figure 6.**

*Microscopic findings for lung adenocarcinoma case. (a) Cytological findings (Papanicolaou). (b) Cell block findings (hematoxylin and eosin). (c) Cell block findings (immunohistochemistry for CEA). (d) Cell block findings (immunohistochemistry for ADC cocktail (TTF-1 and Napsin A)). Excerpt from ref. [24], partially modified.*

#### **Figure 7.**

*The cell block producing method. (a) Sample is fixed with 15% formalin solution in tube. (b) Cut the tip of the disposable tube using scissors. (c) The tip of a tube is placed in a cassette. Excerpt from ref. [24], partially modified.*
