Collecting, preparing and fixing the cellular samples

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Sources of error

A chapter has been dedicated to guidelines for taking a cellular sample, fixing and processing conventional and liquid-based preparations.  Here we discuss sources of error during this stage in the process.

Clinical condition of the patient at the time of sampling

• Samples taken during, or in the few days before or after, menstruation may contain degenerate endometrial cells that are a potential pitfall for misreporting cytological slides. 
• Post-natal cytological appearances may be misleading. 
• Active infections, such as Trichomomas, are potential sources of cytological error.
• Symptoms suggesting cervical cancer should be investigated and managed clinically whatever the cytology result: clinical cancer may lead to false negative cytology results.

Visualising the cervix and collecting the sample

• Incomplete sampling of the cervix is an important cause of false negative cytology and is regarded as the reason for true false negative slides in the presence of a known abnormality on the cervix (Renshaw 2000).
• The cervix may be difficult to visualise, in which the sample may be taken ‘blind’ and not be representative of the transformation zone.
• If the mucus plug is not wiped away, to make sure the spatula/brush is in direct contact with the epithelial surface, mucus and inflammatory cells may obscure the cells resulting in an inadequate sample.  
• If five 360 degree rotations of the collecting device are not made, the sample may not be representative of an abnormality on the cervix

Loss of cells from the sampling device

• When preparing a conventional smear, the cells on the spatula are directly transferred to the glass slide but much of the sample may remain on the spatula and be discarded with it (Huchinson et al.1999).
• Liquid-based cytology largely eliminates this problem because the entire sample is transferred to the liquid medium. Some cells may be discarded on the SurePath broom if it is not left in the vial as recommended with that technique (Bigras et al. 2003). 
• Cells may be lost on the ThinPrep broom (Umana et al. 2013) and care must be taken to rinse the broom carefully before discarding it, which is the recommended procedure for that technique.

Lesions within the endocervical canal or small lesions

• Small lesions may be located high up into the endocervical canal and may be out of reach of the spatula, brush or broom. 

Preparation and fixation of conventional smears

• Interpretation of abnormal cells will be compromised or may even be impossible if a conventional smear is not fixed before the cells become air-dried, which affects the staining and preservation of nuclear detail.  Liquid-based cytology avoids this problem and is one of its main advantages.

Labelling the sample (slide or vial) and completing the request form

• Accurate cytology results require accurate clinical information: errors of cytological interpretation and management recommendations may result from the laboratory being provided with inaccurate information
• The source of the sample, the name of the patient (correctly spelt), date of birth, date of last menstrual period, screening history and symptoms must be recorded correctly on the request form and match the information on the slide or vial.

Methods for QC and QA of collecting, preparing and fixing the slides

• Training and supervision of new recruits is essential to ensure proper collection of cellular samples and fixation of conventional smears. 
• Training should include access to written, illustrated guidelines such European and NHSCSP guidelines (Arbyn et al. 2007; NHSCSP 2006b). 
• Supervision should be available on a daily basis for instances where there is difficulty in visualising the cervix with access to specialist clinics if necessary (QC).
• Feedback about incorrectly completed request forms or mismatches with samples – if necessary asking for tests to be repeats (QC).
• Feedback of cytology results should be provided for clinics and individual smear takers to monitor rates of inadequate samples (QA). 
• Monitoring inadequacy rates is useful for conventional smears but less so for liquid-based cytology since most samples will have sufficient cells for a report to be issued (QA). 
• Rates of slides with and without evidence of transformation sampling may be used to monitor performance of liquid-based and conventional smear sample takers (Faraker & Greenfield 2013) (QA).