Sample Processing


Cervical Cytology Practice Guideline

Approved by the ASC Executive Board November 10, 2000



IV. Laboratory Sample Processing

Laboratory sample processing includes steps from the receipt of the specimen in the laboratory to the delivery of a stained slide ready for microscopic examination.  The information is based upon practices cited in standard cytology references.53545556  Throughout processing, the integrity of the specimen must be maintained57 and the principles of universal precautions followed.58 No result is to be released unless the system is functioning properly.

IV.A.  Receipt and Identification of the Specimen

The laboratory should confirm the integrity of the specimen received.  Specimens are accepted only when ordered by physicians or other persons authorized by law.  To process, each sample must have an accompanying request form completed by the authorized provider.  The laboratory should have a procedure in place for handling oral requests.  The provider must properly label specimens.

IV.A.1.  Requisition Requirements

The requisition accompanying the specimen should be completed with the patient’s first and last name and the age or date of birth at a minimum.43  The date the sample was collected, the source of the material and the name, location and telephone/FAX number of the requesting physician should be included on the requisition.  A medical record number or any other unique identifier may also be included.  These elements are required to ensure that specimen results are linked with the appropriate patient.  They also make it possible for the laboratory to make prior and/or concurrent results available at the time of cytologic interpretation if necessary. 

Ideally, the following information should also be provided on the requisition form as applicable: LMP, pregnant, postmenopausal, estrogen therapy, other hormonal therapy, IUD, DES exposure, chemotherapy, radiation therapy, GYN surgeries, history of cancer, previous abnormal cervical cytology, clinical findings such as infection or a grossly visible lesion and any factors that place the patient at increased risk for developing cervical cancer.

Clinical history is important and should be correlated with the type of specimen submitted.  For example, if the history states that the patient has had a total hysterectomy and the specimen is a cervical sample, clarification and resolution of the discordance should be undertaken before interpretation of the slide(s) is attempted.  All available patient information should be included in the demographic and clinical history sections of the report and archived database for current and future use.

A written procedure must be in place to handle specimens that are received without adequate information on the request form.

IV.A.2.  Glass Slides

Written criteria for the rejection of specimens must be available in each laboratory and should address unlabeled slides, slides labeled with non-permanent writing utensils or paper labels, broken slides, and slides with any piece of the cellular portion missing.  Any slides that are broken beyond repair should not be accepted.  The submitting clinician should be notified and the notification documented in the laboratory.  For slides that can be repaired, a comment regarding the sample condition should be noted in the report.

IV.A.3.  Liquid Based Specimens

The specimen vial should be received tightly closed with no leakage of the preservative and with patient identification on the vial (not the lid).  If the preservative has leaked into the transport container, this should be documented and every reasonable effort should be made to salvage the sample.  However, if an excessive amount of the preservative has been lost, the specimen may not be sufficient for evaluation; in which case, the clinician should be notified and the notification documented in the laboratory.

IV.B.  Accessioning

When the specimen and requisition are removed from the transport container, the specimen identifiers on the requisition form and sample must match.  Any variation in the spelling of the name or in the medical record number or other unique identifier should be questioned and verified.  The requesting physician or designee may rectify variations; the laboratory must keep a record of all changes made, according to the lab’s standard operating procedure.  When all specimen identifiers match, the specimen is accessioned; that is, assigned a unique number which identifies this specimen as belonging to this patient.  The number may be generated manually or electronically.  This unique number is placed on the slide and on the requisition using a material or marking device such that the number will withstand subsequent processing.  Following staining and coverslipping, a label may be affixed over a handwritten name and number.

IV.C. Staining


IV.C.1.  Smears

Any slides fixed with spray fixatives that contain Carbowax should be soaked in ethanol or water before beginning the staining process.  Carbowax is a water-soluble substance that is removed with soaking.  Carbowax left on the slides will impede stain uptake.

IV.C.2.  Liquid Based Specimens

Liquid based specimens should be processed according to the manufacturer’s instructions for transfer of cells from the liquid medium to a glass slide labeled with the patient’s name and accession number.  A written procedure should be in place for rejection of liquid based specimens that are not collected following the manufacturer’s guideline.  Refer to additional discussion in section IX, “Enhancements to Cervical Cytology Testing”.

IV.C.3.  Staining Procedure

The modified Papanicolaou method is recommended for the staining of gynecologic cytology slides.445559  The Papanicolaou method uses a standard nuclear stain, hematoxylin, and two cytoplasmic counterstains, OG-6 and EA.  The value of this method is transparency of the cytoplasm, which allows the examiner to clearly visualize cellular morphology.  Either a progressive or regressive technique may be used for nuclear staining.  Several automatic programmable stainers are available.  Each laboratory should develop a staining protocol for manual, automated, or for both methods, which results in the optimum staining of the specimen.

Maintenance of consistently good staining requires that the stains are filtered and changed on a regular schedule, determined either by the number of slides processed or the length of time elapsed since stains were last changed.  Furthermore, the quality of the stain should be monitored daily and the results documented.  Deviations from optimum quality should be addressed immediately, the problem identified and corrective action(s) taken.  The laboratory must document all problems and corrective action taken.  If the stain quality is acceptable, the remaining smears are stained and submitted for screening.

To prevent cross-contamination, gynecologic smears are usually stained separately from non-gynecologic smears.  If a single staining setup is used, solutions should be changed or filtered between gynecologic and non-gynecologic specimens.  In any staining configuration, samples with a high potential for cross-contamination must be stained separately from the remainder of the laboratory’s cases.6061

IV.D.  Dehydration, Clearing and Coverslipping

IV.D.1.  Dehydration and Clearing

After staining, the sample is dehydrated using a series of increasing concentrations of alcohol followed by baths in clearing solutions.  The last must be colorless and its refractive index must be close to that of the coverslip, slides and mounting medium.  While xylene (dimethyl-benzene) is the most commonly used clearing agent, others derived from citrus terpenes and other sources have found some use.  If using xylene, clearing should be performed in a well-ventilated area or fume hood to limit exposure to xylene fumes.62  Slides should remain in the clearing agent until coverslipping is performed.

IV.D.2.  Coverslipping

Mounting medium used to bond the slide and the coverslip must be compatible with the clearing agent, must be transparent, and should have a refractive index that is similar to the glass slide and the specimen.  The boundaries of chromatin particles are the most distinct when the specimen is mounted in a medium of similar refractive index.  Glass slides according to the American Society for Testing and Materials (ASTM)63 specifications have a refractive index of 1.515.  The refractive index of cells is similar to that of glass.  Most commercially available mounting media have refractive indices that range from 1.49-1.57+.  Mountants that exceed this range should not be used.  Ideally, the refractive index should be 1.52-1.54.

Adequate mounting medium should be applied to protect the cellular material from air-drying and shrinkage, and to form a protective seal to prevent fading of the cell sample.  The cellular material should be completely covered by a suitably sized coverslip or covering material of appropriate quality.  The ASTM requires that coverslips have a refractive index of 1.523+ .005.  Microscope manufacturers recommend a total thickness of mountant and coverslip between 0.17 and 0.18mm.  Therefore, No. 1 coverslips (0.13-0.17mm) should be used.

Coverslipping requires good light, ventilation and eye protection.  Slides should be removed from xylene one at a time to avoid drying of the cell surface.  Different methods used to coverslip include placing the mounting medium on the coverslip, then inverting the coverslip onto the slide surface, or lowering the slide onto a coverslip containing adequate mounting medium.  Glass coverslips, coverfilm and automated coverslippers are available.  The mounting medium should be allowed to dry before the slides are reviewed to reduce movement of cellular material during the slide examination.

Chemical waste collected throughout the staining, dehydration, clearing and coverslipping processes should be disposed of according to the OSHA Guideline.62

IV.E.  Destaining and Restaining

Destaining a slide is a stepwise process, beginning with removal of the coverslip and mounting medium, and proceeding backward through the staining steps, omitting the stains themselves.  Alternatively, once the coverslip and mountant are removed the slide can be soaked in acid alcohol until the slide is colorless.  The process is completed by thoroughly rinsing the slide in water baths. Once destaining is complete, restaining can begin at the nuclear stain step.56

IV.F.  Collation of Slides and Requisitions

The stained and labeled slide should be matched with its requisition or other laboratory document that displays the same information.  The information on the slide must correspond to the information on the requisition or lab document.  If there are any discrepancies, this must be noted and resolved BEFORE the report is released.

IV.G.  Configuration of Laboratory Space According to Function

The laboratory must have adequate space to ensure that the quality of preparatory work, interpretive services and the safety of laboratory personnel are not compromised.5861646566

IV.H.  Variability in Practice

The criteria for accepting/rejecting specimens vary among laboratories.  Minimum requirements for patient information differ as well.  See section VI.A. for more specific examples of clinical information.

There are several methods used for handling broken slides when a piece of the cellular portion is missing.  Some laboratories will not process the sample; others report the slide as “Satisfactory but limited by…” and comment on the condition of the smear when it was received.

There are currently two different FDA approved methods to collect and process liquid-based specimens.  See also section IX.  The protocols are not interchangeable; therefore, the manufacturer’s Guideline in the operator’s manual of the method chosen must be followed.

Accessioning specimens can be performed with a hard copy of the patient requisition or requisitions can be received electronically.

Sixty millimeter coverslips are recommended for conventional Pap smears as they consistently cover the entire smeared area.  Shorter coverslips are acceptable for conventional smears and for liquid based preparations as long as the cellular material is covered.

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