III Laboratory Sample Processing

Nongynecological Cytology Practice Guidlines

prepared by the American Society of Cytopathology, Cytopathology Practice Committee.

Adopted by the ASC Executive Board, March 2, 2004


III 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 for achieving this is based upon practices cited in standard cytology references. 12,22,23,24 Throughout processing, the identity and integrity of the specimen must be maintained,25 and the principles of universal precautions followed.9

III.A. Receipt and Identification of the Specimen

The laboratory should confirm the identity and integrity of the specimen received. Specimens are accepted only when ordered by physicians or other persons authorized by law. Each sample must have a request completed by the authorized provider prior to processing. The laboratory must have a procedure in place for handling verbal requests.26

III.A.1. Requisition

The specimen requisition (whether electronic or manual) must provide space for the patient’s first and last name or other unique identifier and the date of birth (or age.) The requisition must also provide space for the date the sample was collected, the test to be performed, the source of the material, and the name and address or other suitable identifiers of the authorized person requesting the test.26 Methods of rapid communication such as telephone or FAX numbers are also recommended. These elements ensure that specimen results are linked with the appropriate patient.

Ideally, the following information should also be provided on the requisition form as applicable: pertinent physical findings, radiographic findings, history of environmental exposure, possible exposure to infectious agents, immunosuppression, chemotherapy, radiation therapy, surgical operations, history of cancer, previous surgical or cytologic abnormalities.

Clinical history is important and should be correlated with the type of specimen submitted. For example, if the history states that the patient has a left lung mass and the specimen is a right bronchial washing clarification and resolution of the discordance should be undertaken before interpretation of the specimen 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.6

III.A.2. Accessioning

When the specimen and requisition are removed from the transport container, the specimen identifiers on the requisition form and sample must match. The requesting physician or designee may rectify variations; the laboratory must keep a record of all changes made, according to the laboratory’s standard operating procedure.27 When all specimen identifiers match, the specimen is accessioned. Accessioning assigns a unique laboratory identifier linking the sample to the patient. The laboratory identifier may be generated manually or electronically and may be numeric or alphanumeric and may also be bar coded. This unique identifier is placed on the slide and on the requisition with a material or marking device that will withstand subsequent processing.

III.A.3. Specimen Condition

Written criteria for the rejection of specimens must be available in each laboratory.6,28

III.A.3.a. Glass Slides

Criteria for rejection of glass slides should address unlabeled slides, slides labeled with non-permanent writing utensils or paper labels and broken slides. For slides that can be salvaged, a comment regarding the sample condition should be noted in the report. The number of slides received, their method of fixation, and body site should be documented.

III.A.3.b. Liquid Specimens

Liquid specimens should be received in tightly closed containers. Patient identification must be on the specimen container. If the sample has leaked into the transport container, a reasonable effort should be made to salvage the sample and this should be documented in the report. The volume, color, clarity, other pertinent gross characteristics, presence of fixative, presence of heparin, and other additives should be documented.

III.A.3.c. Brushing Specimens

Brushing samples may be received as direct smears, fluid-filled containers with brushes enclosed, or liquid specimens in which the sample was removed from the brush.  III.A.2 and III.A.3  should be followed as dictated by the sample(s) submitted. The number of slides and the variety of specimens should be documented.

III.A.3.d. Fine Needle Aspirations

Fine needle aspirates are usually received as direct smears and may be accompanied by a liquid specimen collected as a needle rinse. Alternatively, needle aspiration biopsies may be submitted entirely in liquid. III.A.2 and III.A.3 should be followed as dictated by the samples(s) submitted.29


III.B. Specimen Preparation


III.B.1. Smears

The preparation objective of direct smears is a slide with an evenly and thinly applied cellular specimen that is free of mechanical distortion and free of drying artifact when the slide is fixed in alcohol. Smears fixed in alcohol (wet fixation) are usually stained by the Papanicolaou method; air-dried smears are usually stained with a Romanowsky stain. Smears preserved with spray fixatives that contain CarbowaxTM should be soaked in 95% alcohol. CarbowaxTM left on the slides will impede stain uptake and must be completely removed before beginning the staining process.12

III.B.2. Liquid Specimens

Liquid specimens should be processed according to the manner in which they are submitted. Liquid specimens may be received fresh, with heparin, with preservative (alcohol or other fixative), or with physiologic solution or tissue culture medium. Additional processing should be considered for grossly bloody specimens prior to slide preparation. Blood clots should be removed and processed as a cell block. The addition of red blood cell lysing agents and density gradient centrifugation are both methods that sufficiently remove blood while retaining diagnostic cells.12,30,31

III.B.2.a. Liquid specimens received without preservative

Specimens of low cellularity and low volume may be cytocentrifuged directly. High volume specimens are usually concentrated prior to preparation. Centrifugation is frequently used with the re-suspended pellet used for direct smears, preparation of slides using automated liquid-based methods, filtration or subsequent cytocentrifugation. A cell block can also be prepared from the centrifuged sample.

III.B.2.b. Liquid specimens received with preservative

Specimens submitted in commercially available preservative products should be processed as indicated by the manufacturer.30,31 Specimens submitted in alcohol should be processed as indicated in III.B.2.a. Cell blocks can be prepared from these specimens.32,33,34,35

III.C. Staining Procedure

The Papanicolaou stain is recommended for the staining of alcohol fixed cytology slides.24,36,37 Romanowsky stains may also be used for wet fixed slides, but are primarily applied to air-dried smears.14

III.C.1. Papanicolaou Stain

The Papanicolaou stain uses a standard nuclear stain, hematoxylin, and two cytoplasmic counterstains, OG-6 and EA. The outcome of this method is crisp nuclear detail and 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 must develop a written staining protocol for manual, automated, or for both methods, which results in the optimal staining of the specimen.6,38

III.C.2. Romanowsky Stain

A Romanowsky stain is recommended for air-dried smears. Romanowsky stains, mixtures of eosin and methylene blue, are a family of polychrome stains that produce their effect by the production of azure dyes as a result of demethylation of thiazines and the acidic component eosin. Unlike the Papanicolaou stain they are metachromatic.39 Most Romanowsky stains used in cytology are aqueous stains as opposed to the methyl alcohol based stains of hematology. Many commercial stains are available, and most consist of a methanol-based fixative, and two dyes which result in differentiation of cytoplasmic and nuclear components. Most Romanowsky stains are rapid and are useful in enhancing pleomorphism, and distinguishing extracellular from intracytoplasmic material.

III.C.3. Stain Quality Control

Maintenance of good staining requires that the stains are filtered and replaced on a regular schedule, determined either by the number of slides processed or the time elapsed since stains were last replaced.40,41 Furthermore, the quality of the stain should be monitored daily and the results documented. Deviations from optimal quality should be addressed immediately, the problem identified and corrective action(s) taken. The laboratory must document all problems and corrective action taken.6

To prevent cross-contamination, non-gynecologic preparations are usually stained separately from gynecologic specimens. If only one Papanicolaou staining setup is used, solutions should be changed or filtered between gynecological and nongynecological staining runs. Samples with a high potential for cross-contamination must be stained separately from the remainder of the laboratory’s cases.6,42

III.D. Dehydration, Clearing and Coverslipping

III.D.1. Dehydration and Clearing

After staining, the sample is dehydrated by a series of increasing concentrations of alcohol followed by rinsing in clearing solutions. The last clearing solution should be colorless and its refractive index should 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. Xylene clearing must be performed in a well-ventilated area or fume hood to limit exposure to xylene fumes.43 Slides should remain in the clearing agent until coverslipping is performed.

III.D.2. Coverslipping

Mounting medium used to bond the slide and the coverslip should be compatible with the clearing agent, transparent, and have a refractive index similar to the glass slide and the stained specimen. Glass slides have a refractive index of 1.515, according to the American Society for Testing and Materials (ASTM) specifications.44 The refractive index of cells is similar to that of glass. Ideally the refractive index of the mounting medium should be 1.52-1.54. Most commercially available mounting media have refractive indices that fall within the range of 1.49-1.57.

Adequate mounting medium should be applied to protect the cellular material from air-drying and shrinkage, and to prevent fading of the cell sample. The cellular material should be 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. Mounting medium and coverslip should have a total thickness of 0.17 to 0.18 mm according to microscope manufacturers and this number is usually indicated on the microscope objective. No. 1 coverslips (0.13-0.17 mm) meet these specifications.

Coverslipping requires good light, ventilation and eye protection. Slides should be removed from the clearing agent one at a time to avoid drying of the cell surface and the resultant brown “corn flaking” artifact. 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. Ideally, 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 must be disposed of or recycled according to OSHA, state and local regulations.43

III.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 mounting medium are removed, the slide can be soaked in acid alcohol until the cells are 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.

III.F. Confirming Identification of the Specimen

The stained and labeled slide(s) 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 laboratory document. If there are any discrepancies, these must be noted and resolved before the report is released.11

III.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.6,25,45,46,47

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