May 2010
Feature Story

Andrew H. Fischer, MD

How can labs with multiple stain setups for immediate evaluation of adequacy comply with CAP checklist question CYP.7680, which asks: “Are there procedures to prevent cross-contamination of specimens during processing and staining?”

This checklist question addresses CLIA regulation 42CFR493.1274(b)(2-3):

The laboratory must have available and follow written policies and procedures for each of the following, if applicable:

1. All gynecologic slide preparations must be stained using a Papanicolaou or modified Papanicolaou staining method.


2. Effective measures to prevent cross-contamination between gynecologic and nongynecologic specimens during the staining process must be used.


3. Nongynecologic specimens that have a high potential for cross-contamination must be stained separately from other nongynecologic specimens, and the stains must be filtered or changed following staining.

For immediate evaluation stain setups, part three of the CLIA regulation is relevant. For labs that add drops of toluidine blue (or equivalent) for stain-ing immediate evaluation slides (rather than immersing the slide in a stain solution), the regulation is “not applicable.” However, for the CAP to have deemed status for laboratory accreditation, CAP checklist questions (unfortunately) sometimes have to slightly exceed the CLIA mandate. The actual checklist question does require a “procedure to prevent cross-contamination.” The procedure can simply state that drops of toluidine blue are used for immediate evaluations.

For stain setups using slide immersion, CLIA gives some room for interpretation of what constitutes “high potential for cross-contamination.” The CAP checklist offers guidance that “Direct smears made from the sediment of highly cellular cases should be stained after the other cases, and the staining fluids must be changed or filtered between each of the highly cellular cases.” Immediate evaluation does not involve “sediment” but rather direct smears from FNAs, brushings, or touch preps from core biopsies. Further, air-dried specimens are well known to have high adhesion to glass slides. A written procedure for using Diff-Quik setups would put a lab in compliance with the CAP checklist question. Good laboratory practice should document changing each Diff-Quik stain setup at regular intervals (for example, every week if there is an average of about five evaluations per week).

For immediate evaluation on wet fixed samples, an immersion stain setup could pose some threat of cross-contamination. A procedure that includes documenting a change or filtering of the stain solution after every positive case would be reasonable. For labs that use a frozen section H&E stain setup for immediate evaluation of adequacy, the lab should cross-reference an anatomic pathology policy for regularly changing or filtering the stains.

Cross-contamination poses a real threat of a false-positive diagnosis in cytology samples, and vigilance and common sense are probably at least as important as a “policy.” With the society-level need to decrease costs, and with increasing awareness of the environmental costs of discarding large volumes of stain solutions, a delicate balance has to be found. Gary Gill has elegantly demonstrated the inability of standard filter paper to trap malignant cells www.cytopathology.org/website/download.asp?id=867). While filtering may not remove all positive cells, in practice the chance of a residual cluster of cells to eventually lodge on another slide appears negligible. However, the staining properties of filtered stain solutions can diminish, and good laboratory procedures should include a documented policy of regular replacement of filtered stain solutions.

There are multiple ways to comply with this checklist question, mandated by CLIA. The key is to have a procedure describing what the laboratory does and have appropriate documentation that it is followed.