VIII AncillaryTesting

Nongynecological Cytology Practice Guidlines

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

Adopted by the ASC Executive Board, March 2, 2004


VIII Ancillary Testing

Ancillary testing is used in cytology at the discretion of the pathologist and according to the clinical circumstances. Light microscopy alone may not always yield sufficient information to render a specific diagnosis. Tumor cells may be poorly differentiated and their origin morphologically obscured. Microorganisms may be difficult to identify by routine cytologic stains. There are special tests that can assist in rendering the most specific diagnosis possible.

VIII.A. Cytochemical Stains

Cytochemical stains can help in the identification of and discrimination between

  1. Organisms (e.g. bacteria, fungi)
  2. Enzymes (e.g. chemical substances, such as acid phosphatase)
  3. Other intra and extracellular substances (e.g. fat, mucin, amyloid, collagen, melanin)

The appropriate use of controls is of utmost importance in cytochemical stains. Refer to VIII.G  for further discussion.

VIII.B. Immunocytochemical Stains

Immunocytochemical stains use monoclonal and polyclonal antibodies to detect antigens. These antigens may indicate the tissue of origin for poorly differentiated neoplasms, may identify infectious organisms, and may aid in distinguishing malignant from benign processes. In rare instances it may be appropriate to use a single immunocytochemical stain; however, it is much more common to use a panel of immunocytochemical stains to arrive at the correct diagnosis. Immunocytochemical stains can be used on cytologic samples, including all forms of slide preparations and cell blocks, with appropriate controls (refer to section IX.G ). This is a rapidly evolving and growing field. Some stains are commercially available; some are available for research use only.82,83,84

VIII.C. Transmission Electron Microscopy

Although used extensively in the 1970’s and 1980’s, immunocytochemical stains have supplanted transmission electron microscopy as an ancillary test, except in certain circumstances. Cytologic preparations are ideally suited for this study because of the large number of cells they contain and because they are often a single cell type. Certain neoplasms have ultrastructural qualities that may help in their identification. The high magnification of electron microscopy elucidates specific ultrastructures85 such as,

  1. Extracellular features (e.g. amyloid)
  2. Cell surface features (e.g. microvilli, intercellular junctions)
  3. Cytoplasmic features (e.g. mitochondria, lysosomes, neurosecretory granules, melanosomes)
  4. Nuclear features (e.g. inclusions)
  5. Other (e.g. viral particles)

Special preparation of the cells, such as fixation in glutaraldehyde, is needed.

VIII.D. Flow Cytometry/ Image Analysis

Flow cytometric analysis is used primarily in the analysis of suspected hematologic malignancy. Characterization of cell surface markers and intracytoplasmic markers are performed on live cells in single cell suspensions. Cells must be submitted in tissue culture medium, e.g. RPMI, to maintain viability necessary for processing.

DNA analysis can also be performed by flow cytometry on fixed or fresh cells, although this application is less popular than in past decades.

VIII.E. DNA or RNA Amplification (Polymerase Chain Reaction (PCR))

Another ancillary test available, but not widely used in cytologic preparations, is DNA or RNA amplification. This technique is used to identify genetic abnormalities or certain DNA or RNA markers. This is a highly specific ancillary study that can be performed with only a small amount of sample. Specific mutations or specific DNA gene sequences may identify certain disease processes. DNA or RNA amplification can be used to rapidly determine the presence of viruses or other organisms, such as Mycobacterium tuberculosis.86

VIII.F. Fluorescence In Situ Hybridization (FISH)

Fluorescence in situ hybridization (FISH) is an ancillary test that allows the visualization of genetic alterations on a cell-by-cell basis.87 Current cytopathology textbooks include sections regarding FISH applications.88,89 Monolayer cell preparations facilitate the application of FISH in cytopathology. Optimal results are obtained on specimens with moderate cellularity;90 however, successful application has been demonstrated on specimens of low cellularity. FISH can be applied to fine-needle aspirates, smears, tissue imprints and disaggregated whole cells from paraffin sections.91

VIII.G. Ancillary Test Limitations

Ancillary tests have their own limitations. Each of these studies should be used in conjunction with initial morphologic evaluation along with clinical information. False positive and false negative results can occur.

Immunocytochemical tests may cross-react with antigens that are not intended to be reactive, or may have high background staining, which renders the test less interpretable. Positive and negative controls should be performed with each sample. An antibody specific and method specific control tissue, cell block or cytology preparation simultaneously stained with the unknown or target specimen is required.92 The ideal control in cytology is a cytology sample; however, tissue or cell block controls are widely used for practical purposes.

Electron microscopy utilizes a minute sample of tissue and is subject to sampling error. Sub-optimal specimen preservation and areas of necrosis can contribute to problems in diagnosis.93

Flow cytometry requires a sufficient number of viable cells and sometimes only limited panels are possible.

DNA quantitation by image analysis is time consuming and requires a skilled morphologist.93

PCR is very sensitive; however, there is a high risk of contamination.

Although FISH is becoming more widely available, it is still expensive and time consuming.93

VIII.H. Variations in practice

Several different techniques can be used in the preparation of the ancillary studies. The range of available ancillary tests will vary among laboratories. Some of these ancillary studies may not be available in every institution and may be referred to laboratories that offer these specialized services.

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82     Color Atlas of Differential Diagnosis in Exfoliative and Aspiration Cytology, Sudha R. Kini, Williams and Wilkins, 1999, Chapter 1.

83     Frisman D, ImmunoQuery website,

84     Dabbs DJ, Diagnostic Immunochemistry, Philadelphia: Churchill Livingstone, 2002.

85     Comprehensive Cytopathology, 2nd edition, Marluce Bibbo, W.B. Saunders Company, 1997, Chapter 36.

86     Advanced Diagnostic Methods in Pathology, TJ O’Leary, Saunders, 2003, Chapter 5.

87     Waters, JJ et al. Demystified…FISH. J Clin Pathol:Mol Pathol 1998;51:62

88     Bergeron C, Ferenzcy A. In situ hybridization. Bibbo, M. Comprehensive Cytopathology, Philadelphia: W.B. Saunders Company, 1995;1053-1060.

89     DeMay, R.M. The Art & Science of Cytopathology, Chicago:ASCP Press, 86-87

90     Abati A et al, Fluorescence in situ hybridization (FISH): A user’s guide to optimal preparation of cytologic specimens. Diagnostic Cytopathology 1995;13:486-492

91     Wolman SR et al, Genetic probes in cytology: principals and applications. Diagnostic Cytopathology 1995;13:429-435

92     Diagnostic Immunohistochemistry, Dabbs, Churchill Livingstone, 2002, Chapter 19

93     Ross, JS. Ancillary Techniques in Diagnostic Cytopathology: Uses and Limitations.  Short course #60, 2001: United States and Canadian Academy of Pathology, 1-6