Basic Template

Background

The basis of any good pathologic diagnosis is entirely dependent upon the quality of the microscopic section which is made. Histotechnologists are the skilled personnel who prepare the tissue and mount them on glass slides, staining them, and present them to the pathologist for diagnosis. In addition, special stains and immunoperoxidase studies are also performed that may assist in the final diagnosis.

Deepers and levels are terms that are used by every pathologist.

OUTLINE

Analytic Methods

Differential Diagnosis

Commonly Used Terms

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ANALYTIC METHODS

ULTRASOUND FIXATION

Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation.

Chu WS, Furusato B, Wong K, Sesterhenn IA, Mostofi FK, Wei MQ, Zhu Z, Abbondanzo SL, Liang Q.

[1] 1Department of Scientific Laboratories, Armed Forces Institute of Pathology, Washington, DC, USA [2] 2Bio-Quick Inc., Silver Spring, MD, USA.

Mod Pathol. 2005 Jun;18(6):850-63. Abstract quote

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16-24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5-15 min.

Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses.

Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.

DIFFERENTIAL DIAGNOSIS KEY DIFFERENTIATING FEATURES

TISSUE FLOATERS

A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks.

Hunt JL, Swalsky P, Sasatomi E, Niehouse L, Bakker A, Finkelstein SD.

Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA.
Arch Pathol Lab Med 2003 Feb;127(2):213-7 Abstract quote
CONTEXT: A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications.

OBJECTIVES: To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues.

MATERIALS AND METHODS: Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-microm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity.

RESULTS: Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability.

CONCLUSIONS: Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.

Identification of mismatched fixed specimens with a commercially available kit based on the polymerase chain reaction.

Shibata D.

Department of Pathology, University of Southern California School of Medicine, Los Angeles.

Am J Clin Pathol 1993 Dec;100(6):666-70 Abstract quote
Specimen mix-ups inevitably occur and have the potential for great harm. The ability to investigate mix-ups objectively and assign fixed tissues to patients correctly is unfortunately limited, as most such assays require fresh specimens.

A commercial kit based on the polymerase chain reaction (PCR) can be applied to the molecular genetic analysis of fixed tissues. This kit, which can amplify and distinguish 21 different genotypes at a polymorphic human leukocyte antigen (HLA) locus, was applied to investigate 16 cases of potential specimen mismatches. The majority of tissues were small and essentially irreplaceable biopsy specimens, and four cases involved minute fragments of potential "floaters." Data were successfully obtained from all 16 cases despite the collection from several different hospitals and the small quantities of tissue.

The assay required approximately 2 days for completion; therefore, data were returned within a clinically useful time period. This study provided evidence that molecular genetic assays based on the PCR can be applied to routinely obtained fixed-tissue specimens to investigate potential mismatches.

Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.
Rosai J. Ackerman's Surgical Pathology. Ninth Edition. Mosby 2004.
Sternberg S. Diagnostic Surgical Pathology. Fourth Edition. Lipincott Williams and Wilkins 2004.
Robbins Pathologic Basis of Disease. Seventh Edition. WB Saunders 2005.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 5th Edition. McGraw-Hill. 1999.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fourth Edition. Mosby 2001.

Commonly Used Terms

Basic Principles of Disease
Learn the basic disease classifications of cancers, infections, and inflammation

Commonly Used Terms
This is a glossary of terms often found in a pathology report.

Diagnostic Process
Learn how a pathologist makes a diagnosis using a microscope

Surgical Pathology Report
Examine an actual biopsy report to understand what each section means

Special Stains
Understand the tools the pathologist utilizes to aid in the diagnosis

How Accurate is My Report?
Pathologists actively oversee every area of the laboratory to ensure your report is accurate

Got Path?
Recent teaching cases and lectures presented in conferences

Internet Links

Last Updated May 25, 2005

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DIFFERENTIAL DIAGNOSIS	KEY DIFFERENTIATING FEATURES
TISSUE FLOATERS
A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks. Hunt JL, Swalsky P, Sasatomi E, Niehouse L, Bakker A, Finkelstein SD. Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA.	Arch Pathol Lab Med 2003 Feb;127(2):213-7 Abstract quote CONTEXT: A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. OBJECTIVES: To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. MATERIALS AND METHODS: Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-microm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. RESULTS: Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. CONCLUSIONS: Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.
Identification of mismatched fixed specimens with a commercially available kit based on the polymerase chain reaction. Shibata D. Department of Pathology, University of Southern California School of Medicine, Los Angeles.	Am J Clin Pathol 1993 Dec;100(6):666-70 Abstract quote Specimen mix-ups inevitably occur and have the potential for great harm. The ability to investigate mix-ups objectively and assign fixed tissues to patients correctly is unfortunately limited, as most such assays require fresh specimens. A commercial kit based on the polymerase chain reaction (PCR) can be applied to the molecular genetic analysis of fixed tissues. This kit, which can amplify and distinguish 21 different genotypes at a polymorphic human leukocyte antigen (HLA) locus, was applied to investigate 16 cases of potential specimen mismatches. The majority of tissues were small and essentially irreplaceable biopsy specimens, and four cases involved minute fragments of potential "floaters." Data were successfully obtained from all 16 cases despite the collection from several different hospitals and the small quantities of tissue. The assay required approximately 2 days for completion; therefore, data were returned within a clinically useful time period. This study provided evidence that molecular genetic assays based on the PCR can be applied to routinely obtained fixed-tissue specimens to investigate potential mismatches.

Analytic Methods
Differential Diagnosis
Commonly Used Terms
Internet Links