Vol. 18 • Issue 5 • Page 24
The Molecular Edge
Molecular testing within anatomic pathology has increasingly become incorporated into routine practice and is used to improve the accuracy of pathology diagnosis and predict response to specific therapies and disease outcomes. Genetic identity testing, also known as DNA fingerprinting or DNA typing, utilizes differences in DNA sequences between individuals to distinguish one human being from another. It is most widely used in forensic science; however, it also has numerous applications in anatomic pathology, including detection of tissue contaminants or "floaters" in paraffin-embedded tissue blocks and establishing the identity of switched or mislabeled surgical pathology specimens.
Sources of Error
Surgical pathology specimens are procured and handled by numerous medical personnel, and despite their best efforts to adhere to strict policies and precautions, human errors do occur and specimens may be delivered to a pathologist as mislabeled or unlabeled. The College of American Pathologist's study of 417 institutions reported that patient identification errors were detected in 4,800 surgical pathology cases out of 1 million cases studied.1Another recent study reported analysis of 272 surgical pathology legal claims and found that 13 (5 percent) claims involved allegations that specimens had been mixed up between patients.2The National Patient Safety Goal for the Joint Commission (Laboratory Goals) has established as their primary goal the improvement of the accuracy of patient identification by confirming that the individual is, in fact, the person for whom the service/test has been requested.
Errors in specimen identification may be suspected by a pathologist based on discrepancies in histopathologic findings with clinical diagnosis or based on unusual case numeration or labeling. However, the most reliable method for specimen identification is DNA identity testing, which can help distinguish the origin of two samples as coming from the same or different individuals based on the molecular profile.
Tissue contaminants or paraffin tissue block floaters are other challenging problems in anatomic pathology.3Small fragments of tissue can be carried over from one surgical specimen to another during the multi-step laboratory processing of tissues. Tissue of one patient may become embedded within an unrelated paraffin tissue block or fragments of tissue sections may become intermingled on the microscope slide. Most tissue contaminants can be recognized by a pathologist based on microscopic or immunohistochemical characteristics. However, some cases are difficult to resolve, especially if the fragments are from small biopsies or if a smaller piece of contaminant tissue becomes embedded within a larger block of tissue. Failure to identify a specimen floater may result in incorrect diagnosis and potential significant adverse effects on a patient. For resolution of such cases, DNA identity testing is the method of choice.
Principles of DNA Identity Testing
Tissue identity testing is performed using PCR amplification of DNA isolated from the "correct" or "known" tissue fragment (or from the patient's blood or buccal swab) and from the tissue fragment in question. Tissue fragments may be microdissected either manually or using laser capture microscopy. In manual microdissection, the tissue areas are marked on the hematoxylin and eosin (H&E) stained slides, then tissue targets are manually removed under microscopic observation from unstained and deparaffinized tissue sections. Laser capture microdissection is used for isolation of extremely small tissue targets (less than 1 mm in diameter) and utilizes a laser beam technology for dissecting tissue from the slide. Standard DNA isolation methods are used and there are numerous commercially available kits. The PCR-based tissue identity test requires very small amounts of DNA and can be successfully performed using formalin-fixed paraffin embedded (FFPE) tissue samples or even H&E stained slides.
The DNA tissue identity method is based on comparison of the length of multiple hypervariable DNA regions, such as microsatellite repeats, between the two specimens.4-6 Microsatellite repeats, also known as short tandem repeats (STRs), are short sequences of DNA (2-7 base pairs length) that are repeated multiple times. The number of repeats varies from person to person and most individuals will have two alleles of different sizes inherited from their parents (one from the mother, the other from the father). The number of repeats is determined based on the size of PCR products following amplification of the dissected tissue fragments and/or other specimens.
To improve test reliability, multiple STRs are tested for the same patient. Typically, multiplex PCR is performed and up to 16 different microsatellite loci are amplified in the same reaction. Most frequently used microsatellite loci are tetranucleotide repeats, which offer a larger difference in allele sizes and allow for ease of interpretation. Markers on chromosome X and Y are included for gender determination. Multiple PCR products are amplified with primers labeled with different fluorophores and separated by capillary gel electrophoresis. The size of each PCR product is identified and compared between the two samples (Figure). Since microsatellites are highly polymorphic in the population, the test is typically informative and able to distinguish if the two DNA profiles are identical (i.e., belong to the same individual) or different (i.e., belong to different patients) (Figure).
DNA-based tissue identity testing is a useful tool in anatomic pathology for identification of specimen mix-ups and detection of tissue contaminants in paraffin-embedded tissue blocks, especially if it cannot be resolved based on microscopic or immunohistochemical characteristics. It can be successfully used on different tissue types and fixatives and requires minimal amount of material for the analysis. As this test becomes more available for pathologists, it can be used as a standard work-up for detection of tissue contaminants, determination of identity of switched or mislabeled specimens and will help to improve the quality of pathology service.
Dr. Nikiforova is associate director, Molecular Anatomic Pathology, Department of Pathology, University of Pittsburgh (PA). Dr. Mantha is program manager, Molecular Anatomic Pathology Laboratory, University of Pittsburgh Medical Center.