Guidance for fluorescence in situ hybridization testing in hematologic disorders

Abstract

Fluorescence in situ hybridization (FISH) provides an important adjunct to conventional cytogenetics and molecular studies in the evaluation of chromosome abnormalities associated with hematologic malignancies. FISH employs DNA probes and methods that are generally not Food and Drug Administration-approved, and therefore, their use as analyte-specific reagents involves unique pre- and postanalytical requirements. We provide an overview of the technical parameters influencing a reliable FISH result and encourage laboratories to adopt specific procedures and policies in implementing metaphase and interphase FISH testing. A rigorous technologist training program relative to specific types of probes is detailed, as well as guidance for consistent interpretation of findings, including typical and atypical abnormal results. Details are provided on commonly used dual-fusion, extra signal, and break-apart probes, correct FISH nomenclature in the reporting of results, and the use of FISH in relation to other laboratory testing in the ongoing monitoring of disease. This article provides laboratory directors detailed guidance to be used in conjunction with existing regulations to successfully implement a FISH testing program or to assess current practices, allowing for optimal clinical testing for patient care.

Figures

Figure 1

Representative interphase nuclei showing D-FISH signal patterns in cells with various Ph chromosomes. A: Nuclei with two red (ABL1) and two green (BCR) signals are normal. B: Nuclei that have one red, one green, and two fusion signals have a balanced t(9;22). C Nuclei that have one red, one green, and three yellow fusion signals have an extra Ph chromosome. D: Nuclei that have amplification of the yellow BCR/ABL1 fusion signal will show multiple copies of the BCR/ABL1 fusion signal. E: Nuclei that have two red, two green, and one yellow fusion signal have a complex Ph chromosome. F: Nuclei that have one red, one green, and one fusion signal either have a masked Ph chromosome or have loss of the ABL1 and BCR hybridization sites that are normally observed on the abnormal chromosome 9. G: Nuclei that have two red, one green, and one yellow fusion signal have loss of the BCR hybridization site that is translocated to the abnormal chromosome 9. H: Nuclei that have one red, two green, and one fusion signal have loss of the ABL1 hybridization site that normally remains on the abnormal chromosome 9.

Figure 2

Probe patterns observed at diagnosis in the bone marrow of six patients with childhood B-lineage ALL. In each case, marrow slides were prepared by standard cytogenetic techniques for chromosome analysis and hybridized with Vysis’ TEL/AML1 ES probe for FISH analysis. Results for A–F (upper left through lower right), probe pattern first and then interpretation: A: Red, Red, Green, Green; normal, no evidence of abnormality; B: Red, residual Red, Green, Fusion; typical pattern for t(12;21)(p13;q22); C: Red, residual Red, Fusion; t(12;21) with loss of the TEL (ETV6) locus from the uninvolved 12; D: Red, Red, Green; no evidence of translocation, loss of one TEL (ETV6) signal consistent with del(12p); E: Red, Red, Red, Red, Red, Green, Green; no translocation, this pattern was found in a patient with high hyperdiploidy with five copies of chromosome 21; and F: A partial metaphase spread from a patient with no evidence of t(12;21) but multiple AML1 (RUNX1) signals on a single marker chromosome (AML1 amplification).

Figure 3

G-banded metaphase spread [47,XY,+11,t(11,21)(q23;q22)] on the right with arrows showing the two normal copies of chromosome 11 and derivative chromosomes 11 and 21. MLL break-apart probe FISH analysis on the same metaphase cell (left) showing the two normal copies of chromosome 11 (yellow), the red derivative chromosome 21 [der(21)] signal, and the derivative chromosome 11 (arrow).