A multicenter investigation with interphase fluorescence in situ hybridization using X‐ and Y‐chromosome probes

Abstract

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98-99% XY with no XX cells, and 97-98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96-97% XX with 0.03% XY cells, and 94-96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service.

Guidance for Fluorescence in Situ Hybridization Testing in Hematologic Disorders

“…An adequate number of cells and loci should be scored to ensure that the probe has the sensitivity and specificity required for the clinical testing being performed. 9 Probes used for hematologic malignancy studies should have a high analytic sensitivity and specificity (Ͼ95%), particularly if they are to be used for minimal residual disease assessment.…”

Preclinical validation of fluorescence in situ hybridization assays for clinical practice

“…The normal cutoff is calculated by using the maximum number of false-positive cells for any normal sample and using a binomial statistical formula to project the upper bound of the 95th percentile. 13 This computation will help predict whether the new FISH assay will meet clinical expectations (more details on determining the normal cutoff are provided in Table 4 and in the clinical evaluation study).…”

“…10,11 In a series of three independent studies, a group of cytogenetic laboratories worked together to evaluate the efficacy of FISH proficiency testing on metaphase and interphase preparations, and identified methods to calculate specific analytic parameters. [12][13][14] Dewald 15 published a book chapter on a procedure for validation of BCR/ABL fusion in interphase nuclei; this method has considerable application for other quantitative interphase FISH assays as well. In 2004, the National Committee on Clinical Laboratory Standards (NCCLS) (now known as the Clinical and Laboratory Standards Institute) published a comprehensive description of processes to validate FISH assays.…”

Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers

“…[10][11][12][13][14] Established procedures are commercially available, thus making standardization of the methods possible and ensuring comparable results. 15 In sexmatched transplantation, PCR-based procedures using amplification of polymorphic DNA sequences, namely short tandem repeat (STR) or variable number of tandem repeat (VNTR) markers, have become a widely used approach. These assays are attractive, because they need only minimal amounts of material and are not dependent on a sex mismatch.…”