Preclinical validation of fluorescence in situ hybridization assays for clinical practice.
OBJECTIVE
Validation of fluorescence in situ hybridization assays is required before using them in clinical practice. Yet, there are few published examples that describe the validation process, leading to inconsistent and sometimes inadequate validation practices. The purpose of this article is to describe a broadly applicable preclinical validation process.


METHODS
Validation is performed using four consecutive experiments. The Familiarization experiment tests probe performance on metaphase cells to measure analytic sensitivity and specificity for normal blood specimens. The Pilot Study tests a variety of normal and abnormal specimens, using the intended tissue type, to set a preliminary normal cutoff and establish the analytic sensitivity. The Clinical Evaluation experiment tests these parameters in a series of normal and abnormal specimens to simulate clinical practice, establish the normal cutoff and abnormal reference ranges, and finalize the standard operating procedure. The Precision experiment measures the reproducibility of the new assay over 10 consecutive working days. To illustrate documentation and analysis of data with this process, the results for a new assay to detect fusion of IGH and BCL3 associated with t(14;19)(q32;q13.3) in lymphoproliferative disorders are provided in this report.


RESULTS
These four experiments determine the analytic sensitivity and specificity, normal values, precision, and reportable reference ranges for validation of the new test.


CONCLUSIONS
This report describes a method for preclinical validation of fluorescence in situ hybridization studies of metaphase cells and interphase nuclei using commercial or home brew probes.
Wiktor
Anne E
AE
Division of Laboratory Genetics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Van Dyke
Daniel L
DL
Stupca
Peggy J
PJ
Ketterling
Rhett P
RP
Thorland
Erik C
EC
Shearer
Brandon M
BM
Fink
Stephanie R
SR
Stockero
Kimberly J
KJ
Majorowicz
Jason R
JR
Dewald
Gordon W
GW
D000081242:B-Cell Lymphoma 3 Protein; D002883:Chromosomes, Human, Pair 14 / Q000235:genetics; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics; D019411:Clinical Laboratory Techniques; D015342:DNA Probes; D005456:Fluorescent Dyes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008232:Lymphoproliferative Disorders / Q000175:diagnosis* / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D010865:Pilot Projects; D011237:Predictive Value of Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics; D015203:Reproducibility of Results; D014157:Transcription Factors; D014178:Translocation, Genetic
D000081242:B-Cell Lymphoma 3 Protein; C000631344:BCL3 protein, human; D015342:DNA Probes; D005456:Fluorescent Dyes; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors