Identification of clonal IgH rearrangement in diagnostic lymphoblasts and detection of leukemia/patient-specific IgH rearrangement in blood samples of archival newborn screening cards.

(A) Genomic DNA extracted from diagnostic lymphoblasts was amplified using a panel of 6 V_H-segment family primers (V_H 1-6) and a J_H-segment consensus reverse primer (see “Patients, materials, and methods”). Lane 1 represents a patient/leukemia-specific clonal IgH gene rearrangement that was excised and sequenced. (B) Seminested PCR testing the specificity and sensitivity of the patient-specific primers designed across the D_H-N-J_H and N-D_H-N segments based on the clonal IgH rearrangement identified in panel A. Lane 1 represents amplification of 100 ng of the patient's diagnostic genomic DNA; lanes 2 through 5 represent serial 1:10 dilutions of the patient's genomic DNA with genomic DNA extracted from peripheral blood lymphocytes of a healthy control. Lane 6 represents a PCR using control genomic DNA, and lanes 7 and 8 represent blank controls from the first and second rounds of the seminested PCRs, respectively. (C) PCR amplification of 2-mm punches obtained from the patient's newborn screening card blood sample using a seminested PCR with primers based on the IgH sequence identified in panel A. Lanes 2, 5, 6, and 7 represent punches in which the patient/leukemia-specific IgH rearrangement was amplified by seminested PCRs using a set of patient/leukemia-specific primers as described in “Patients, materials and methods.” Lane 8 represents a PCR using a punch from an artificial control newborn card and lanes 9 and 10 represent blank controls. (D) DNA sequence identifying identical IgH genomic sequences from the patient's newborn screening card blood samples amplified in panel C (lower sequence) and from lymphoblasts obtained at diagnosis (upper sequence).