High-resolution HLA-A*0201 subtyping using directed heteroduplex analysis.

HLA-A02* has become an important target for cytotoxic T lymphocyte-based immunotherapy reflecting the high prevalence of this allele in patient populations. There are at least 26 different A*02 alleles, and their subtype specificity has significant functional implications for T-cell-mediated recognition of immunologic targets. We have developed a novel method for HLA-A*02 allelic screening using directed heteroduplex analysis (DHDA). DNA samples from Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (EBV-B) representing 10 different HLA-A*02 alleles (0201, 0202, 0204, 0205, 0206, 0208, 0210, 0211, 0216, 0217) were prepared. In addition, DNA was prepared from 81 individuals representing a wide variety of A*02 subtypes previously determined by sequence specific primer (SSP) polymerase chain reaction (PCR) including individuals heterozygous for two A*02 specificities. Probes and samples were generated by PCR amplification using HLA-A*02 specific primers encompassing exons 2 and 3, where most of the functionally significant allelic polymorphism is clustered. DHDA was performed by generating heteroduplex molecules composed of a fluorescein-labeled allelic probe sequence and an unlabeled allelic PCR product. Gel retardation was consistent for allele-probe combinations. We were able to identify several A*02 alleles prepared from EBV-B cell lines that, when used as probes, had very impressive specificity and sensitivity. Combinations of two probes were identified (0205 + 0211 and 0208 + 0211) that allowed differentiation of A*0201 alleles from all other A*02 alleles tested. All samples typed by probe combinations had DHDA typing and SSP typing confirmed by DNA sequencing. This study expands the molecular typing repertoire available to the modern HLA laboratory, and shows that DHDA has significant promise as a reliable screening method for HLA A*02 subtyping.

Pathological aspects of transcutaneous cardiac pacing.

Although electrical energy has the potential to produce myocardial injury, the risk of tissue damage from transcutaneous cardiac pacing is largely unknown. This study reports the anatomical findings of a canine transcutaneous stimulation study. Ten dogs had 100-mA, 20-msec (pulse duration), transcutaneous impulses delivered across the thorax for 30 minutes at a rate of 80 stimuli per minute. Seventy-two hours later the animals were sacrificed, and the heart, lungs, and tissues of the chest wall were examined for pathological changes. Gross and microscopic lesions consistent with electrically induced myocardial damage were found in all hearts examined. These lesions included myocardial pallor and focal myofibril coagulation necrosis in the right ventricular outflow tract and perivascular microinfarcts in the posterior left ventricular myocardium. These lesions were not extensive; less than 5% of the right ventricular free wall and less than 1% of the left ventricular posterior wall were involved. Lesions of this extent would not be expected to cause clinically detectable changes in cardiovascular status. Short-term use of transcutaneous pacing appears to be safe. Determination of the potential for clinically significant injury with long-term use requires further study.