Daniel H. Farkas

Specimen Stability for Dna-based Diagnostic Testing

The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4°C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4°C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.

Use of Molecular Diagnostic Methods for Lymphoma Staging in Bilateral Bone Marrow Biopsies.

Background: Testing for immunoglobulin and T-cell-receptor gene rearrangement is a useful adjunct to morphologic evaluation of bone marrow involvement by non-Hodgkins lymphoma. At this institution, each sample from a patient undergoing bilateral bone marrow sampling for lymphoma staging is assayed seperately to avoid possible false negatives from dilution, if, for example, only one sample is positive. Methods and Results: Gene rearrangement results were reviewed for 140 patients, to assess if pooling the two aspirate samples from bilateral biopsies could be implemented. Discordant results between right and left iliac crest samples were found in 4 of 140 (2.9%) patients. Morphologic assessment was performed for 138 of the 140 patients. In 64 cases there were no abnormal findings. Four of these 64 (6.3%) had monoclonality demonstrated by genotyping or immunophenotyping. Of 134 patients for which both tests were performed, 17 (12.7%) showed discordance: 6 were negative by immunophenotyping, positive by genotyping (3 cases with malignant morphology, 1 with suspicious morphology, and 2 without evidence of malignancy); 11 positive by phenotyping but not genotyping (6 cases with malignant morphology, 3 with suspicious morphology, and 2 without evidence of milignancy). Conclusions: These findings support the role for these ancillary studies; in some cases both may be needed to demonstrate monoclonality. Based on this low occurence of discordant findings between right and left samples, we feel that bilateral specimens can be pooled without unacceptable risk of false negatives due to possible dilution.

Novel chemiluminescent substrate and probe systems for the identification of CFΔF508 genotypes

Background: Chemiluminescence detection systems are rapidly gaining popularity as safer alternatives to isotopic methods in molecular diagnostics with equal sensitivity and specificity. In addition, they offer versatility of detection because of the availability of different haptens for labeling the probes, the antihapten antibodies conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP), and their respective chemiluminescent substrates. A novel dual chemiluminescent substrate (AP and HRP based) and probe systems to distinguish genotypes of cystic fibrosis DeltaF(508) mutation are described. Methods and Results: Two methodologies have been formulated to identify positively the genotypes of the cystic fibrosis DeltaF(508) mutation. In method 1, a pair of oligonucleotides designed to anneal to the fanking regions of DeltaF(508) mutation are differentially labeled with the hapten biotin or fluorescein and ligated using the template DNA of wild-type (N/N), heterozygous (N/DeltaF(508)), and homozygous (DeltaF(508)/DeltaF(508)) genotypes. The ligated product containing both labels is detected by first binding with avidin-HRP and anti-fluorescein-AP followed by reaction with the dual substrate. As expected, the ligation products are detected only in n/DeltaF(508) and DeltaF(508)/DeltaF(508) genotypes but not in N/N, where the ligation is precluded by the presence of three intervening nucleotides. In method 2, the three genotypes are hybridized on a membrane simultaneously with uniquely labeled (biotin or digoxigenin) oligonucleotides each designed to bind either the normal or the mutant allele. On treatment with HRP- and AP-conjugated antibodies followed by reaction with the dual substrate, only the band from N/DeltaF(508) genotype emitted a strong signal because of the binding of both oligonucleotides. Conclusions: The ligation and hybridization methods in conjunction with the dual substrate can detect and differentiate the genotypes with the DeltaF(508) mutation. These formats may be valuable for distinguishing normal individuals from carriers in population screening and fetuses that are heterozygous, from those that are homozygous for cystic fibrosis DeltaF(508) in prenatal and neonatal diagnosis.