Biomedical subjects
K J Skogerboe
Publications and source records attributed to K J Skogerboe.
Screening for alpha-thalassemia. Correlation of hemoglobin H inclusion bodies with DNA-determined genotype.
We evaluated potential screening protocols for alpha-thalassemia in a group of 80 patients whose genotypes were determined by Southern blot analysis with alpha- and zeta-globin DNA probes. Erythrocyte inclusion bodies were measured by a modified brilliant cresyl blue test. Erythrocyte indices and iron status were also measured. The brilliant cresyl blue test reliably detects couples at risk for hemoglobin Bart's hydrops fetalis. Measurement of the number of inclusion bodies differentiates the alpha-thalassemia genotypes in the absence of a coincident beta-chain synthesis deficiency, such as hemoglobin E or beta-thalassemia. The test appears to identify patients, such as those with the Thai and Filipino deletion variants, whose alpha-thalassemia cannot be definitively characterized by DNA testing when only alpha- and zeta-globin probes are used in the analysis. We also found evidence of elevated serum ferritin levels in many patients with deletion of two or three alpha-globin genes. This study shows that most routine screening for alpha-thalassemia can be performed with three simple tests: (1) the brilliant cresyl blue inclusion study, (2) erythrocyte indices, and (3) iron studies. Analysis with DNA probes is needed in only some circumstances.
Genetic screening of newborns for sickle cell disease: correlation of DNA analysis with hemoglobin electrophoresis.
Although DNA analysis based on the polymerase chain reaction (PCR) offers potential advantages for screening newborns for sickle cell disease, few data are available concerning the reliability of PCR-based tests for such screening. We describe a protocol for detecting the A, S, and C alleles of the beta-globin gene in dried blood from phenylketonuria screening cards. This method is based on PCR and detection with allele-specific oligonucleotide probes. Results of a blind comparison of PCR analysis of the dried blood with hemoglobin electrophoresis of whole-blood samples agreed for 80 of 81 samples. The single discrepancy is probably not attributable to a failure of the PCR method, but rather to limitations of the electrophoresis method. The PCR method should be a highly accurate means of detecting beta-globin alleles in routine genetic screening with dried blood already collected for (e.g.) phenylketonuria screening.
Laser photobioactivation mechanisms: in vitro studies using ascorbic acid uptake and hydroxyproline formation as biochemical markers of irradiation response.
Clinical investigations of laser photobioactivation, or biostimulation, might be differently designed and more fruitful if knowledge of basic biochemical mechanisms were better understood. In this investigation, biochemical events identified as responses to 904 nm irradiation included increased ascorbic acid uptake by fibroblasts. These cells also showed increased hydroxyproline formation, and this was increased several-fold by the addition of proline to the medium. Maximum biochemical responses were observed at a pulse frequency of 67 Hz and a pulse width of 150 nsec with an energy density of approximately 7 mJ/cm2 per exposure. Elements in the mitochondrial cytochrome system are proposed as the radiation absorbing chromophore(s). Hypothetically, the energy generated is linked to ascorbic acid uptake, which in turn stimulates collagen synthesis.
Development and evaluation of a simplified dot-blot method for detecting the delta F508 mutation in cystic fibrosis.
The recent discovery of the cystic fibrosis gene enables DNA-based testing for the direct identification of the deletion of three basepairs coding for phenyalanine at codon 508, the major mutation responsible for the disease. This mutation can be detected by analysis of amplified DNA with allele-specific oligonucleotide probes. We have simplified the procedure of Kerem et al. (Science 1989;245:1073-80) so that the assay can be routinely completed in one working day, starting with an extracted DNA sample. Addition of salmon-sperm DNA to the product of the polymerase chain reaction greatly improved the quality of the hybridization signal. The precision of the method was evaluated by blind analysis and interpretation of results for 100 specimens from 25 patients. The same result was obtained for each patient analyzed separately four times, and four independent observers agreed on the interpretation of results for all 100 specimens. No specimen required repeat analysis to produce interpretable results. We conclude that this method is reliable and convenient for routine clinical laboratory use.
Death following accidental sodium azide ingestion.
Two college students developed symptoms of poisoning following ingestion of a salt solution during a college physiology laboratory exercise. Symptoms included nausea, vomiting, diarrhea, and altered consciousness. The ingested solution was identified as isotonic buffered saline containing sodium azide in a concentration of 1.0 g/L. The solution was commercially prepared for instrumentation use only and was used inadvertently for the exercise instead of freshly preparing sodium chloride in water. One student drank three sips of the solution and survived. The other student drank 700 to 800 mL and over several days became progressively ill, suffering myocardial damage and cardiac dysrhythmias, and, finally, died. Toxicologic studies confirmed the presence of azide in an antemortem urine sample from the deceased. Sodium azide is an uncommon but potent poison which can cause serious illness and death.
Chemiluminescent measurement of total urinary nitrogen for accurate calculation of nitrogen balance.
This instrumental method for total urinary nitrogen (TUN) is based on the principle of gas-phase chemiluminescence. Results correlate well with measurements of TUN by the Kjeldahl method, which has long provided the means to calculate nitrogen balances for nutritional management. In recent years, because of speed and convenience of measurement, determination of urinary urea nitrogen (UUN) has been substituted for Kjeldahl TUN. However, in patients requiring aggressive nutritional support, the UUN may not be a valid indicator of total nitrogen excretion. We compared nitrogen balances calculated for patients, using both UUN and chemiluminescence TUN data. For both normal and hospitalized populations, nitrogen balance calculated from UUN data exceeded that calculated from TUN data. We show that use of UUN data in calculating nitrogen balance may result in an incorrect assessment of many patients as being in positive nitrogen balance. TUN determined by chemiluminescence evidently provides a simple means of calculating nitrogen balance more nearly accurately.
PCR dot blots: large signal differences between sense and anti-sense probes.
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Contributions of analytical chemistry to the clinical laboratory.
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