Clark A. Rundell

Laboratory Diagnosis of Cystic Fibrosis

(1982). Laboratory Diagnosis of Cystic Fibrosis. CRC Critical Reviews in Clinical Laboratory Sciences: Vol. 18, No. 4, pp. 313-338.The demonstration of abnormally high concentrations of electrolytes in eccrine sweat is still the only practical laboratory procedure available for diagnosis of cystic fibrosis. Properly performed, the sweat test is very reliable, but there are many published reports that all of the methods in current use frequently generate incorrect diagnoses. Analysis of potential for error in sweat test methods shows that of the three essential phases involved, stimulation, collection, and analysis, the major cause of intrinsic inaccuracy occurs in the collection process. In this case the problem is due to condensate formation, which leads to the subsequent analysis of nonrepresentative sweat. Human error is also an important cause of false results and is a direct function of the number of critical manual operations involved in the technic. This review provides a critical examination of sweat test methods, identifying problem areas and suggesting ways to improve procedures in the interests of clinically reliable laboratory data in support of diagnosis.

Relationship Between Lipoprotein- and Oxidation-Related Variables and Atheroma Lipid Composition in Subjects Undergoing Coronary Artery Bypass Graft Surgery

The relationship between atheroma lipid composition and serum lipoprotein and oxidation measurements has not been fully explored. To address this question, we studied serum, plasma, and aortic wall specimens from 66 subjects undergoing coronary artery bypass graft surgery. The lipid composition of aortic specimens was characterized in terms of cholesterol ester and cholesterol crystal plus phospholipid by using hot-stage polarizing light microscopy; tissue oxidation status was assessed by measuring conjugated dienes. Serum lipoprotein-related measurements included total cholesterol, triglyceride, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, apolipoproteins B and AI, and lipoprotein(a). Oxidation status was assessed by measuring LDL mobility, thiobarbituric acid-reactive substances, LDL conjugated dienes, and IgG and IgM autoantibodies against oxidized LDL. Fasting blood glucose was also determined. Lesion cholesterol crystal plus phospholipid content was associated inversely with serum HDL cholesterol levels (r=-0.279, P=0.029) and positively with fasting blood glucose (r=0.359, P=0.016), LDL mobility (0.276, P<0.05), and IgM autoantibodies against oxidized LDL (r=0.272, P=0.037). There was also a significant relationship between the level of aortic tissue conjugated dienes and plasma LDL mobility (r=0.332, P=0.007). In multivariate analysis, IgM autoantibodies against oxidized LDL, fasting blood glucose, and LDL mobility, in descending order of significance, together accounted for 35% of the variability in aortic lesion cholesterol crystal plus phospholipid content. These data support direct and independent roles for oxidation and hyperglycemia in the pathophysiology of atherosclerosis.

Evaluation and use of a synthetic quality control material, included in the European external quality assessment scheme for cystic fibrosis.

Assuring high quality within the field of genetic testing is fundamental, as the results can have considerable impact on the patient and his or her family. The use of appropriate quality control (QC) samples is therefore essential. Diagnostic laboratories mainly use patient samples as QC material, which of course include a maximum of two mutations per sample. Bearing in mind that some assays (such as for cystic fibrosis [CF] testing) can test for more than 100 mutations, multiplex QC materials including more than two mutations could save valuable time and reagents. Based on this need, synthetic multiplex controls have been developed by Maine Molecular Quality Controls, Inc. (MMQCI) for CF. A synthetic control, containing six homozygous mutations and one polymorphism for CF transmembrane conductance regulator (CFTR), was evaluated by distributing it through the CF external quality assessment (EQA) scheme, along with the EQA samples in 2005. A total of 197 participants returned results of the yearly EQA scheme and 133 laboratories participated in the evaluation of the synthetic sample. Respectively, 76% and 73% of the participants were assigned as successful. This evaluation study revealed that the multiplex QC material performed well in the majority of assays and could be useful in method validation, as a tool to challenge interpretation skills, and as potential proficiency testing (PT) material. Hum Mutat 0, 1–8, 2008.

Amniostat-FLM: An initial clinical trial with both vaginal pool and amniocentesis samples

Amniostat-FLM, a new rapid slide agglutination test, was compared with thin-layer chromatography as a method for detecting phosphatidylglycerol in amniotic fluid. This is the first reported use of Amniostat-FLM to evaluate vaginal pool and contaminated vaginal pool amniotic fluid. One hundred one of 161 amniotic fluid samples were collected from the vaginal pool. Thirty-nine of these were contaminated. Vaginal pool amniotic fluid, whether contaminated or not, did not adversely effect the ease of performance, reliability, or interpretation of Amniostat-FLM. This test seems ideally suited to institutions where 24-hour availability of thin-layer chromatography is not available. In institutions where it is available, Amniostat-FLM could be used as a screening test. Amniostat-FLM was found to be significantly less sensitive when compared with thin-layer chromatography in detecting the presence of phosphatidylglycerol. At our institution the screening of amniotic fluid samples with Amniostat-FLM before use of thin-layer chromatography was only cost effective at greater than or equal to 34 weeks of gestation.

Allele-specific hybridization of lipoprotein lipase and factor-V Leiden missense mutations with direct label alkaline phosphatase-conjugated oligonucleotide probes.

Direct label alkaline phosphatase (AP) conjugated oligonucleotide probes (AP-DNA) were prepared to assess their utility for allele-specific detection of single base substitutions. Oligonucleotide conjugates were designed to detect point mutations in the genes for lipoprotein lipase (LPL) and coagulation factor-V (FV). Genomic DNA samples, including ones known to harbor point mutations in the genes for LPL and FV, were prepared from whole blood and subjected to polymerase chain reaction (PCR). PCR products were analyzed by Southern hybridization with the allele-specific AP-DNA probes and restriction endonuclease analysis. Thermal profiles for hybridization indicate optimal allele-specific selectivity was achieved with temperatures ranging from 45 degrees C to 55 degrees C at a total Na divided by concentration of 150 mM. Under these conditions the base changes studied were easily discriminated with allele specific hybridization signals in excess of 200:1 as estimated by scanning densitometry. Complete concordance was observed between hybridization and restriction analyses for 175 LPL and 201 FV clinical and reference samples. The total time for analysis of the PCR products was less than 2 h with a dot blot hybridization protocol.

Direct measurement of serum low-density lipoprotein cholesterol in patients with acute myocardial infarction on admission to the emergency room

Measurement of low-density lipoprotein cholesterol during acute myocardial infarction in nonfasting patients on initial presentation to an emergency room by any of 3 methods (ultracentrifugation, immunoseparation, or the Friedewald estimate), identifies patients eligible for antilipemic interventions. Although slightly less sensitive, the conventional Friedewald estimate of low-density lipoprotein cholesterol levels provides clinicians good correlation with ultracentrifugation.

Fluorescence Polarization Testing of Amniotic Fluid as Predictor of Neonatal Respiratory Outcome

In the limited terms of neonatal respiratory distress syndrome (RDS), neonatal respiratory outcome has been correlated with fluorescence polarization assays of amniotic fluid. Respiratory outcome in terms of the severity of RDS, as well as the duration of respiratory support, has not.During a 4-year period, 179 patients delivering within 72 h of amniotic fluid analysis by fluorescence polarization were reviewed for neonatal respiratory outcome. The degree of respiratory distress and respiratory support was determined and stratified by TDx-FLM values.Overall risk of RDS for patients delivering within 72 h of fluid sampling was 13.8%. Risk of RDS for FLM values >50 mg/g was 5.3%, with an average duration of respiratory support of 2.1 days. No long-term RDS-related sequelae were encountered in samples in which FLM values exceeded 50 mg/g. RDS occurred in 4.0% of patients with FLM >70 mg/g; in all but one case, however, simultaneous chromatographic analysis (lecithin/sphingomyelin and phosphotidylglycerol) in...