Cystic fibrosis: molecular approaches to diagnosis.

Whole blood collected on filter paper (Guthrie cards) has provided an excellent means for screening inborn errors of metabolism in neonates. Traditional biochemical methods adapted for use with this collection device have proven instrumental in the detection of many congenital defects such as phenylketonuria, galactosemia, hypothyroidism and hemoglobinopathies. The advent of molecular techniques, specifically polymerase chain reaction (PCR), has resulted in unparalleled advances in diagnostic sensitivity. Because of its ability to amplify small quantities of deoxyribonucleic acid (DNA), PCR has proven particularly successful for use with Guthrie card bloodspots in the identification of many genetic disorders including cystic fibrosis, sickle cell anemia and muscular dystrophy. Furthermore, it has been suggested that Guthrie cards represent a vast archive of genomic material yet to be explored. In this article we review our experience using Guthrie card bloodspots for PCR amplification of the cystic fibrosis gene, describe the advantages and limitations of this technology and speculate on future prospects for molecular diagnostics over the next 100 years.

Polymerase chain reaction amplification of Guthrie card deoxyribonucleic acid: extraction of nucleic acid from filter matrices.

This study evaluated two deoxyribonucleic acid (DNA) extraction methods for Guthrie card bloodspots. Our water-based extraction technique was compared with a commercial kit (GFX extraction system). In contrast to our nondenaturing method, the GFX system utilizes chaotropes to extract DNA. Extracted DNA is subsequently purified by selective elution from a glass fiber matrix. To evaluate DNA extraction, polymerase chain reaction (PCR) for a 491 bp region encoding the cystic fibrosis delta F508 mutation was performed and PCR products electrophoresed on polyacrylamide gels and stained with ethidium bromide. Amplification of GFX-extracted DNA required low sample volume (1 to 5 microL) indicating the presence of residual PCR inhibitors. The GFX volumes (1/20 to 1/4 punch) were comparable to our standard conditions and represented 0.16-0.80 microL whole blood (20 to 40 ng DNA). The GFX-extracted DNA was found to be stable (6 mo, -15 degrees C). Performance time for the GFX method was less than our standard water-based extraction (about 30 min), but more costly. The GFX extraction kit was adaptable for limited sample (i.e., extraction of a 3 mm punch yields 20 amplifications). The GFX extraction kit provides a useful means to standardize Guthrie card DNA extraction.

Amplification of Guthrie card DNA: effect of guanidine thiocyanate on binding of natural whole blood PCR inhibitors.

Amplification of DNA from whole blood collected on Guthrie card filter paper presents considerable technical obstacles due to the presence of natural PCR inhibitors (protein, heavy metals, heme, and heme degradation products) and low copy number of genomic material. For this purpose we evaluated guanidine thiocyanate-impregnated filter paper (GT-903), a DNA collection device designed specifically to bind PCR inhibitors and preserve DNA in an aqueous extractable form. Compared to standard 903, which retains DNA and elutes inhibitors during aqueous extraction, we found GT-903 retained 90% of protein, hemoglobin, and iron. SDS-PAGE analysis indicated that the majority of the protein released from standard 903 corresponded to albumin (70-) and globin (15-kDa); negligible levels of these proteins were eluted from GT-903. To evaluate PCR efficiency, we amplified the 491 bp region encoding the cystic fibrosis delta F508 mutation. Using comparable template, we found GT-903 amplification more efficient than standard 903 following qualitative (TBE-PAGE) and quantitative (anti-dsDNA EIA) determination. We conclude that GT-903 provides a good DNA collection device and addresses the complications associated with natural PCR inhibitors.

Outcomes assessment of a residency program in laboratory medicine.

During a down-sizing of residency programs at a State University Medical School, hospital based residents' positions were eliminated. It was determined to find out the characteristics of the residents who graduated from the Laboratory Medicine Program, to compare women graduates with men graduates, and to compare IMGs with United States Graduates. An assessment of a 25 year program in laboratory medicine which had graduated 100 residents showed that there was no statistically significant difference by chi 2 analysis in positions (laboratory directors or staff), in certification (American Board of Pathology [and subspecialties], American Board of Medical Microbiology, American Board of Clinical Chemistry) nor in academic appointments (assistant professor to full professor) when the male graduates were compared with the female graduates or when graduates of American medical schools were compared with graduates of foreign medical schools. There were statistically significant associations by chi 2 analysis between directorship positions and board certification and between academic appointments and board certification. Of 100 graduates, there were 57 directors, 52 certified, and 41 with academic appointments. Twenty-two graduates (11 women and 11 men) attained all three.

The effect of storage on Guthrie cards: implications for deoxyribonucleic acid amplification.

The effect of storage on (1) amplifiability of nucleic acid (present at low level) and (2) properties of whole blood polymerase chain reaction (PCR) inhibitors (present at high levels) in Guthrie card bloodspots was evaluated. Natural PCR inhibitors (protein, hemoglobin, iron) were selectively eluted from Guthrie cards (1 to 30 mo storage) under nondenaturing conditions and quantitated. The PCR was performed by direct amplification. It was found that PCR inhibitors become increasingly resistant to elution ("fixed") over time. For example, 600 micrograms protein, 1.87 au hemoglobin, and 374 ng iron were solubilized from 1 mo bloodspots. In contrast, only 137 micrograms protein (22 percent), 0.34 au hemoglobin (18 percent), and 147 ng iron (39 percent) were solubilized from 30 mo bloodspots. Fixation does not result from excessive desiccation since bloodspot weight 2.20 mg +/- 0.21 (1 mo) and 1.92 mg +/- 0.31 (30 mo) was not significantly changed (p > 0.05). The majority of protein was characterized as albumin, and two rbc metal-containing proteins, carbonic anhydrase and hemoglobin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite the presence of "fixed" PCR inhibitors, it was found that bloodspots stored 1 to 30 mo could be amplified for two regions (98 bp and 491 bp amplicons) encoding the delta F508 cystic fibrosis mutation. It is suggested that nucleic acid also becomes "fixed" to the filter paper matrix and accounts, in part, for the ability to amplify Guthrie cards by direct PCR and low yield of deoxyribonucleic acid (DNA) reported for microextraction methods.

Detection of cystic fibrosis delta F508 mutation by anti-double-stranded DNA antibody.

This study evaluated an enzyme immunoassay (EIA) as a screening tool for detection of the most common mutation (delta F508) in cystic fibrosis (CF). Guthrie card bloodspots were extracted to remove whole blood polymerase chain reaction (PCR) inhibitors. The washed filter paper was directly amplified under standard (98 bp amplicons) or modified PCR conditions (491 bp amplicons) for the delta F508 mutation. Monoclonal anti-double stranded deoxyribonucleic acid antibody was used to detect competent hybrid formation between PCR products and normal (N) and mutant (M) cDNA (deoxyribonucleic acid) probes coated to microtiter plate wells. Under standard conditions, mean relative color production (N/M) via an enzyme-linked horseradish peroxidase secondary antibody was 8.8, 0.6 and 0.04 for individuals normal, heterozygous and homozygous for the CF delta F508 mutation, respectively (n = 27). Comparison of EIA results to those obtained by tris-borate-EDTA/8 percent polyacrylamide gel electrophoresis (TBE-PAGE) yielded excellent correlation (100 percent) for all three genotypes (n = 27). In comparison to TBE-PAGE, the EIA was 5 to 10 fold more sensitive when serially diluted PCR samples were evaluated. Larger PCR products (491 bp amplicons) for the CF delta F508 mutation obtained under modified conditions were not resolved by TBE-PAGE. The EIA, however, demonstrated equal sensitivity to the 98 bp and 491 bp amplicons. Performance time for TBE-PAGE analysis was substantially shorter (25 percent) than the EIA (3.5 to 4 h and 4.5 to 5 h, respectively) when small batches of samples (n = 5) were analyzed. The TBE-PAGE was not, however, convenient for screening large numbers of PCR-amplified samples (n > 15).

Intraarticular carcinogenesis bioassays of CoCrMo and TiAlV alloys in rats.

Wear-debris powders of cobalt-chromium-molybdenum (CoCrMo) and titanium-aluminum-vanadium (TiAlV) alloys, which are widely used for orthopedic implants (eg, hip and knee prostheses), were tested for carcinogenic activity following intraarticular administration (20 mg/rat) to groups of 44 male Fischer-344 rats (Charles River Breeding Laboratories, North Wilmington, MA). Control groups received similar intraarticular injections of either a noncarcinogen (manganese powder, negative control rats) or a potent carcinogen (nickel subsulfide powder, positive control rats). The experimental groups of 8-12 rats were observed for 24 months after injection. No local tumors developed at the injection site in the negative control rats or in rats that received the CoCrMo or TiAlV powders; poorly differentiated or pleomorphic sarcomas developed at the injection site in 10 of the 12 positive control rats that were treated with nickel subsulfide. Incidences of primary tumors distant from the injection site did not differ significantly among the experimental groups. This study shows that, under experimental conditions, any carcinogenic activity of CoCrMo or TiAlV wear-debris powders is weak in comparison to nickel subsulfide. Based on this study and observations in other laboratories, intraarticular administration of test materials to rats provides a practical, reliable, and biologically relevant method for carcinogenesis testing of biomaterials used for orthopedic implants.

In vitro characteristics of white cell-reduced single-unit platelet concentrates stored in syringes.

BACKGROUND: Platelet concentrates (PCs) for premature infants may be subjected to filtration, centrifugation, and various storage conditions before transfusion. STUDY DESIGN AND METHODS: As there are few data on the cumulative effect of these procedures on PCs, platelet properties (including biochemical and functional in vitro assays) were evaluated after the processing of single units of PCs through a 1-unit-capacity high-efficiency white cell (WBC)-reduction filter followed by syringe storage at either 22 or 37 degrees C for 6 hours. Two- and 5-day-old PCs, volume-reduced PCs, and prestorage WBC-reduced PCs were evaluated. RESULTS: WBC filtration consistently resulted in a 3 to 4 log10 reduction in WBCs, with less than 15-percent platelet loss. No adverse effects of platelet function or evidence of increased platelet activation as determined by the percentage of P-selectin positivity were noted. A decrease in pH associated with increased lactate production and consumption of glucose was observed following syringe storage under all conditions tested. Such changes were most pronounced, however, with volume-reduced PCs stored at 37 degrees C (pH 6.31 +/- 0.15, lactate 23.0 +/- 3.06 mmol/L). All pH levels at the end of storage were above the minimum Food and Drug Administration requirement (pH 6.0). CONCLUSION: The in vitro data suggest that single units of PCs can undergo WBC filtration followed by syringe storage for up to 6 hours and still maintain acceptable storage characteristics. The practice of maintaining volume-reduced PCs in syringes for 6 hours at 37 degrees C in isolettes during transfusion should, however, be avoided.

Malformations persist after metamorphosis of Xenopus laevis tadpoles exposed to Ni2+, Co2+, or Cd2+ in FETAX assays.

This study was performed to determine whether malformations induced in Xenopus laevis embryos by exposures to divalent nickel, cobalt, or cadmium chlorides in FETAX assays persist after the tadpoles undergo metamorphosis to juvenile frogs. Embryos were exposed for four days to EC50 concentrations of Ni2+, Co2+, or Cd2+ under the standard conditions of FETAX assays; thereafter, the exposures were discontinued and the tadpoles were kept in aquaria through metamorphosis. Controls were treated similarly, without exposure to metals. At 13 weeks of age, surviving frogs were killed and examined for malformations. Control and metal-exposed groups of Xenopus did not differ significantly in their median ages at metamorphosis, mean body weights, or survival at 13 weeks. Overall incidences of malformations found in Ni(2+)-, Co(2+)-, or Cd(2+)-exposed frogs at 13 weeks of age were 55, 40, and 51%, respectively (P < 0.01 vs. 3% in controls). The malformations of metal-exposed frogs included retinal depigmentation, diastematomyelia, scoliosis, kyphosis, phocomelia, sacro-pelvic and hind-limb deformities, and dysplasia of the heart, kidney, ovary and gut.

Ocular malformations of Xenopus laevis exposed to nickel during embryogenesis.

The pathogenesis of eye anomalies induced by exposure to Ni2+ (as nickel chloride) during embryogenesis was studied in the frog, Xenopus laevis. Eyes of control and Ni(2+)-exposed tadpoles were examined without staining using a dissecting microscope, by light microscopy of histological sections, and by electron microscopy. The ocular abnormalities of Ni(2+)-exposed tadpoles included (a) microphthalmia, (b) hypopigmentation, (c) hernias and cysts of the choroid and retina, and (d) iris coloboma; cataracts were uncommon. The pathogenesis of the ocular lesions appears to involve diffuse or focal dysplasia and loss of the retinal pigment epithelium, with dystrophy of photoreceptor outer segments and protrusion of neuroretina through gaps in the pigment epithelium. This study confirms that Ni2+ is a potent ocular teratogen for Xenopus embryos and points to the retinal pigment epithelium as a primary cellular target for Ni(2+)-induced embryotoxicity.

Mg(2+)-deprivation enhances and Mg(2+)-supplementation diminishes the embryotoxic and teratogenic effects of Ni2+, Co2+, Zn2+, and Cd2+ for frog embryos in the FETAX assay.

The influence of Mg2+ on the embryotoxicity and teratogenicity of Ni2+, Co2+, Zn2+, and Cd2+ for Xenopus embryos was studied by an adaptation of the FETAX protocol. In seven assays, 25 groups of embryos were grown from 5 to 101 hours post-fertilization in FETAX media that contained five graded MgCl2 concentrations (0, 6.2, 62, 620, or 6,200 mumol per L), with or without added NiCl2 (56 mumol per L), CoCl2 (1,800 mumol per L), ZnCl2 (300 mumol per L), or CdCl2 (18 mumol per L). In FETAX assays performed with the standard Mg2+ concentration (620 mumol per L), the incidence of malformations in control embryos averaged 5.4 (SD +/- 1.3) percent; the incidence of malformations in the controls was increased at low Mg2+ concentrations (32 +/- 7 percent at 62 mumol per L; 100 percent at greater than or equal to 6.2 mumol per L). The specified additions of Ni2+, Co2+, Zn2+, or Cd2+ caused death in < 10 under standard conditions (620 mumol Mg2+ per L). Mg(2+)-deprivation greatly enhanced and Mg(2+)-supplementation significantly reduced the incidence and severity of the teratogenic and embryotoxic effects of Ni2+, Co2+, Zn2+, and Cd2+ (p < 0.0001 by analysis of variance [ANOVA]). To explain these findings, the authors speculate that Mg2+ competes with the other divalent metal ions for a carrier mechanism involved in metal absorption or cellular uptake, or for binding to critical molecular targets.

Embryotoxicity and teratogenicity of Cu2+ and Zn2+ for Xenopus laevis, assayed by the FETAX procedure.

Cupric chloride (CuCl2) and zinc chloride (ZnCl2) were tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. The median teratogenic concentrations (EC50) of Cu2+ and Zn2+ were 2.5 and 40 mumol per L. The median embryolethal concentrations (LC50) of Cu2+ and Zn2+ were 22 and 850 mumol per L. The teratogenic indices (TI = LC50/EC50) were 8.8 for Cu2+ and 21 for Zn2+. Both metal ions were shown to be potent teratogens for Xenopus, causing concentration-related increases of eye, gut, facial, notochord, and cardiac anomalies.

pNiXa, a Ni(2+)-binding protein in Xenopus oocytes and embryos, shows identity to Ep45, an estrogen-regulated hepatic serpin.

A Ni(2+)-binding protein (pNiXa, 45 kD, pI 8.5) discovered in Xenopus embryos, was isolated from oocytes. Based on amino acid sequences, pNiXa belongs to the serpin superfamily and shows identity to the cDNA sequence of Ep45, an estrogen-regulated hepatic serpin that contains an (HX)n-motif found in eukaryotic transcription factors. Nondenatured pNiXa, purified by Ni-affinity chromatography, inhibited bovine alpha-chymotrypsin. The presence of pNiXa in embryos when they are susceptible to Ni2+, the high avidity of pNiXa for Ni2+, and the (HX)n-motif point to pNiXa as a molecular target of Ni(2+)-teratogenesis.

Teratogenicity of cadmium chloride in the South African frog, Xenopus laevis.

The teratogenicity of cadmium chloride was tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure. In five assays, groups of Xenopus embryos were grown in media containing concentrations of 0.75-56 mumol/l; controls were incubated in medium without cadmium chloride. Exposures began 5 h post-fertilization and ended 101 h post-fertilization. In control groups, > 95% of embryos survived at 101 h and the incidence of malformations was < 7%. In Cd(2+)-exposed groups, concentration-dependent mortality and numerous malformations were observed, including gut malrotation, ocular anomalies, bent notochord, misshapen fin, facial dysplasia, cardiac deformities and dermal blisters. Other abnormalities included stunted growth and hypopigmentation. The minimum concentration of cadmium chloride that inhibited growth was 18 mumol/l. The median embryolethal concentration (LC50) was 32 (SE +/- 4) mumol/l; the median teratogenic concentration (EC50) was 3.7 (SE +/- 1) mumol/l; the teratogenic index (TI = LC50/EC50) was 8.6. This study demonstrates that cadmium chloride is teratogenic for Xenopus laevis and provides a standardized experimental model for studying the molecular mechanisms of cadmium teratogenesis.

Embryotoxicity and teratogenicity of cadmium chloride in Xenopus laevis, assayed by the FETAX procedure.

The embryotoxicity and teratogenicity of cadmium chloride (CdCl2) were tested by the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure in the South African frog, Xenopus laevis. In five assays, groups of Xenopus embryos were grown in media that contained CdCl2 at concentrations ranging from 0.75 to 56 mumol per L; control groups were incubated in the same medium without added CdCl2. The exposures to CdCl2 were begun at the blastula stage (five hours post-fertilization) and were terminated 96 hours later (101 hours post-fertilization). The embryos were counted, fixed in formalin, and examined by microscopy to score malformations and measure head-to-tail lengths. In control groups, greater than or equal to 95 percent of the embryos survived at 101 hours post-fertilization, and the incidence of malformations was less than or equal to 7 percent. In Cd(2+)-exposed groups, there was concentration-dependent mortality, and the embryos showed a concentration-related pattern of malformations, including gut malrotation, ocular anomalies, bent notochord, misshapen dorsal fin, facial dysplasia, cardiac deformities, and dermal blisters. Other abnormalities, not categorized as malformations, included stunted growth and hypopigmentation. The minimum concentration of CdCl2 that inhibited growth (MCIG) was 18 mumol per L. The median embryolethal concentration (LC50) of CdCl2 was 32 (SE +/- 4) mumol per L; the median teratogenic concentration (EC50) was 3.7 (SE +/- 1) mumol per L; the teratogenic index (TI = LC50/EC50) was 8.6. This study demonstrates that CdCl2 is teratogenic for Xenopus laevis and provides a standardized experimental model for studies of the molecular mechanisms of cadmium teratogenesis.