Prenatal diagnosis of hemoglobin Bart's hydrops fetalis by HPLC analysis of hemoglobin in fetal blood samples.

Since HbF and HbA are not found in fetuses with Hb Bart's hydrops fetalis the feasibility of prenatal diagnosis of homozygous alpha-thalassemia 1 by fetal hemoglobin typing was examined. Blood samples were obtained from fetuses at 18 to 22 weeks of gestation by cordocentesis in 32 pregnant women at risk of having a child with homozygous alpha-thalassemia 1 (alpha-thal-1). The samples were analyzed by a PCR-based method for the diagnosis of alpha-thal-1 (SEA type) and the proportion of hemoglobin fractions were determined by automated HPLC. DNA analysis showed that 8 of the 32 fetuses were homozygotes for alpha-thal-1, 17 were heterozygous for alpha-thal-1 (alpha-thal-1 trait), and a normal complement of four a-globin genes was found in 7 cases. The Hb typing in fetuses with homozygous alpha-thal-1 showed a peak of unbound Hb (Hb Bart's and Hb Portland) and no HbF, HbA and HbA The alpha-thal-1 trait chromatograms showed unbound Hb, pre HbF, HbF and HbA peaks. The chromatogram of normal fetuses showed HbF and HbA peaks without HbA2. In these cases the HbA proportion is between 3% and 10% with no apparent differences between the 18h and 22nd week of gestation. As the analysis of fetal Hb types by HPLC is facile and speedy and the results correspond with those obtained by DNA analysis, fetal Hb typing by automated HPLC is a convenient prenatal diagnostic method for homozygous alpha-thal-1. The method is recommended for prenatal diagnosis in populations with a high frequency of alpha-thal-1.

Prenatal diagnosis of beta-thalassemia major by high-performance liquid chromatography analysis of hemoglobins in fetal blood samples.

In Thailand and adjacent countries, most of the beta-thalassemia genes are beta(0)-thalassemia mutations that prevent the production of Hb A. We propose the quantitation of the Hb A fraction in fetal blood in the mid-trimester of pregnancy by automated high performance liquid chromatography as a reasonable prenatal diagnostic method to be applied in areas with limited laboratory facilities. Forty pregnant women at risk of delivering a child with beta-thalassemia major were identified using an erythrocyte osmotic fragility test and quantitation of Hb A2. Cordocentesis was performed at the gestational age of 18-22 weeks and fetal blood was analyzed for hemoglobin fractions by automated high performance liquid chromatography. The beta-globin gene mutations were characterized by beta-globin gene sequencing. The 4 bp deletion at codons 41/42 (-TTCT) was the most frequent of the 40 beta-thalassemia mutations observed (20/40 = 50%), followed by the splice site mutation IVS-I-1 (G-->T) (7/40 = 17.5%), the nonsense mutation at codon 17 (A-->T) (7/40 = 17.5%), the nonsense mutation at codon 35 (C-->A) (3/40 = 7.5%), and the beta(+)-thalassemia promoter mutation at -28 (A-->G) (3/40 = 7.5%). High performance liquid chromatography revealed nine fetuses which had only Hb F and no Hb A. All were homozygotes or compound heterozygotes for beta(0)-thalassemia mutations. In the remaining 31 fetuses, a Hb A peak was present in the chromatograms. One fetus with 0.5% Hb A was a compound heterozygote for the -28 (A-->G) and codons 41/42 (-TTCT) mutations. In the remaining 30 fetuses, the Hb A values ranged between 0.8 and 7.4%. Twenty of these, with a Hb A concentration of 1.82 +/- 0.49% (range 0.8-2.8%), were beta-thalassemia heterozygotes. The remaining 10 fetuses had Hb A values of 4.89 +/- 1.47% (range 2.9-7.4%) and normal beta-globin genes. The absence of Hb A in homozygotes or compound heterozygotes for beta(0)-thalassemia mutations and the presence of measurable amounts of Hb A in heterozygotes and normal homozygotes, permits the diagnosis of fetuses expected to develop postnatal beta-thalassemia major.

Prenatal eradication of Hb Bart's hydrops fetalis.

OBJECTIVE: To evaluate the effectiveness of prenatal prevention of Hb Bart's hydrops fetalis. STUDY DESIGN: The study was a prospective descriptive analysis of pregnant women attending an antenatal clinic between June 1990 and June 1998. The study consisted of two periods, the first half with no prenatal diagnosis (PND) (1990-1994) and the second half with PND. During the study period, all cases of Hb Bart's hydrops fetalis were prospectively collected and postnatally confirmed. In the second period, prenatal strategy to control severe thalassemia was introduced. The strategy included (1) carrier identification by retrospective (history review for known risk) and prospective screening (simple erythrocyte osmotic fragility test) in women without known risks, (2) the couples at risk were offered genetic counseling and cordocentesis, (3) analysis of fetal blood for diagnosis, and (4) counseling for termination of pregnancy. RESULTS: During the first half of the study, the prevalence of Hb Bart's hydrops fetalis was 0.305 (89 in 29,399 deliveries). There were no fetuses with Hb Bart's hydrops fetalis among 16,360 screened pregnancies in the second half. However, of 6,856 pregnancies in the second half not screened due to a late first visit, 10 (0.15%) fetuses had Hb Bart's hydrops fetalis. Among the screened group, cordocentesis was performed in 361 pregnancies at risk, 170 and 191 from retrospective and prospective screening, respectively; and 75 (20.8%) were proven to have Hb Bart's disease, which was diagnosed before hydropic changes occurred. CONCLUSION: The strategy proved effective in preventing Hb Bart's hydrops fetalis, and extensive experience with it suggests that it be considered an effective way to control severe thalassemia.

The distribution of D1S80 and VWA alleles in a Karen population from Northern Thailand.

The DIS80 and VWA loci were studied in a Karen population from Northern Thailand by the polymerase chain reaction and polyacrylamide gel electrophoresis. Twelve DIS80 and six VWA alleles were found. No deviations from the Hardy-Weinberg and linkage equilibrium were observed. The power of exclusion (PE) from the analysis of the DIS80 and VWA locus is 0.67 and 0.45, respectively, the power of discrimination (PD) is 0.95 and 0.85, respectively, with a combined PD of 0.99 and PE of 0.82.

Screening for alpha-thalassemia-1 heterozygotes in expecting couples by the combination of a simple erythrocyte osmotic fragility test and a PCR-based method.

OBJECTIVE: To develop a simple method for the prospective identification of couples at risk of homozygous alpha-thalassemia-1 (Hb Bart's hydrops fetalis) in pregnancy. METHODS: Antenatal care (ANC) women and their husbands were analyzed using a simple erythrocyte osmotic fragility (EOF) test and a PCR-based method for the detection of the mutation leading to alpha-thalassemia-1 of the Southeast Asian type (SEA). RESULTS: Heterozygosity for the alpha-thalassemia-1 (SEA) deletion was found to correlate with an EOF value <60%. For a prospective screening, ANC women and their husbands are analyzed with the EOF test and only those having a value <60% were further checked by PCR. Of 2,769 cases analyzed during a 6-month period, 24 couples in which both partners are heterozygotes could be identified for genetic counseling and prenatal diagnosis. The application of the EOF test decreased the workload for PCR by approximately 80%. CONCLUSION: Prospective screening for alpha-thalassemia-1 (SEA) heterozygotes in northern Thailand is becoming easier to realize by the combination of EOF test and PCR.

An investigation of the TH01 locus in a population from northern Thailand.

The STR locus HUMTH01 was studied in 110 unrelated Thais from the area of Chiang Mai in North Thailand. By using PCR and vertical PAGE, six alleles were identified and the frequencies ranged from 0.005 to 0.400. The allele frequency distribution in this population showed significant differences from a Japanese population and other ethnic populations but was similar to Asians in the USA and Australia. The genotype distribution meets Hardy-Weinberg expectations. The average power of exclusion (in no-parent and one-parent cases) and the discriminating power (DP) were calculated to be 0.3020, 0.4761 and 0.8722 respectively.

Frequency distribution of D1S80 alleles in the northern Thai population analyzed by amplified fragment length polymorphism technique.

A population study on the D1S80 locus in 100 northern Thais was performed using polymerase chain reaction (PCR) and high resolution polyacrylamide gel electrophoresis. Sixteen alleles and 39 genotypes were observed with a heterozygosity of 82%. In contrast to other studies, the allele with 31 repeat units was found to be the most common, followed by alleles 24 and 18. Alleles with more than 41 repeat units were also observed in this study. When applying statistical tests for Hardy-Weinberg equilibrium, no significant deviations were found in this Thai population. The average power of exclusion (in no-parent and one-parent cases) and the discriminating power (DP) was calculated to be 0.52, 0.69 and 0.94, respectively.

Frequency of alpha-thalassemia-1 of the Southeast Asian-type among pregnant women in northern Thailand determined by PCR technique.

Five hundred pregnant women were analyzed for the presence of alpha-thalassemia-1 of the Southeast Asian (SEA)-type by polymerase chain reaction (PCR) technique at the Maharaj Nakhon Chiang Mai University Hospital in Chiang Mai during the period from April to June 1995. Forty-four of them (8.8%) were recognized as carriers, corresponding to a frequency of 0.044. Homozygous alpha-thalassemia-1 of the SEA-type, the fatal condition of hemoglobin Bart's hydrops fetalis, has an expected frequency of 0.00194, or about 2 hydrops fetalis cases per 1,000 births in this population.

Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae.

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.

Import of ADP/ATP carrier into mitochondria: two receptors act in parallel.

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.

Import pathways of precursor proteins into mitochondria: multiple receptor sites are followed by a common membrane insertion site.

The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1-complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.

Validation of accuracy by interlaboratory programme.

A statistical design is proposed for assessing the accuracy of an analytical method by its application to a certified reference material in an interlaboratory programme. The validation of accuracy is based on the difference between the certified value and the overall mean of the test programme and is linked to the concept that below a certain limit this difference has no practical significance. It is shown that a certified reference material cannot be used to detect bias in a method if the bias is smaller than the confidence interval of the certified value.

The use of certified reference materials in the verification of analytical data and methods.

An experimental design is proposed for the verification of the accuracy and precision of an analytical method by its application to certified reference materials.

Effect of EDTA/NaF solutions on the orion Cu(II) ion-selective electrode.

The titration of ethylenediaminetetra-acetic acid in fluoride medium with Cu(II) solution with the Orion Cu(II) ion-selective electrode as indicator, results (after several titrations) in a broad end-point which is unsatisfactory for exact analytical purposes. This effect has been found to arise from an enhanced rate of response to changes in EDTA concentration when the electrode has been exposed to an EDTA/NaF medium for prolonged periods.

Titrimetric determination of aluminium in zinc-aluminium alloys, with edta and a cu(II)-selective electrode.

The end-point for the titration of EDTA with Cu(II), as measured by a Cu(II)-selective electrode, varies with pH and temperature. Moreover, the effect of pH and temperature on the behaviour of this electrode differs according to whether fluoride is present. As a consequence, the determination of aluminium in zinc-aluminium alloys by the Freegarde and Allen method with use of a Cu(II)-selective electrode must be performed with close control of pH and temperature to maximize accuracy and repeatability.

Methodological information from the certification of CCRMP ores and concentrates.

In the course of 90 certifications for 27 elements in 26 reference ores and concentrates, carried out by the Canadian Certified Reference Materials Project, much methodological information has been documented and is now made available to analysts for the selection of suitable methods for the analysis of specific materials. Information is presented for copper, gold, lead, silver, sodium, potassium, tin, tungsten, uranium and zinc. A relationship between the average coefficient of variation and element concentration makes it possible to make some generalizations about the precision to be expected for a given concentration of an element in ores and concentrates.

Oxidation of sulphide minerals-VI Ferrous and ferric iron in the water-soluble oxidation products of iron sulphide minerals.

A pseudo-kinetic method has been developed for determining the ferrous and ferric iron in the water-soluble oxidation products of pyrrhotite, pyrite and chalcopyrite, and ores and concentrates containing them. Two determinations are required for each material. In one, the total iron is determined with 1,10-phenanthroline after reduction to Fe(II). In the other, the reduction of Fe(III) is retarded by complexation with fluoride. The difference in the amount of ferrous phenanthranoline complex produced in these two determinations is a function of the original FE(III) concentration and of time.

A case study of the ambient oxidation of two zinc-lead (sulphide) reference ores.

The history of two lead-zinc (sulphide) reference ores is presented to show quantitatively the serious effects of ambient oxidation on unprotected samples. This study should serve as a warning to the users and producers of sulphide-bearing reference ores and concentrates. Suggestions are given for overcoming or diminishing the oxidation problem.

The drying of sulphide-bearing materials in a microwave oven: A caveat.

The drying of sulphide-bearing materials in a microwave oven should be undertaken with caution. Several sulphide minerals and sulphide-bearing materials have shown a susceptibility to oxidation (which may even be violent) during microwave drying and this could affect the results of subsequent work on these materials.

Oxidation of sulphide minerals-III determination of sulphate and thiosulphate in oxidised sulphide minerals.

A method has been developed to determine sulphate and thiosulphate in small amounts of the oxidation products of sulphide minerals. The sample is treated with ammonium sulphide solution to promote ion-exchange between sulphide ion and the sulphur-bearing anions of the oxidation products. Sulphate is determined alone and then all other sulphoxy anions are oxidized to sulphate and determined as such. The non-sulphate anions are reported as thiosulphate. The relative error is about 10% or less for 2 mg or more of sulphoxy anion. Although this method does not yield exact results with respect to sulphite or polythionates, a clearer understanding of the oxidation of sulphide minerals is now available.