Fetal RHD detection from circulating cell-free fetal DNA in maternal plasma: validation of a diagnostic kit using automatic extraction and frozen DNA.

OBJECTIVE: This study aimed to validate non-invasive RHD genotyping of cell-free fetal DNA (cff-DNA) using different DNA extraction methods and of fresh and frozen extracted cff-DNA. BACKGROUND: Non-invasive RHD genotyping of cff-DNA predicts fetal RhD phenotype, allowing for the rational implementation of antenatal immunoprophylaxis and representing a big step forward in the management of RhD-immunised women. Validation of a diagnostic method is mandatory before its clinical application. METHODS: RhD-negative pregnant women were recruited at different gestational ages. The cff-DNA extraction was carried out using manual and automatic methods in order to improve cff-DNA yield and optimise the extraction. Fetal RHD genotyping was performed using a commercial real-time polymerase chain reaction (PCR) kit, and the results were compared with postnatal serological RhD determination on cord blood. RESULTS: Overall, 133 plasma samples were examined for the validation process, and a total of 423 tests were performed. No differences have been observed between the two extraction methods or between fresh or frozen cff-DNA regarding cff-DNA stability and quality parameters. There was 100% concordance between fetal RHD genotyping of cff-DNA and RhD phenotype on cord blood for both extraction methods on both fresh and frozen cff-DNA. CONCLUSION: Our study shows the reliability of automatic and manual cff-DNA extraction methods and the possibility of freezing extracted cff-DNA when performing RHD genotyping. This result might be relevant for improving laboratory work and organisation through the development of a standardised procedure for fetal RHD genotyping on cff-DNA, laying the foundations for evidence-based use of anti-D Ig prophylaxis in RhD pregnant women.

Abnormal arachidonic acid content of red blood cell membranes and main lithogenic factors in stone formers.

BACKGROUND: Increased arachidonic acid content in red blood cell membranes of stone formers (SF) has recently been reported and is hypothesized as representing the underlying causal factor for both hyperoxaluria and hypercalciuria. We performed the present study to see whether we could confirm this finding and to test whether any relationship exists between the fatty acid composition of red blood cell membranes and the main metabolic factors involved in stone formation. METHODS: In 21 SF and 40 healthy controls subjects the fatty acid composition of red blood cell membranes was assessed. In addition, the following parameters were evaluated in SF: daily and fasting urinary calcium excretion, fractional intestinal calcium absorption, 1,25-dihydroxy-vitamin D, intact parathyroid hormone, hydroxyproline in fasting urine, daily urinary excretion of oxalate, citrate, urate, electrolytes, urea, sulphate, relative supersaturation for calcium oxalate monohydrate. RESULTS: The red blood cell membrane of SF had a lower content of arachidonic acid, linoleic acid, and docosahexaenoic acid than that of control subjects. Arachidonic acid content was not correlated with any of the parameters studied. However, when patients were grouped according to the degree of oxalate excretion, hyperoxaluric SF had a higher arachidonic acid content and arachidonic/linoleic acid ratio than SF with normal oxalate excretion. CONCLUSIONS: Our results do not confirm the finding of an increased arachidonic acid content of red blood cell membrane in SF. On the contrary, reduced arachidonic acid levels were found in our patients. However, hyperoxaluric SF had a relatively higher arachidonic acid content than SF with normal urinary oxalate excretion.

Severe energy impairment consequent to inactivation of mitochondrial ATP synthase as an early event in cell death: a mechanism for the selective sensitivity to H2O2 of differentiating erythroleukemia cells.

Irreversible damage to Friend's erythroleukemia cells was caused by induction of endogenous heme biosynthesis with the differentiating agent N,N'-hexamethylene bisacetamide followed by a 30-min exposure to 0.25 mM H2O2. Early irreversible ATP depletion was observed concomitant with oxidative inactivation of the mitochondrial ATP synthase. Cell proliferative capacity was also impaired within 2 h of the treatment, and progressive delayed cell lethality, starting 2 h after the insults, was also found. Based on the prevention provided by specific antioxidants and on the absence of malodialdehyde production, all the effects were ascribed to the oxidant action of .OH radicals, or closely related species, generated through iron-catalyzed reactions of H2O2, which apparently caused site-directed oxidative modifications of iron-binding proteins, in particular mitochondrial ATP synthase, rather than peroxidation of membrane lipids. Similar effects were mimicked even in the parental cell line when oligomycin was used to inhibit selectively mitochondrial ATP synthase activity, thereby lowering the enzyme activity to a level similar to that found in H2O2-damaged differentiating cells. Hence, induction of erythroid differentiation makes the mitochondrial ATP synthase a major target of H2O2 by enhancing the availability of redox-active iron in the local environment of the enzyme. Subsequent oxidative inactivation of the mitochondrial ATP synthase, resulting in severe energy impairment, leads to loss of cell growth capacity. Erythroleukemia cells may serve as a model system for the combination of two selective properties: (1) the capacity for carrying out efficient heme synthesis and/or for undergoing iron overload-like state; and (2) subsequent enhanced sensitivity to reactive oxygen species generators. Early severe mitochondrial dysfunction and energy impairment may be a major part of the mechanism of the sensitivity.

New micromethod for the determination of lamotrigine in human plasma by high-performance liquid chromatography.

An extraction procedure and reversed-phase high-performance liquid chromatographic assay is described and validated for the determination of lamotrigine in human plasma. The method involves extraction with chloroform-isopropanol after alkalinization with a carbonate buffer, back-extraction into 0.05% phosphoric acid and separation by reversed-phase HPLC using a 5-micron Supelco diphenyl column (150 x 4.6 mm I.D.). Quantitation was performed by measurement of the UV absorbance at a wavelength of 265 nm. This method was carried out on 50-200 microliters samples of plasma, depending on whether they were pediatric or adult samples. The lower limit of quantitation was 0.2 microgram/ml using 200 microliters of plasma. A linear response was tested from 0.5 to 20 micrograms/ml. Within- and between-day accuracy and precision were always below 10.0% at all analysed concentrations. The method selectivity towards the most used antiepileptic drugs has been proven. Satisfactory performances were obtained in the evaluation of samples from epileptic patients.

Automated high-performance liquid chromatographic separation with spectrofluorometric detection of a malondialdehyde-thiobarbituric acid adduct in plasma.

An automated HPLC separation method for the detection of malondialdehyde (MDA) in plasma samples of a healthy population is described. The procedure involves a first step of acidic hydrolysis and complex formation with thiobarbituric acid (TBA), then a protein precipitation step, and finally the separation of TBA-MDA adducts through a complete HPLC apparatus with spectrofluorometric quantification. This procedure is very useful for a routine laboratory because of its rapidity (tR = 1.7 min), simplicity and applicability, and the method gives very good results with respect to linearity (0.1-12 mumol/l), precision (within-assay C.V.%: 8, between-assay C.V.%: 10) and accuracy (recovery average: 91.66%).

Longitudinal evaluation of prostate-specific antigen levels in a case-control study.

BACKGROUND: Over the last period--late 1970 to early 1990--the incidence of prostate carcinoma has nearly doubled, even though many more patients die with prostate cancer rather than of it. This finding, together with the slow growth of this tumor and the absence of a controlled trial, makes early diagnosis for this pathology quite questionable. On the other hand, it is well known that prostatic carcinoma is curable as long as it is intracapsular and that there is an ever increasing encouragement for early detection in all diseases. Since prostatic pathology increases its incidence as age advances, the first step in early diagnosis is to be able to discriminate between healthy, benign prostatic hyperplasia and cancer cases with a well-accepted and easily understandable method. The problem is to find the best method to do it. METHODS: We measured serum prostate-specific antigen (PSA) levels in 435 men participating in an epidemiological study, at first in 1987 and again in 1992. Men with PSA levels above 4 ng/ml (in 1992) were invited to undergo other diagnostic tests (digital rectal examination, transrectal ultrasonography, biopsy) in sequence on the basis of the results of the previous tests. The pathological findings from biopsies were the reference test to determine the presence or absence of prostate cancer (2.5% of the population). RESULTS: We divided PSA concentrations into three categories, according to the most used cutoffs ( < 4, 4-10, > 10 ng/ml); in the meantime, we took into account the change rate in PSA concentration in time, defined as delta PSA. By the comparison between PSA categories and delta PSA, we found out that the first one does not discriminate between benign and malignant pathologies, while the use of delta PSA strongly discriminates them (p < 0.001). CONCLUSION: On the basis of our results, we think that delta PSA might be the best parameter to indicate the presence of prostate cancer cases in an asymptomatic population.

Is serum PSA measurement a good test for early diagnosis of prostate cancer? Results of a pilot study and cost analysis.

Over the last years--late 1970s to early 1990s--the incidence of prostate carcinoma has nearly doubled, even though many more patients die suffering from prostate cancer than because of it. This finding, together with the slow growth of this tumor and the absence of a controlled trial that would suggest a benefit from screening, makes early diagnosis of this disease quite questionable. On the other hand, it is well known that prostatic carcinoma is curable as long as it is intracapsular, and that there is an ever increasing encouragement to early detection in all diseases. The costs of screening and the difficulty in balancing the benefits of screening against its negative effects, such as psychological impact and overtreatment, must be taken into account as well. In our opinion, one of the advantages of early diagnosis should be that the patients' quality of life improves, because the stage at diagnosis and, as a result, the number of patients suffering from bone metastasis decrease, and unknown benign pathologies can be cured. These observations are not at all negligible. Our study aims to demonstrate that by using PSA as an initial test, the screening costs are reasonable and the disease incidence is just as expected.

H2O2-induced damage to beef heart mitochondria F0F1 ATP synthase complex: differential sensitivity of the F1 and F0 moieties.

Exposure of purified mitochondrial F0F1 ATP synthase to H2O2 resulted in a marked inhibition of the ATPase activity, irrespective of the purification procedure used and of the incorporation of the enzyme complex into phospholipid vesicles. The inactivation appeared consequent to oxidative modifications of the F1 moiety, whereas damage to the F0 sector, leading to low enzyme activity through impaired binding with F1, seemed not to occur. In fact, when H2O2-inactivated complex was deprived of F1, no loss of the capacity of the F0 sector thus obtained to properly reassemble with untreated purified F1 was apparent, because the resulting enzyme complex showed full activity and oligomycin sensitivity. On the contrary, the exposure of the isolated components F1 or F0 to H2O2, followed by reassembly with untreated F0 and F1 respectively, resulted in both cases in lower catalytic activity of the reconstituted complexes, whereas low oligomycin sensitivity was exhibited only in the case of F0 treatment, suggesting the inactivation in this case as due to oxidative modifications leading to impaired binding with F1.