Nonradioactive quantification of low concentrations of hemoglobin A by HPLC for midtrimester prenatal diagnosis of beta-thalassemia.

The usual methods for prenatal diagnosis of beta-thalassemia and other hemoglobinopathies by assay of fetal blood erythrocytes are either complex (analysis of globin chains synthesis by carboxymethylcellulose chromatography) or only semiquantitative [isoelectric focusing of hemoglobin (Hb)]. To further simplify the diagnostic procedure and to obtain quantitative data, we measured the small concentrations of Hb A in fetal erythrocytes by using a high-pressure liquid chromatography (HPLC) instrument (DIAMAT-TM; Bio-Rad) equipped with the new column proposed for measuring Hb A2. We analyzed 212 uncontaminated fetal blood samples obtained by cordocentesis between the 18th and 22nd weeks of pregnancy, using the HPLC procedure, and compared the results with those obtained by the above-named methods.

Heterozygous beta-thalassemia: relationship between the hematological phenotype and the type of beta-thalassemia mutation.

In this study we have correlated the severity of the hematological features to the type of the beta-thalassemia mutation [codon 39 (C----T), IVS-I nt 110 (G----A), IVS-I nt 1 (G----A), IVS-I nt 6 (T----C), IVS-II nt 745 (C----G), -87 (C----G) and beta 6 (-1 bp)], in a group of beta-thalassemia heterozygotes of Italian descent in whom we excluded the presence of iron deficiency or deletion alpha-thalassemia. The beta-thalassemia mutation was defined by dot blot analysis on amplified DNA with allelic specific oligonucleotide probes. We found that a) heterozygotes for beta+ IVS-I nt 6 and beta+ -87 mutations produce larger and better hemoglobinized red blood cells, and b) heterozygotes for beta+ IVS-I nt 6 and beta+ IVS-I nt 110 mutations have a less marked increase of Hb A2 levels as compared to heterozygotes for the other mutations investigated.

IHP entrapment into human erythrocytes: comparison between hypotonic dialysis and DMSO osmotic pulse.

Three different blood units were treated separately by the hypotonic dialysis (HD) and the dimethylsulphoxide osmotic pulse (DMSO) method, in order to load the erythrocytes with inositol hexaphosphate. A detailed comparison between the two loading techniques was performed by monitoring the red cell distribution patterns on discontinuous Percoll density gradients, the RBC oxygen affinity and the amount of the main intracellular organic phosphates with the 31P-NMR. The results obtained showed that: (1) The HD loading produces a redistribution of the RBC fractions with a concomitant smoothing of the relative differences among distinct fractions (2) only a minor portion of erythrocytes (from 8.

Clinical utility of fractionating erythrocytes into "Percoll" density gradients.

Two rapid methods for fractionating the RBC into five or nine layers of increasing density are reported. These procedures have been used to monitor the decline of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activity during the process of red cell aging in normal subjects and in beta-thal carriers, to study transfused patients with G6PD and pyruvate kinase (PK) deficiency and to test the effects of inositol hexaphosphate (IHP) encapsulation on RBC subpopulations.

Preliminary experience with the differential pH technique for glucose-6-phosphate dehydrogenase (G6PD) measurement in whole blood: application to an area with high prevalence of thalassaemia and G6PD deficiency.

A new differential pH technique for glucose-6-phosphate dehydrogenase quantitative determination in whole blood has been evaluated. It is a rapid (90 s/analysis), reproducible (C.V.

Whole blood L-lactate assay by a new differential pH method: application to metabolic investigations.

We propose a new quantitative method for L-lactate assay in whole blood, based on the measurement of pH variation caused by specific and irreversible oxidation of L-lactate to pyruvate in the presence of an electron acceptor (hexacyanoferrate) and of the enzyme cytochrome b2 (EC 1.1.2.

[Endogastric migration of an Angelchik prosthesis. A case report and review of the literature].

At first, the authors mention indications and way of placement of the Angelchik prosthesis. Then after a wide review of the literature, they describe the complications and results. Finally a case of penetration into the stomach of an Angelchik prosthesis is reported.

Chromatofocusing and isoelectric focusing in immobilized pH gradients compared for characterization of human hemoglobin variants.

We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb Köln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.

Double heterozygosity for hemoglobin Malmö [beta 97 (FG 4) His----Gln] and beta-thalassemia traits.

A 32-year-old Sicilian man had marked erythrocytosis (Hb = 23.0 g/dl, RBC = 10.5 x 10(12)/l, MCV = 71 fl, Hct = 84-92%, a 4.

An evaluation of the Diamat HPLC analyser for simultaneous determination of haemoglobins A(2) and F.

The authors describe a modification of the instrumental parameters of the Diamat fully automated HPLC system for Hb A(2) assay (Bio-Rad Laboratories, Milan, Italy) in order to obtain simultaneous determination of Hb A(2) and Hb F.Hb A(2) and Hb F measurements are reproducible (within-run CV 2.6%, with Hb A(2)2.

Different priming on human neutrophil respiratory burst by lipopolysaccharides from phase I and phase II Coxiella burnetii.

Human neutrophils produce small amounts of superoxide anion when stimulated with the chemotactic peptide FMLP; preincubating neutrophils with low concentrations of lipopolysaccharides (LPS) markedly increases this response, an effect referred to as priming. In this work LPS from Coxiella burnetii either phase I (virulent) or phase II (avirulent) were examined for their ability to induce priming. Results clearly show that only LPS from phase II microorganism was able to increase the release from neutrophils upon subsequent stimulation with FMLP.

Possible role of plasminogen activator content of the palmar nodules in recurrence of Dupuytren's contracture.

Increased amounts of plasminogen activator enzymes were found in the large Dupuytren's nodules in the so-called active phase of the disease. A prospective study in 15 patients who had operations investigated possible relationships between fibrinolytic capacity of the palmar nodules (assessed by the fibrin plate method) and the recurrence of contracture. There were substantial analogies and suggestive connections with the results of previous electron microscopic studies.

Urea determination in dialysis, based on a differential pH technique.

The application of a new technique, based on differential measurements of pH, to determine urea concentration in patients of a dialysis center, is reported. Urea in plasma, whole blood or dialysis fluids is measured by an enzymatic reaction, with urease; the procedure, requiring 10 microL of sample, is simple, fast and correlates well with a reference spectrophotometric method, in the 0-300 mg/dL concentration range, according to the equation y = 1.0291 X -0.

Use of chlamydiazyme for the detection of Chlamydia trachomatis in genital infections.

48 endocervical specimens from women with cervicitis and 82 urethral specimens from men with urethritis were investigated for the presence of Chlamydia trachomatis by either the cell culture method or enzyme immunoassay (Chlamydiazyme, Abbott Laboratories). The overall sensitivity and specificity of Chlamydiazyme assay were 84.6% and 95.

Glycated proteins in serum: effect of their relative proportions on their alkaline reducing activity in the fructosamine test.

The fructosamine test is considered clinically useful for assessing short-term integrated control of blood glucose, but there are few published data to support this hypothesis. We fractionated glycated and nonglycated proteins by affinity chromatography on phenylboronate columns and, with specific immunochemical methods, determined in the eluted fractions the following proteins, selected according to their biological half-lives and relative concentrations in serum: albumin, IgA, IgG, IgM, apolipoprotein B, haptoglobin, transferrin, alpha 1-antitrypsin, and alpha 2-macroglobulin. We found the following correlations between fructosamine (mmol/L) and, respectively, glycated albumin, IgG, and (albumin + IgG) (each in grams per liter): r = 0.

Ethylene glycol-stabilized haemolysates for glycated haemoglobin assay. Further analytical investigations and applications to quality control programmes.

The direct effect of the ethylene glycol on the quantitation of glycated haemoglobins by ion-exchange minicolumn chromatography, affinity chromatography and high performance liquid chromatography was investigated. No evident effect was found for ethylene glycol concentrations between 0.0 and 0.

Plasma protein glycation as measured by fructosamine assay.

The fructosamine test for assessing control of glucose in blood has been extensively evaluated, but some questions remain regarding its validity. From the analytical and clinical evaluation we present here, we conclude that: the test is sensitive to variations in the composition of the sample protein; the fructosamine reaction is almost completely unaffected by labile fractions; the concentrations of fructosamine correlate well with the degree of glycation of total serum proteins, especially with glycated albumins and glycated immunoglobulins, as determined by affinity chromatography; the correlation with glycated hemoglobin (Hb A1c), measured as the stable fraction, is very poor, in diabetics treated with insulin (r = 0.373), or with oral hypoglycemic agents (r = 0.

Adenosine induced production of a soluble factor affecting lymphocyte activation.

We show that a brief exposure of human peripheral blood mononuclear cells (PBMC) to adenosine or to theophylline results in a mitomycin C resistant regulatory activity. Adenosine induced suppression is also detectable in a lymphocyte subpopulation (T4+ enriched, originally described as helper inducer) resistant to the theophylline induced loss of capacity to form spontaneous rosettes with sheep erythrocytes (TTR). This activity is apparently dependent on the production of a soluble factor(s) since supernatants from adenosine treated TTR (SnA) exert a significant inhibition on the proliferative response of resting lymphocytes.

Measurement of erythrocyte acetylcholinesterase and plasma cholinesterase activity by a differential pH technique.

We describe a new electrochemical method for the determination of erythrocyte acetylcholinesterase activity (EC 3.1.1.