Semi-automated carbohydrate-deficient transferrin in primary biliary cirrhosis: a pilot study.

Primary biliary cirrhosis (PBC) is one of the few non-alcohol induced liver pathologies which causes false positive results in the evaluation of carbohydrate-deficient transferrin (CDT) for the diagnosis of alcohol misuse. This phenomenon has only been observed when using the CDTect assay (Pharmacia & Upjohn, Uppsala, Sweden). In this study, we evaluated CDT in female PBC patients (n = 14) by a new CDT procedure, the %CDT turbidimetric immunoassay (TIA, Axis Biochemicals, Oslo, Norway) using the isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD, Specialty Laboratories, Santa Monica, CA, USA) procedure as the gold standard. One of the PBC patients tested CDT+ by IEF/IB/LD (cut-off >9 densitometry units, DU) and %CDT TIA (cut off >6%); one patient tested at the cut-off point of the IEF/IB/LD and another one tested at the cut-off point of the %CDT TIA. Thus, unlike CDTect, the %CDT TIA is a procedure that produces few false positives in PBC.

Soluble highly coloured phenylboronic acids and their use in glycohemoglobin quantification.

Water soluble dye-phenylboronic acid conjugates (dye-PBAs) possessing strong absorption of visible light are introduced as new reagents for the determination of glycohemoglobin. Their functionality and prospective use are demonstrated in a semi-homogenous glycohemoglobin assay. The assay is based on cis-diol esterification of dye-PBA to glycohemoglobin followed by selective precipitation of hemoglobin from solution, co-precipitating bound dye-PBA. Quantification of the molar "dye-PBA/Hb"-ratio in redissolved precipitates using either absorption or fluorescence spectroscopy, reflects the glycation level of the blood samples used. Future development of the assay principle is illustrated in a filter based assay, collecting the precipitated hemoglobin on a filter followed by reflectometric readings directly on the precipitate. The significance of this work lies first, in the demonstration of a new principle for the determination of glycohemoglobin, and second, as an illustration of the prospective use of water soluble, signal-forming non-immobilised boronic acids in the determination of cis-diol containing analytes.

Semiautomated procedures for evaluation of carbohydrate-deficient transferrin in the diagnosis of alcohol abuse.

Carbohydrate-deficient transferrin (CDT) may now be the most valuable biological marker for diagnosis of alcohol abuse. We compared the diagnostic performance of two new CDT tests, Axis %CDT turbidimetric immunoassay (TIA) and Axis %CDT HPLC, against Specialty Laboratories' isoelectric focusing/immunoblotting/laser densitometry (IEF/IB/LD). Both Axis tests include one-half the concentration of trisialotransferrin isoforms in their CDT quantitation schemes. Considering an alcohol abuse prevalence of 7%, Axis %CDT TIA shows a sensitivity of 87% at 98% specificity and a positive predictive value (PPV) of 0.75; %CDT HPLC shows a sensitivity of 87% at 100% specificity for a PPV of 1, and the IEF/IB/LD shows 81% sensitivity at 94% specificity for a PPV of 0.5. All three CDT tests show the same negative predictive value (0.98). Both Axis procedures perform better than IEF/IB/LD in the diagnosis of alcohol abuse; %CDT TIA is available in several semiautomated, cost-effective formats.

Selective precipitation of human hemoglobin by organic solvents and metal cations.

The interaction of human hemoglobin with several organic solvents and metal cation has been studied in order to obtain selective precipitation of hemoglobin from solution. Alcohols, and preferably the mixture ethanol: butanol added to a final concentration of 8% (v/v) 1-butanol was found to be superior in this respect, giving close to selective precipitation of hemoglobin from whole blood lysates. An equally specific precipitation was achieved by using zinc-chloride in 10-15 molar excess to hemoglobin. Contrary to organic solvents, complex formation with Zn2+ resulted in a reversible precipitation enabling renaturation using strong chelating agents. Specificity of the hemoglobin-precipitating agents was verified by chromatographic and electrophoretic studies. Applications of the presented methods in analytical chemistry and in the isolation and purification of blood proteins are discussed.

Units, mg/l or % CDT for detection of harmful alcohol consumption?

Recent studies have demonstrated that the diagnostic performance of CDT measurements is influenced by variations in total transferrin concentration. Improved specificity is obtained with relative values, either %CDT or units CDT per total transferrin concentration. Whether relative or absolute values are used, confirmatory methods by HPLC or IEF should be available, and definition of the CDT values and correlation to HPLC or IEF should be given.

Glycohemoglobin filter assay for doctors' offices based on boronic acid affinity principle.

We present a new filter assay for the determination of glycohemoglobin as a unique application of the boronic acid affinity principle. With the use of a water-soluble blue-colored boronic acid derivative and a specific precipitation method for hemoglobin, total hemoglobin including bound boronic acid is precipitated and collected on a filter strip before quantification. Hemoglobin and boronic acid are quantified by a dual-wave-length reflectometric measurement, and the result is reported directly as percent glycohemoglobin. The test is simple, quick, and designed as a doctors' office test for the monitoring and management of diabetes. The imprecision of the assay is < 4% over the range 3-18% Hb A1c, and the method is linear up to at least 20% Hb A1c. Comparisons with four well-established glycohemoglobin methods yielded correlation coefficients ranging from 0.94 to 0.99, with slopes from 0.94 to 1.01.

Should tri-sialo-transferrins be included when calculating carbohydrate-deficient transferrin for diagnosing elevated alcohol intake?

CDT (carbohydrate-deficient transferrin) has been identified as a specific marker for chronically elevated alcohol consumption. We investigated the sensitivity and accuracy of using relative concentrations of different isotransferrins in serum for diagnosis of chronically elevated alcohol consumption. The different transferrin variants (isoforms) were quantified by HPLC. Including the trisialo-transferrin fraction into the definition of %CDT resulted in an increased accuracy in the detection of chronically elevated alcohol intake in a study among 17 heavy drinkers, 25 healthy individuals with moderate alcohol consumption and nine total abstainers. The results also suggest that desialylation of transferrin is a gradually continuing process, rather than one leading to a single end-result separating asialo-, mono- and disialo-transferrins from trisialo-, tetrasialo-, pentasialo- and higher sialo-transferrins.

Carbohydrate-deficient transferrin as a screening marker for drinking in a general hospital population.

We investigated the usefulness of the laboratory marker of alcohol consumption carbohydrate-deficient transferrin (CDT) in 101 consecutively admitted patients in a surgical and internal medical ward of a hospital in a rural wine-growing area. Four major aspects were considered: the influence of liver disease, the method of expression of CDT values (relative % vs absolute units/1), level and pattern of alcohol consumption and comparison with y-glutamyl transferase (GGT). The results show that %CDT is a more valuable discriminating marker of high alcohol consumption than absolute CDT values and its usefulness in this respect is independent of changes in serum total transferrin levels, as in liver disease. Sensitivity and specificity of % CDT were 70 and 98% respectively, compared with 65 and 83% respectively for GGT.

Carbohydrate-deficient transferrin as a marker of alcohol intake: a study with healthy subjects.

This paper reports the results of a 3-week drinking experiment in 51 healthy male subjects, examining the value of %CDT (carbohydrate-deficient transferrin) in the context of different levels of alcohol intake. All healthy persons were urine-tested drug-free and underwent daily breath alcohol tests for the 7 days preceding, and during the whole 3 weeks of, the experiment. Subjects were divided into five groups, consuming different amounts of alcohol daily over a 3-h period in the presence of the investigators. The five groups consisted of 10, 9, 10, 16 and 6 subjects respectively and consumed a daily dose of ethanol of 20, 40, 60, 80 and 80 g respectively for 3 weeks. No significant changes in %CDT were detected in most subjects, even in the 80 g alcohol-consuming groups. The results suggest that CDT is not sensitive for the detection of short-term heavy drinking by healthy subjects.

Failure of glucose-binding lectins Con A and Lentil Lectin to identify glycation of haemoglobin.

We have studied the interaction of Concanavalin A and Lentil Lectin with glycohaemoglobin by a nephelometric lectin-glycogen/dextran precipitation system and monitored the inhibitory effect of glycohaemoglobin on the precipitation. Although inhibitory effects were clearly demonstrated using simple sugars and transferrin, no effect was observed by glycohaemoglobin in relevant concentrations. This is compared to affinity chromatography, binding studies using gel filtration and electrophoresis, and affinity studies using Concanavalin A immobilised on magnetisable polymer particles. Lack of interaction between glycohaemoglobin and lectins is discussed in view of steric constraints and reduced availability of the glycated residues and the stereochemical form of the glycated 1-amino-1-deoxy-fructosyl residues in glycohaemoglobin.

Radiolabelling of human haemoglobin using the 125I-Bolton-Hunter reagent is superior to oxidative iodination for conservation of the native structure of the labelled protein.

Both disruption of the native protein structure and oxidation of iron in the haem/iron(II)-proto-porphyrin-IX residues were observed using the Iodo-gen (1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycouril) method in 125I-labelling of haemoglobin. The reactions taking place affect the native structure of haemoglobin and result in a more acidic molecule. The detrimental effects were unaffected by the presence of iodine. Electrophoretic studies demonstrate that 125I-labelling of haemoglobin using the Bolton-Hunter reagent is the method of choice in order to preserve the native protein.

Protein-boronic acid conjugates and their binding to low-molecular-mass cis-diols and glycated hemoglobin.

Different methods for covalent linkage of phenylboronic acid (PBA) to structural proteins and enzymes are presented. Protein-PBA conjugates, free in solution or immobilised on magnetizable polymer particles, were tested for their binding of D-sorbitol, D-mannose and glycohemoglobin (GHb). Similarly, alkaline phosphatase-PBA conjugates were used in an attempted enzyme-linked sorbent assay for the detection of GHb. Affinity chromatography on immobilised D-mannose and gel chromatographic studies of protein-PBA complexes with [14C]sorbitol, clearly illustrated a low affinity of the interaction studied. Glycated hemoglobin could not be detected using the enzyme-linked sorbent assay approach. However, GHb was found to be specifically retained on columns filled with protein-PBA-coated particles as affinity matrix, enabling the glycation level of blood samples to be determined.

Pulmonary, hepatic and splenic sequestration of technetium-99m labelled autologous rabbit granulocytes: scintigraphic cell distributions after intravenous and intraarterial injections, exsanguination and intraarterial injection of cells passed through an intermediary host.

In man, about half the intravascular granulocytes are not freely circulating, but temporarily sequestered ('marginated'), so that they cannot be retrieved by bleeding. Where and how the sequestration occurs is not settled and is the subject of the present report. Isolated autologous rabbit granulocytes, labelled with two different 99mTc methods, were reinjected and followed with external scintigraphy. Intraarterial as well as intravenous injection led to rapid accumulation of radioactivity over the lungs. This finding was corroborated and extended by similar experiments, where the labelled cells had firstly been passed through an intermediary rabbit host to remove altered cells, i.e. cells damaged, 'primed' (pre-activated), or activated. In the final autologous host about two thirds of the label rapidly localized to the lungs and liver, and a few per cent to the spleen (which is very small in the rabbit). Even though more than half of the intermediary rabbit's calculated blood volume was removed, the blood sample contained only a few per cent of the rabbit's radioactivity; consequently, many of the labelled leucocytes had marginated during the bleeding. The proportional distribution of radioactivity over lungs, spleen, kidneys, and the rest of the intermediary animal was not markedly changed by this exsanguination, but there was a 4-20% decrease over the liver. Taken together, our findings indicate that normal granulocytes marginate in lungs, liver, and spleen--apparently explicable by the effects of cell size, vessel diameter, cell stiffness (visco-elastic properties) and size of the arterio-venous hydrostatic pressure difference. The liver and spleen seemed to play additional roles, since radioactivity over these organs decreased much slower than expected from reported blood half-times of intact and slightly damaged rabbit granulocytes. This led to a suggestion that macrophages exposed to blood normally phagocytose apoptotically dying granulocytes.

Migration of 99mTc-labelled syngeneic lymphocytes in the rat. Biological and theoretical models predict radiation damage and poor scintigraphic detectability.

The possibility of obtaining useful scintigrams of secondary lymphoid organs after infusion of syngeneic lymphocytes labelled with technetium-99m (99mTc) was explored in a rat model. Thoracic duct lymphocyte (TDL) accumulation in various organs was measured with both 99mTc and 51Cr labelled cells, the latter processed with a method that has been shown not to damage lymphocytes. 99mTc labelled TDL did not localize properly in the lymph nodes and spleen. We could not visualize lymph nodes in scintigrams, neither could we demonstrate any difference between normal and hyperplastic spleens. Our conclusion is that radiation from the 99mTc label readily influences lymphocyte migration so that useful scintigraphy in rats and other small experimental animals becomes impossible. This was supported by results from culture experiments with 99mTc labelled, radiosensitive mouse haemopoietic progenitor cells. Theoretical considerations, including the calculations of lymphocyte self-irradiation and signal/noise ratios during scintigraphy of rat tissues, supported our conclusion that scintigraphy in small animals, to disclose the physiological migration of lymphocytes, may be impossible with the present sensitivity of gamma cameras.

Radiolabelling of platelets with technetium-99m.

A method for labelling of platelets with technetium-99m (Tc-99m) is presented. In principle, aminobenzoic acid and tartaric acid are used as reagents, allowing Tc-99m complexes of intermediate chemical stability to be formed. These complexes react rapidly with proteins, such as platelet proteins, when added. We have examined the isolation procedure for the platelets and the labelling procedure using residual aggregational ability and residual content of beta-thromboglobulin (beta-TG) as indicators of damage to the platelets. In its final version the method allowed a 32.6 +/- 2.7% (mean +/- SD) incorporation of Tc-99m into platelets which again showed a 66 +/- 15% residual aggregational ability, tested by 50 mumol/l of ADP, and a 79 +/- 17% residual content of beta-TG releasable by 10 IU/ml of thrombin. In a pilot clinical study involving 28 patients we found labelled autologous platelets useful in detecting lung embolism and deep vein thrombosis.

Whole body distribution of 99mTc labelled autologous human granulocytes and radiation dose to cells and organs.

The whole body distribution of radioactivity as a function of time after infusion of 99mTc labelled autologous granulocytes was measured in three volunteers by means of a scanning bed, a scintillation camera and a minicomputer. Labelling was performed with a bisalt method without pretinning. There was a considerable initial lung sequestration (22%-31%) of the injected activity, which disappeared with an effective half life of 42 min. One h after infusion the activity was found mainly in the liver (41%), spleen (8%), lungs (9%) and kidneys (5%). Urine excretion amounted to 30% during the first 32 h after infusion. An injected activity of 100 MBq caused a radiation dose of 4.4 m Gy to the liver, 6.3 m Gy to the spleen, 3.7 m Gy to the kidneys, and 0.2 m Gy and 0.1 m Gy to the ovaries and testes respectively. The labelling procedure and the subsequent decay within the granulocytes gave them an absorbed radiation dose of 1.8 Gy after 25 min (i.e., at completion of the infusion) and 8.4 Gy after 4 h (i.e., the normal imaging time). In vitro tests revealed no signs of radiation damage to the cells.

Determination of the oxidation state of 99mTc in complexes with MDP and DTPA.

The oxidation state of technetium-99m, reduced by concentrated hydrochloric acid and evaporation until dryness, was determined by radiochromatography. The dry deposits of reduced 99mTc were allowed to react with DTPA and MDP with and without antioxidants present. The formation of 99mTc(IV)-DTPA and 99mTc(IV)-MDP complexes and the influence of the antioxidants gentisic and ascorbic acids were studied by radiochromatography and by biodistributional studies in rabbits. 99mTc(IV)-MDP complexes thus formed were demonstrated to have the same biodistribution in rabbits as 99mTc-MDP complexes formed by a conventional stannous reduction kit technique.

A new method for granulocyte labeling with technetium-99m: preliminary results in abscess detection.

A new method for 99mTc-labeling of granulocytes for clinical routine use has been developed. The labeling is simple to perform by means of a kit of radiopharmaceutical quality, utilizing dihydroxy-benzoic acid. Pretinning techniques are avoided. The technique has been applied clinically in 15 patients with indications of intra-abdominal abscess. In six patients, [99mTc]granulocyte scintigraphy at 3 hr and/or 24 hr after i.v. administration, correctly depicted the abscess, as verified by subsequent surgery. In the remaining patients, who were negative at surgery or recovered without operation, all scans were negative.

A new method for rapid technetium-99m labelling of leucocytes: functional cell studies in vitro.

The aim of this study was to assess the cytotoxicity of a new leucocyte-labelling method, which may be used clinically to localize inflammatory and immune reactions. Human blood leucocytes, their mononuclear sub-population, and mouse mononuclear bone marrow cells were labelled with 99mTc for 30-45 min, washed once, and then evaluated in various functional assays. The new procedure includes [99mTc]-labelling with a bisalt method, in the presence of dihydroxybenzoic acid as an intermediate antioxidant-complexing stabilizer, and a carboxylic acid salt of stannous ions as a reducing agent. To challenge the method, cells were labelled about two orders of magnitude more heavily in these initial methodological studies than in on-going clinical trials. Labelled leucocytes ingested latex beads as readily as the controls, but migrated chemotactically and randomly somewhat slower than the control cells. The lymphocytes were triggered by PHA and Con A in a normal way. However, lymphocytes and haemopoietic progenitor cells exposed to radiation for several days, were killed by the isotope doses used, of which about 2% (i.e. 20 MBq) were bound per million cells. All deleterious effects were apparently due to irradiation, and the labelling procedure itself did not damage the cells.

A new technique for formation of 99mTc-labelled blood leucocytes and platelets.

99mTc -platelets and 99mTc -leucocytes have been formed at neutral pH in presence of gentistic acid using 99mTc pretreated with concentrated hydrochloric acid and vacuum evaporation. High radiochemical purities and specific radioactivities were obtained. The complexes between 99mTc and the blood cells were found remarkably stable, and further studies for nuclear medicine applications are recommended.