[Identification of elevated carbohydrate-deficient transferrin (CDT) serum level as transferrin (Tf)-D-variant by means of isoelectric focusing]. / Identifizierung eines mittels Immunturbidimetrie nach Anionenaustausch-Chromatographie erhöhten Carbohydrate-Deficient-Transferrin-(CDT-)Serumspiegels als Transferrin (Tf)-D-Variante.

Many studies have shown carbohydrate-deficient transferrin (CDT) to be a sensitive and specific marker of chronic alcohol abuse. We present the case of a 23-year-old, healthy professional soccer player who caused a car accident due to alcohol consumption. Several CDT test results were elevated above the laboratory reference range and were considered to be caused by alcohol intake at a level commensurate with misuse and thus license reapplication was refused. In addition, assuming chronic alcohol abuse, the young man suffered from increasing social isolation. He was finally referred to our out-patient clinic for further evaluation on the assumption of a liver disease. Since chronic alcohol consumption was denied, and there was no evidence of liver disease, a qualitative characterization of the transferrin isoforms was performed. Isoelectric focusing of serum transferrin revealed a pattern atypical for chronic alcohol intake but detected a genetically determined transferrin (Tf)-D-variant. The changed amino acid sequence caused an overlapping of transferrin isoforms with different degrees of sialylation, thus revealing false-positive serum CDT values. Determination of this Tf-D-variant heterozygosity resulted in his social rehabilitation and license reinstatement. Thus, where the evidence for alcohol dependency is either uncertain or uncorroborated, qualitative isoelectric focusing of transferrin is a useful method for analyzing unexplained CDT elevations, thus increasing the value of CDT as a marker for chronic alcoholic abuse.

[CDG (congenital disorders of glycosylation). Differential hereditary ataxia in adulthood diagnosis]. / CDG (congenital disorders of glycosylation). Zur Differenzialdiagnose hereditärer Ataxien im Erwachsenenalter.

Congenital disorders of glycosylation (CDG) are a group of hereditary multisystem diseases due to different defects of enzymes or transport molecules involved in the synthesis of glycoproteins. CDG-la is the most common subtype, with cerebellar ataxia as the main neurological symptom. Currently there is little information about CDG-la manifestation in adulthood. Here we present two sisters in whom the diagnosis of CDG-la was made in the fourth decade of life and who to our knowledge are the oldest known patients with the disorder in Germany. The clinical course of the disease was typical, although less severe than previously described. The carbohydrate-deficient transferrin (CDT) level was increased but lower than in other CDG patients. Isoelectric focusing of transferrin revealed changes typical of CDG, whereas those of alpha 1-antitrypsin were only moderately pathologic. This might be due to the milder manifestation of the disease in our patients or it could be indicative of a stabilization of the disease after puberty. The CDG should be included in the differential diagnostic workup of hereditary cerebellar ataxia in adults.

A new case of CDG-x with stereotyped dystonic hand movements and optic atrophy.

We report the clinical findings and the diagnostic work-up of a 17-month-old girl with CDG-x. Predominant clinical signs were, besides psychomotor retardation and truncal hypotonia, stereotyped dystonic hand movements and ophthalmological abnormalities such as optic atrophy, nystagmus and strabismus. Other symptoms that are often found in patients with CDG were not present, such as seizures, microcephaly, cerebellar hypoplasia, dysmorphic features, hepatointestinal disease, coagulopathy or multiorgan involvement. Isoelectric focusing (IEF) of the patient's serum showed a marked elevation of disialotransferrin, thus confirming an IEF type 1 pattern. A generalized glycosylation defect was confirmed also by IEF of a further glycoprotein (alpha1-antitrypsin), an increased carbohydrate deficient transferrin (CDT) serum concentration and an increased CDT/transferrin ratio. All known types of CDG-I, secondary glycosylation abnormalities and variants of amino acid sequence were excluded.

Congenital disorder of glycosylation IId (CDG-IId) -- a new entity: clinical presentation with Dandy-Walker malformation and myopathy.

A 1.5-year-old boy with macrocephaly due to a Dandy-Walker malformation presented with progressive hydrocephalus, extensive muscular hypotonia, transient cholestatic syndrome, extensive coagulation abnormalities and elevated creatine kinase indicating myopathy. Diagnostic work-up indicated a congenital disorder of glycosylation (CDG, formerly carbohydrate deficient glycoprotein syndrome). The serum transferrin pattern obtained by automated isoelectric focusing (IEF) showed an hitherto unreported pattern with strongly elevated tri-, di-, mono- and asialotransferrin bands, increasing in this order together with markedly decreased tetrasialotransferrin. Investigation of two additional glycoproteins, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, confirmed a generalised defect of glycosylation. All known glycosylation defects could be ruled out by enzymatic analyses in either leukocytes or fibroblasts or by the results obtained by IEF. SDS-electrophoresis demonstrated a marked difference in the molecular weight of transferrin, suggesting the lack of parts or of all oligosaccharide chains. The defect could be delineated to a deficiency of beta-1,4-galactosyltransferase (E.C.2.4.1.38) due to a homozygous insertion (1031 - 1032 insC). Details of the biochemical and molecular findings will be described elsewhere.

A new subtype of a congenital disorder of glycosylation (CDG) with mild clinical manifestations.

A boy with an unspecific symptomatology consisting of mental retardation, strabismus, hypotonia and mild ataxia was diagnosed with a congenital disorder of glycosylation (CDG). Neither cerebellar atrophy nor dysmorphic features were present. The serum transferrin band pattern obtained by isoelectric focusing(IEF) showed a strongly elevated disialotransferrin band together with only slightly elevated asialotransferrin, thus a type I pattern. This is a new CDG classified CDG-x since CDG-la, -b, -c, -d and -e were excluded. Quantitative differences to the type 1 pattern of a CDG-la patient with a moderate to severe course were confirmed by densitometric evaluation of the gels and by SDS gel electrophoresis. Liver biopsy showed lysosomal inclusions suggesting a pre-Golgi defect. This patient's case supports the approach to include isoelectric focusing of serum transferrin in the diagnostic work-up of patients with unexplained symptoms.

Outbreak of Legionnaires' disease associated with a display whirlpool spa.

BACKGROUND: Recognized outbreaks of Legionnaires' disease (LD) are rare; when they occur, they provide opportunities to understand the epidemiology of the illness and improve prevention strategies. We investigated a population-based outbreak. METHODS: After the confirmation of LD in October 1996 in five people in neighbouring towns in southwest Virginia, active surveillance for additional cases was undertaken. A case-control study was conducted to identify exposures associated with illness, followed by a cohort study among employees of the facility at which the source of the outbreak was located in order to assess unrecognized exposure and illness. Samples of likely sources of LD in the facility were cultured for LEGIONELLA: RESULTS: In all, 23 laboratory-confirmed cases of LD were eventually identified. Of the 15 cases in the case-control study, 14 (93%) reported visiting a home-improvement store, compared with 12 (27%) of 45 controls (matched odds ratio [MOR] = 23.3; 95% CI : 3-182). Among home-improvement centre patrons, 10 (77%) of 13 cases questioned recalled either visiting or walking by a display whirlpool spa, compared with 3 (25%) of 12 controls (MOR = 5.5; 95% CI : 0.7-256.0). Two cases' sputum isolates were an exact match, by monoclonal antibody subtyping and arbitrarily primed polymerase chain reaction, to a whirlpool spa filter isolate from the store. Employees reporting more exposure to the display spas were more likely to report symptoms of LD or to have an elevated titre. CONCLUSIONS: This investigation shows that LD can be transmitted from a whirlpool spa used for display only, and highlights the need for minimizing the risk of transmission of LD from all water-filled spas. Key messages This paper describes an investigation of a population-based outbreak of Legionnaires' disease (LD). A case-control study first identified a home-improvement store as the likely source of the outbreak. An environmental investigation later confirmed that finding, as two cases' sputum isolates were an exact match, by monoclonal antibody subtyping and arbitrarily primed polymerase chain reaction, to a whirlpool spa filter isolate from the store. The spa was intended and used for display only.

Investigation by isoelectric focusing of the initial carbohydrate-deficient transferrin (CDT) and non-CDT transferrin isoform fractionation step involved in determination of CDT by the ChronAlcoI.D. assay.

BACKGROUND: The introduction of a new set of reagents for the determination of carbohydrate-deficient transferrin (CDT) as a marker of chronic alcohol abuse requires an independent evaluation of the analytic specificity of the test. This information is needed for correct interpretation and classification of test results. METHODS: Isoelectric focusing on the PhastSystem(TM) followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D. assay. RESULTS: The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV < or = 10%). Except for quantitatively unimportant traces of trisialo-Fe(2)-transferrin (<5% of total CDT), only asialo-, mono-, and disialo-Fe(2)-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe(2)-transferrin (during column rinsing) and disialo-Fe(2)-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe(2)-transferrin did not change from >1 (serum) to <1 (eluates) as described for the CDTect assays. The transferrin patterns in the ChronAlcoI.D. eluates were representative of those in serum. Transferrin D variants with isoelectric points close to that of trisialo-Fe(2)-transferrin C1 did not cause overdetermination of CDT by the ChronAlcoI.D. test. CONCLUSIONS: The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.

Semi-automated rapid isoelectric focusing of apolipoproteins C from human plasma using Phastsystem and immunofixation.

Apolipoproteins (apo) C-I, C-II, and C-III play crucial roles in intravascular lipid metabolism. Whereas apo C-II is an obligate cofactor for lipoprotein lipase, apo C-III was shown to inhibit its action. Apo C-I can be a potent cofactor of human lecithin:cholesterol acyltransferase. Structural mutants and deficiencies of apo C-II lead to hypertriglyceridemia. A similar phenotype is associated with apo C-III mutants and is inducible by overexpression of human apo C-III in transgenic animals. No structural variant has so far been reported for apo C-I. The present paper describes a rapid semi-automated procedure for isoelectric focusing analysis of these C-apolipoproteins from whole plasma or serum and their visualization by immunofixation and silver staining. The procedure allows detection of charged variants of C-apolipoproteins. As applied to 295 patients with coronary heart disease and 85 controls, it also serves to detect deficiency syndromes of these apolipoproteins. The procedure provides reliable, easy and quick analysis of C-apolipoproteins applicable as a routine or screening procedure not restricted to specialized laboratories.

Carbohydrate-deficient transferrin is not affected by serum separators.

We studied the possible effects on serum carbohydrate-deficient transferrin (CDT) determination by a CDTect (Pharmacia) method of serum isolation in four different types of blood-collection tubes, namely: (1) glass tubes (glass Vacutainer tubes with no additive); (2) S-Monovette Neutral tubes (plastic tubes with no additive); (3) S-Monovette Serum tubes (plastic tubes with kaolin-coated plastic granulate coagulation accelerator); and (4) S-Monovette Serum/Gel tubes (plastic tubes with kaolin-coated plastic granulate and a polymerized acrylamide resin). Using Passing and Bablok regression analysis, we did not observe significant differences in CDT concentrations determined in 58 serum samples using any of these four blood-collection systems.

Isoforms of murine and human serum amyloid P component.

Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not affect their number. When the acute-phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H/HeN mice showed one more acidic isoform and an increase in the concentration of the most basic isoform. C57BL/6 J mice exhibited two to three new isoforms during the acute-phase response. This appears to be the first demonstration of the physiological existence of SAP isoforms. In contrast, demonstration of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase treatment caused a shift of the isoforms, but no reduction in isoform number. Two-dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.

Validation by isoelectric focusing of the anion-exchange isotransferrin fractionation step involved in determination of carbohydrate-deficient transferrin by the CDTect assay.

Serum concentration of carbohydrate-deficient transferrin (CDT) is used for laboratory diagnosis of chronic alcohol abuse. Using isoelectric focusing for validation of the initial isotransferrin fractionation step involved in the determination of CDT by the CDTect assay, we found a complete in vitro iron saturation of transferrin and sufficient stability of the transferrin iron load during column passage; effective separation of non-CDT-isotransferrins and CDT-isotransferrins at the microcolumns; partial coelution of trisialo-Fe2-transferrin, which did not significantly affect CDT measurement; partial retention of CDT-isotransferrins, especially disialo-Fe2-transferrin, which may cause falsely negative results for CDT at the upper reference limits; good precision of the isotransferrin fractionation step; and no significant effects of low concentrations of serum protein and transferrin. We strongly urge standardization of CDT analysis and suggest isoelectric focusing for validation of CDT analysis methods and verification of odd results.

CDTect-RIA and CDTect-EIA for determination of serum carbohydrate-deficient transferrin compared.

CDTect-RIA and CDTect-EIA for determination of serum carbohydrate-deficient transferrin (CDT) by radioimmunoassay and enzyme immunoassay respectively were tested for equality and precision in four European laboratories. For correlational studies, serum samples with CDT concentrations up to 130 U/l were analysed in accordance with a uniform trial schedule. The regression of CDT values obtained by the two procedures was computed for each laboratory using the method of Passing and Bablok. Slopes and intercepts of the regression functions did not differ significantly from the values 1 or 0, as proved by the corresponding 95% confidence intervals. Precision studies were computed using analysis of variance. For CDT concentrations at the upper reference limit for men, the within-day coefficients of variation (CVs) ranged between 0.7 and 6.4% (median 5.2%) for CDTect-RIA and from 4.3 to 9.2% (median 6.2%) for CDTect-EIA. The corresponding pure between-day CVs were 5.0-18.5% (median 9.8%) and 3.5-14.5% (median 10.9%). The study demonstrates the equality of CDT values obtained by CDTect-RIA and CDTect-EIA. According to this study, the two methods can be used interchangeably without getting fluctuating CDT values, e.g. in longitudinal studies.

Increased serum concentration of carbohydrate-deficient transferrin in patients with combined pancreas and kidney transplantation.

Serum concentration of carbohydrate-deficient transferrin (cCDT) is used for laboratory diagnosis and follow-up of chronic alcohol abuse. In analyzing by CDTect-RIA (Pharmacia) sera from outpatients with combined pancreas and kidney transplantation and no excessive alcohol consumption, we found above-normal values for cCDT and CDT/transferrin ratios (CDT/Tf) in more than half of the samples. Isoelectric focusing of these samples showed distinct bands of carbohydrate-deficient isotransferrins, supporting the abnormal findings from the CDTect assay. In contrast, diabetics and outpatients who had received only kidney transplants showed normal values for cCDT, CDT/Tf, and isotransferrin patterns. Increased serum Tf, sialidase-producing microorganisms, and immunosuppressive medication were eliminated as causes of these abnormal cCDT and CDT/Tf results. Successful pancreas transplantation leads to hyperinsulinemia and normoglycemia, in contrast to hypoinsulinemia and hyperglycemia in the patients who receive kidney transplants alone. These factors may have pathogenic importance for CDT increase, yielding results falsely interpreted as positive with respect to alcohol abuse in patients with combined pancreas and kidney transplantation.

Fibronectin in human bile fluid for diagnosis of malignant biliary diseases.

In a preliminary study, we demonstrated a strong association between the concentration of the glycoprotein fibronectin (FN) in human bile fluid and the presence of malignant biliary diseases. We now present the results of measurements of total FN (tFN) and cellular FN (cFN) within a larger group of 71 patients. Bile fluid was collected during routine endoscopic retrograde cholangiography or by transhepatic puncture, respectively, from patients admitted for examination/treatment of biliary obstruction. Determination of tFN in bile was performed using a previously described time-resolved fluorescence immunoassay (TRFIA). For cFN, a newly developed TRFIA, using a specific monoclonal antibody for the EDA epitope of cFN, was applied. Within the noncarcinoma group of patients (n=50), consistently low concentrations of tFN (median = 5 ng/mL) were found. In most of these cases, the corresponding concentrations of cFN were below the detection limit (2.6 ng/mL) of this assay. Highly significantly elevated concentrations were found for both tFN (median = 1,220 ng/mL) and cFN (median = 243 ng/mL) in the carcinoma group (n = 21) in comparison with the noncarcinoma group (P < or = .01). By adopting cutoff values of 60 ng/mL for tFN and >0 ng/mL for cFN, diagnostic sensitivities for carcinoma of the biliary tract of 0.89 and 0.92, and specificities of 0.96 and 0.98, respectively, were computed. FN in bile fluid is suggested as a sensitive, specific, and easily determined marker for differential diagnosis of malignant and benign diseases of the biliary tract.

Effect of separation conditions on automated isoelectric focusing of carbohydrate-deficient transferrin and other human isotransferrins using the PhastSystem.

To investigate the effect of automated isoelectric focusing conditions in the PhastSystem, e.g., the point of sample application, prerun and separation times, and minimized gels on isotransferrin band pattern, human sera were analyzed with native transferrin iron load, after iron saturation or iron depletion in vitro. Varying the focusing conditions we found (i) Point of sample application (anode, middle of the gel, cathode) strongly affected transferrin iron loss. It was greatest at the anode and least at the cathode. (ii) Without prerun, distinct transferrin iron loss also occurred. A short prerun time prevented iron loss, but increasing it did not improve transferrin iron load stability as stated by others. (iii) An inappropriately long separation time inevitably yielded iron loss. In conclusion, inappropriate isoelectric focusing conditions strongly affect iron load stability of isotransferrins (obviously via low pH within the gel), resulting in transferrin iron release and cofocusing of isotransferrins with different sialic acid or iron contents. For determination of carbohydrate-deficient transferrin, such conditions resulted in overestimation of the marker of chronic alcohol abuse. Our findings may be of guiding importance for isoelectric focusing of protein-ligand complexes. We recommend the procedure described for development of isoelectric focusing of protein-ligand complexes.

Apolipoprotein AI, AII, and AIV isoforms in plasma determined by automated isoelectric focusing with PhastSystem and immunofixation.

Distinct genetic variants of apolipoprotein (apo) AI have been shown to influence concentrations of high-density lipoprotein (HDL) cholesterol. The genetic polymorphism of apo AIV may modulate HDL-cholesterol, plasma triglycerides, and lipoprotein(a) concentrations. There is evidence for an antagonizing role of apo AII in reverse cholesterol transport. Since genetic polymorphisms and variants of these apolipoproteins are detectable by isoelectric focusing (IEF), we developed a rapid and easy automated method for IEF analysis of apos AI, AII, and AIV on self-made or commercially available gels, using the PhastSystem. Diluted plasma or serum samples (1 microL) are applied automatically onto the gel and IEF is carried out for 35-45 min. Afterwards, the apo A bands are precipitated by specific polyclonal antibodies and visualized by automated silver staining. This rapid procedure is suitable as a routine or screening method for IEF analysis of these major HDL apolipoproteins.

Laboratory diagnosis of radicular and pseudoradicular syndromes in cerebrospinal fluid (CSF): reliability of methods in consideration of pathogenetic aspects. Report on section "CSF diagnosis" of 4th Klagenfurter Neurology Workshop Conference on radicular and pseudoradicular syndromes, Klagenfurt, Austria, August 28-29, 1992.

Laboratory tests may be used to confirm the clinical differentiation of pseudoradicular syndromes and radicular syndromes. In the presence of pseudoradicular syndromes, CSF and blood samples yield no positive results with either non-specific or specific methods. Radicular syndromes give rise to positive findings; using non-specific methods they can be subdivided into inflammatory and non-inflammatory forms, with and without blood-nerve barrier impairment. Non-specific quantities of CSF routine diagnosis are total protein, albumin, leukocyte counts and differential cell count, L-lactate, intrathecal -IgG, -IgA, -IgM and immunoglobulin-class oligoclonal bands. Oligoclonal bands enable the highly sensitive differentiation of non-inflammatory from subacute-chronically inflammatory forms of radicular syndromes. Most of the specific quantities are the subject of current research, e.g. bacterial antigens, D-lactate, cultivation tests, polymerase chain reaction tests and pathogen-specific oligoclonal bands. Pathomechanisms affecting the permeability of the blood-nerve barrier to increasing concentrations of protein and to leukocyte subsets possibly explain the CSF findings in radicular and pseudoradicular syndromes.