Multicenter evaluation of Reflotron direct dry-chemistry assay of high-density lipoprotein cholesterol in venous and fingerstick specimens.

The Reflotron HDL Cholesterol test (Boehringer Mannheim GmbH) directly separates and analyzes high-density lipoprotein (HDL) cholesterol in plasma collected with EDTA in an integrated dry-reagent system suitable for alternative site testing of lipoproteins. We describe a multicenter evaluation of this test by two US and six European laboratories experienced in lipid analysis. Each laboratory compared the Reflotron with the same conventional wet-chemistry method, Boehringer phosphotungstate-Mg2+ precipitation with enzymatic cholesterol assay. Imprecision was within accepted guidelines, with CVs of < or = 8% for fresh and frozen plasmas (median CV 1.7-3.9%) and for lyophilized sera (median CV 3.8-4.7%), similar to those of the conventional method. Results of linear-regression analysis were as follows: Reflotron HDL Cholesterol = 1.03 conventional - 3.9 mg/L, r = 0.987. The Reflotron results were somewhat low in the two US laboratories, demonstrating the need for general standardization of methods for measuring HDL cholesterol. Results from capillary fingerstick plasma agreed well with those from venous-derived plasma; capillary = 1.04 venous + 4.5 mg/L, r = 0.967. The system is relatively insensitive to interference from hemoglobin (< or = 0.75 g/L), ascorbic acid (< or = 0.3 g/L), bilirubin (< or = 50 mg/L), cholesterol (< or = 3.5 g/L), and triglycerides (< or = 4 g/L). The relative ease of operation and the rapid availability of results (within 90 s for plasma collected in EDTA) make the method appropriate for use by well-trained, but not necessarily technical, operators in the physician's office or other alternative sites.

IgE and IgG antibodies of patients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa.

IgE and IgG antibody response to birch pollen antigens were studied by means of immunoblotting experiments testing 58 sera from patients with Type I allergy to birch pollen. 56/58 patients showed IgE antibodies reactive with Bet v I, a 17 kilodalton (kD) pollen protein. 2D-electrophoresis/immunoblot revealed a heterogeneity of that protein. Ten spots (pH 4.9-5.9) could be detected, presumably representing differentially glycosylated isoallergens. In 33/58 patients, there was no evidence of IgE antibodies directed against allergens other than Bet v I. However, in 25/58 of patients' sera, 11 minor allergens (13, 15, 18, 27, 29, 32, 39, 44, 57, and 68 kD) with individual incidences from 1.7% to 17.2% were identified. All proteins were also recognized by the patients' IgG antibodies: in the case of Bet v I recognition was weak, whereas the IgG response to the minor allergens was pronounced. Sera from healthy individuals showed similar IgG antibody responses, but no IgG to the 15, 27, and 29 kD proteins. Our results suggest that IgG directed against minor allergens may function as trapping antibodies in healthy individuals. Too low or lacking amounts of anti-Bet v I IgG may facilitate an allergic reaction.

Specificities of IgE and IgG antibodies in patients with birch pollen allergy.

58 sera from patients with established birch pollen allergy showed characteristic antibody-binding patterns in immunoblotting experiments. Regarding IgE, 56/58 patients recognized a protein of molecular weight (MW) 17 kilodaltons (kD), previously defined as Bet v I. 23/58 patients in addition reacted with a variety of 11 minor allergens with MWs ranging from 13 to 68 kD. A 13-kD protein was proved to represent an independent minor allergen. IgG binding in patients and healthy individuals was more pronounced on the minor allergens than on Bet v I. 3 different allergens were not detected by IgG of healthy individuals. In two-dimensional electrophoresis/immunoblot, a monoclonal antibody and human IgE (in both cases directed against Bet v I) detected a very similar cluster of spots, probably representing isoallergens of Bet v I.

High resolution two-dimensional polyacrylamide gel electrophoresis of cerebrospinal fluid in patients with neurological diseases.

The protein pattern of cerebrospinal fluid (CSF) of 334 patients with various neurological and systemic diseases was investigated by high resolution two-dimensional electrophoresis (2-DE). 2-DE gels of normal CSF contain proteins which are not detectable in 2-DE gels of serum. Disturbances of the blood-brain or blood-CSF barrier, and degenerative diseases of the brain and malignant diseases produce specific changes on 2-DE gels of the CSF. The appearance of 10 spot areas in the light chain region of 2-DE gels seem to be connected with the diagnosis of multiple sclerosis. The sensitivity and the specificity of these spot areas for the diagnosis of multiple sclerosis are described. Proteins within the ten spot areas are immunoglobulin light chains or substances which cross-react very strongly with light chain antibodies as demonstrated by immunoblotting and immunoabsorption.

Two-dimensional electrophoresis of proteins in tumours of the lung.

The proteins of solid lung tumours (15 adenocarcinomas and 10 squamous cell carcinomas) were examined by high resolution two-dimensional electrophoresis (2-DE) and compared with the proteins of adjacent lung tissue which demonstrated no histological evidence of malignant transformation, and with the proteins of other malignant tumours and normal tissues. To investigate tumour cell-specific protein synthesis, we isolated malignant and normal cells enzymatically with collagenase, elastase, and DNase. Tissue and tumour cells were enriched in an additional step on a Percoll gradient. The 2-DE gel patterns derived from entire tissue and enriched tissue cell preparations were compared. No specific differences were found between the 2-DE protein patterns from adenocarcinomas and squamous cell carcinomas of the lung, but three proteins identified on the 2-DE gels appeared to be tumour-associated. Spot A is present in non-neoplastic and neoplastic epithelial tissues. Spot B is pronounced in 2-DE gels of sarcomas, but is also present in preparations of other malignant tissues. Spot C is present in all malignant cell preparations. These three spots were also demonstrated in 2-DE protein patterns from tissue cultures of malignant cell lines. Spot B and spot C were also present in some normal tissues.

Two dimensional electrophoresis method for additional characterization of paraproteins in serum. Clinical application of two dimensional electrophoresis, I.

High resolution two dimensional electrophoresis, one of the most potent methods for the analytical separation of proteins, is still used only in specialized laboratories. The present paper describes a two dimensional electrophoresis method, which permits characterization of abnormal serum proteins (paraproteins) and is suitable for application in a clinical laboratory. Both steps of the electrophoresis are performed by the "flat bed technique". The critical transfer from the isoelectric focusing step to the gel of the second dimension is facilitated by use of specially prepared foil stencils. The practicality of the procedure was considerably increased by using commercially available materials such as gels for isoelectric focusing and the silver stain kit. The analysis can be calibrated by measurement of the pH gradient and by using molecular weight protein standards. Moreover, two different samples can be analysed simultaneously in the same gels of the first and the second dimension. This permits a direct comparison of the sample of interest with the sample of reference. Serum samples showing an abnormal band ("M gradient") in routine methods such as agarose gel electrophoresis, were analysed by the described method. Some "M gradients" consisted of single abnormal proteins. Another sample showed a "M gradient" composed of a number of abnormal protein spots in our method. Questions concerning pathobiological and clinical evaluation of the findings justify further studies using this method.

Pharmacokinetics of high-dose methotrexate in dogs. An experimental model with diffusion chambers.

The usefulness of a diffusion chamber method for determination of concentrations of cytostatic drugs in the interstitial fluid of tissues was tested. Chambers with a permeable membrane (pore size: 0.45 micrometer) were implanted in the liver, kidney, bladder wall, and prostate of dogs. After administration of high doses of methotrexate (100 mg/kg body wt) the concentrations in the chamber fluid and in serum were measured simultaneously and repeatedly for 72 h. The method proved to be effective for collecting data on the distribution of drugs in different organs. The results show that knowledge of the serum concentration does not permit predictions of the drug concentration in the interstitial fluid of various tissues to be made.

Mechanism of leukocyte locomotion in hemophilia.

In order to study a relationship between leukocyte locomotion and hemophilia, three assays to investigate cell motility were performed: Investigations were carried out, firstly, on the leukocyte locomotion (LL), secondly, on the activability of sera by zymosan, and thirdly, on the influence of sera upon LL. 17 patients with hemophilia and 16 healthy control persons were included in the study. While LL of patients and controls did not differ significantly with either cytotaxin, zymosan activated serum of patients or controls, or casein, sera of patients were significantly less activatable (p< 0.0001) than control sera. Locomotion of control leukocytes was significantly inhibited (p<0.002) by sera of hemophiliacs.

Collagen types and anticollagen-antibodies in Dupuytren's disease.

The relative proportion of collagen type I and type III in the aponeurosis of twenty-four patients with Dupuytren's disease was determined and compared with the aponeurosis of normal persons. The presence of considerable amounts of type III collagen was found in the Dupuytren's disease patients. The sera of the patients were screened for circulating anti-collagen antibodies using a sensitive radioimmunoassay. In seven out of the twenty-four patients low concentrations of these antibodies were found.

[The influence of surgery upon leucocyte function (author's transl)]. / Der Einfluss von Operationen auf Leukozytenfunktionen.

14 patients who underwent a standardized total hip joint replacement were investigated in order to detect postoperative impairment of chemotactic or phagocytic function of their polymorphonuclear leucocytes. A chemotaxis assay in modified Boyden [3] chambers and the nitroblue-tetrazolium assay were used. 25 healthy control persons served for the assessment of normal values and the reproducibility of the respective test. Patients were investigated preoperatively and on the first, third and sixth postoperative day. While phagocytosis remained unchanged throughout the period of investigation, chemotactic leucocyte function was regularly significantly depressed on the first postoperative day. The defect in postoperative chemotaxis might, despite of an unimpaired phagocytic capacity, contribute towards an increased risk of postoperative infections.