Analytic variability and clinical significance of different assays for serum estradiol.

Recent reports in the clinical laboratory literature have raised questions about the accuracy of different methods of measuring serum estradiol. To determine whether between-method variability was clinically significant, we used five different commercial radioimmunoassay methods to measure the concentration of estradiol in sera from nine patients undergoing induction of ovulation for gamete intrafallopian transfer. There were large differences between the estradiol concentrations measured in the same samples by the different methods. Furthermore, apparent changes in concentration that would be regarded as clinically significant may reflect nothing more than the imprecision of the assay. Physicians using serum estradiol measurements to support therapeutic decisions should be aware that an excessive reliance on those measurements may be misleading.

Evaluation and optimization of variables in immunofixation electrophoresis for the detection of IgG paraproteins.

We examined different antisera and varying serum dilutions for their effects on the sensitivity of immunofixation electrophoresis (IFE) for detecting and characterizing serum IgG paraproteins. Different antisera reacted unequally with a single paraprotein and conversely, a single antiserum reacted unequally with several different paraproteins. The dilution of serum was extremely important; the adoption of a single dilution for all sera could result in failure to detect low-concentration or poorly immunoreactive paraproteins. While the application of a standardized amount of paraprotein is recommended, it must be emphasized that no single antiserum can be relied on to provide unequivocal detection of all paraproteins of that particular isotype.

Analysis of the feline immune response to human myelin-associated glycoprotein.

Elucidation of the pathogenesis of demyelinating peripheral neuropathy associated with myelin-associated glycoprotein (MAG) binding IgM paraproteins requires an in vivo animal model of the syndrome. Multiple immunizations of cats with MAG in Freund's adjuvant did not produce an antibody response but four immunizations with MAG-iscom (Morein, B. et al. (1984) Nature, 308: 457-460) did induce IgM antibodies which bound to human MAG and cat peripheral nerve myelin. Despite the presence of antibody for a 13-month period, no neuropathy developed. At necropsy, the peripheral nerves were ultrastructurally normal and no antibody was detectable in the endoneurium. A competitive ELISA indicated that the cat and human IgM antibodies recognized different epitopes.

Present status of pyoderma gangrenosum. Review of 21 cases.

This article summarizes the management of 22 cases of pyoderma gangrenosum over the past four years at the hospital of the University of Pennsylvania, Philadelphia. Eighteen patients with pyoderma gangrenosum were studied using the most sensitive routine laboratory method for detection of monoclonal immunoglobulins, immunofixation electrophoresis. Four cases of IgA gammopathy were detected, confirming previous reports of the incidence of monoclonal gammopathy in pyoderma gangrenosum. High-dose glucocorticoid therapy (pulse therapy) is an effective treatment for some severe, refractory cases of pyoderma gangrenosum. Eight patients were treated with pulse therapy. Six responded favorably, and none had serious complications.

Significance of low concentrations of creatinine in serum from hospital patients.

We present an analysis of the clinical significance of creatinine concentrations less than or equal to 4 mg/L (35 mumol/L) in serum as measured by a specific enzymatic method. In an unselected hospital patient population, 4% of whom had serum creatinine concentrations this low, a value of 5 mg/L (44 mumol/L) or higher was obtained on repeat analysis for a third of these patients, but the remaining two-thirds had persistently low values. Associated clinical conditions included low body mass, pregnancy, insulin-dependent diabetes mellitus, and total immobilization, but 12% of the patients, all female, had no obvious cause for the persistently low creatinine concentration. We conclude that low concentrations of creatinine in serum have no profound clinical significance.

Sensitivity of immunofixation electrophoresis for detecting IgM paraproteins in serum.

Detection and characterization of small IgM paraproteins by immunoelectrophoresis are often difficult because of (a) slow diffusion of the macromolecular immunoglobulin, and (b) the obscuring of the light chain, especially kappa, by the light chains of background polyclonal immunoglobulin (the "umbrella effect"). Various methods of sample pretreatment have been tried in an attempt to overcome this problem. Using immunofixation electrophoresis (IFE), we were able to detect and characterize IgM paraproteins in serum at concentrations as low as 0.25 g/L, even when the polyclonal immunoglobulin background was increased. With routine IFE, our laboratory identified 58 patients with IgM paraproteinemia in a four-year period. In 36, the paraproteins were present at less than 5 g/L; 10 of these were detectable only by IFE.

A direct electron microscopic immunocytochemical study of IgM paraproteinemic neuropathy.

Direct electron microscopic immunohistochemistry of the sural nerve of a patient with neuropathy and an IgMk paraprotein demonstrated that immunoglobulin was deposited throughout the compact myelin. These findings support the notion that immunoglobulin deposition plays a role in the pathogenesis of the neuropathy in vivo.

Comparative binding of murine and human monoclonal antibodies reacting with myelin-associated glycoprotein to myelin and human lymphocytes.

Human monoclonal IgM antibodies present in the blood of some patients with peripheral neuropathy and murine hybrid IgM antibodies C5 and C6, raised against myelin-associated glycoprotein, and HNK-1, raised against the human T cell line HSB-2, all bind to the carbohydrate moiety of myelin-associated glycoprotein. The relative avidity of the monoclonal antibodies was HNK-1 greater than C5/C6 much greater than human IgM, as determined in a competitive binding radioimmunoassay. HNK-1 bound myelin equally well at incubation temperatures between 4 degrees C and 37 degrees C; the human antibodies bound significantly only at 4 degrees C; and C6 bound best at 4 degrees C, less strongly at 20 degrees C and did not bind at 37 degrees C. All of the antibodies bound to a band corresponding to myelin-associated glycoprotein on immunoblots of human CNS myelin proteins in addition to several other antigens. Flow cytometric studies revealed that the murine but not the human antibodies bind to peripheral blood lymphocytes. Taken together, these data suggest that the antibodies probably recognize the same epitope but bind with different avidity.

IgG heavy-chain (Gm) allotypes in demyelinating polyneuropathy associated with MAG-binding monoclonal IgM autoantibodies.

IgG heavy-chain (Gm) allotypes were determined in 11 Caucasian patients with the syndrome of demyelinating polyneuropathy associated with monoclonal IgM autoantibodies with affinity for myelin-associated glycoprotein. The frequency of Gm phenotypes in the patient group was significantly different from that seen in a normal Caucasian population.

Imprecision of quantification of serum protein fractions by electrophoresis on cellulose acetate.

We studied the precision of densitometric quantification of the protein zones resolved by cellulose acetate electrophoresis. Replicate analyses of patients' samples by a single technologist showed mean CVs ranging from 2.9% for serum albumin to 9.5% for alpha 1-globulin. There were marked differences in measurements obtained by replicate analysis of the same samples by two experienced technologists. We calculated what changes in fractional concentrations would be analytically significant and concluded that densitometry of cellulose acetate electrophoretograms can only be semi-quantitative. We suggest that visual interpretation of high-resolution electrophoretic patterns by a trained observer can replace densitometry in most cases.

Human monoclonal IgM autoantibodies with restricted antigenic specificity for myelin express unrelated idiotypes.

Idiotype-specific polyclonal antisera were prepared against myelin-binding human IgM paraproteins with specificity for the myelin-associated glycoprotein (MAG). Eight anti-idiotypic sera (6 against one monoclonal IgM and 2 against another) were tested by gel precipitation, immunoradiometric and ELISA assays for binding to the myelin-binding IgM paraproteins from 12 patients with demyelinating peripheral neuropathy. Anti-idiotypic antibodies bound only to the MAG-specific IgM used for immunization; there was no evidence of idiotypic cross-reactivity between the different IgM paraproteins.

IgM paraproteins with immunological specificity for a Schwann cell component and peripheral nerve myelin in patients with polyneuropathy.

The sera of 9 patients with benign IgM paraproteinaemia and chronic sensorimotor neuropathy were tested for reactivity to human peripheral nerves by the indirect immunoperoxidase method. They reacted in very high titre (10(-3)-10(-6) with a cytoplasmic Schwann cell component, and to lesser degree, with peripheral nerve myelin (10(0)-10(-3). The Schwann cell staining was in the form of perinuclear cytoplasmic granules and was only seen with adult nerve. The distribution of the antigen was similar to that of the metachromatically staining Pi-granules of Reich, which accumulate in the peripheral nerves with age. Specific activity was present in the IgM and F(ab)2 fractions and could be absorbed out with peripheral nerve tissue, but not with liver. Reactivity is not a simple function of the IgM level, since many IgM paraproteins do not react. The antibody is species specific and binds to human, but not to any component of rabbit, rat or guinea pig sciatic nerves. Antigenicity is removed by pretreatment of the nerve with chloroform-methanol or periodate, but not protease or trypsin. Reactivity is restored, after periodate treatment, by exposure to sodium borohydride. It is suggested that some IgM paraproteins have a specificity for a myelin glycolipid or glycoprotein, which normally accumulates in the Pi-granules of the Schwann cell cytoplasm as a function of age.

Chronic demyelinating neuropathy associated with benign IgM paraproteinaemia.

Twelve cases of chronic sensorimotor neuropathy associated with benign IgM paraproteinaemia are described. The onset was in the sixth or seventh decades and 9 cases were male. Tremor and ataxia were common features. The ESR was raised in 6 cases and the CSF protein content elevated in 10. There was severe reduction of motor nerve conduction velocity. Sera from all cases contained monoclonal IgM antibodies strongly reactive with human peripheral myelin, made up by the paraprotein. Histological studies on nerve biopsies taken from 8 cases all demonstrated a demyelinating neuropathy. Eight biopsies were examined by immunofluorescence. Monoclonal IgM was present on the myelin sheaths of surviving myelinated nerve fibres. The findings suggested that the neuropathy was of autoimmune origin and caused directly by the antimyelin antibody.

Paraproteinaemia in neurological disease: incidence, associations, and classification of monoclonal immunoglobulins.

Fifty-six patients presenting with neurological complaints were found to have paraproteinaemia unassociated with immunocytic malignancy; 16 patients presented with peripheral neuropathy. There was an association between IgM paraproteinaemia and an idiopathic neuropathy with markedly slowed nerve conduction velocities.

Effect of sample preparation on cerebrospinal fluid protein patterns in polyacrylamide gels.

The method of preparing CSF and dilute serum samples for polyacrylamide gel electrophoresis has a marked effect on the pattern of the resolved proteins. The incorporation of sucrose or glycerol into the sample seriously impairs the quality of the resolution and affects the actual number of bands resolved and the relative mobility of certain proteins. The use of sucrose as an anti-convection medium in dense samples such as serum and CSF is not recommended.