Clinical and clonal characteristics of monoclonal immunoglobulin M-associated type I cryoglobulinaemia.

Monoclonal immunoglobulin M-associated type I cryoglobulinaemia is poorly characterised. We screened 534 patients with monoclonal IgM disorders over a 9-year period and identified 134 patients with IgM type I cryoglobulins. Of these, 76% had Waldenström macroglobulinaemia (WM), 5% had other non-Hodgkin lymphoma (NHL) and 19% had IgM monoclonal gammopathy of undetermined significance (MGUS). Clinically relevant IgM-associated disorders (including cold agglutinin disease [CAD], anti-MAG antibodies, amyloidosis and Schnitzler syndrome) coexisted in 31%, more frequently in MGUS versus WM/NHL (72% vs. 22%/29%, p < 0.001). The majority of those with cryoglobulins and coexistent CAD/syndrome had the molecular characteristics of a CAD clone (wild-type MYD88 in 80%). A half of all patients had active manifestations at cryoglobulin detection: vasomotor (22%), cutaneous (16%), peripheral neuropathy (22%) and hyperviscosity (9%). 16/134 required treatment for cryoglobulin-related symptoms alone at a median of 38 days (range: 6-239) from cryoglobulin detection. At a median follow-up of 3 years (range: 0-10), 3-year cryoglobulinaemia-treatment-free survival was 77% (95% CI: 68%-84%). Age was the only predictor of overall survival. Predictors of cryoglobulinaemia-related treatment/death were hyperviscosity (HR: 73.01; 95% CI: 15.62-341.36, p < 0.0001) and cutaneous involvement (HR: 2.95; 95% CI: 1.13-7.71, p = 0.028). Type I IgM cryoglobulinaemia is more prevalent than previously described in IgM gammopathy and should be actively sought.

Thermodynamic and voltammetric characterization of the metal binding to the prion protein: insights into pH dependence and redox chemistry.

The prion protein is a high-affinity copper binding protein that plays a role in the neurodegenerative prion diseases when it is converted into an altered isoform. The function of the protein remains controversial, but its relationship to its metallochemistry has prompted further investigation. While many researchers continue to use short peptide models for binding studies, the clear discrepancy between data obtained with such models when compared to those of full-length recombinant proteins requires clarification with this more appropriate model. Isothermal titration calorimetry was used to assess metal affinity for PrP. Using both full-length native and recombinant prion protein, we have demonstrated that the prion protein binds copper but has little affinity for other metals. Metal binding is highly pH sensitive, being optimal at pH 7.5 for copper, nickel, and zinc and at pH 5.5 for iron. Metal binding affinity for PrP was not altered by protein glycosylation. The use of suitable thermodynamic modeling reveals complex and cooperative copper binding, with evidence of negative cooperativity within the octarepeat region. Cyclic voltammetry was utilized to assess the electrochemistry of copper-charged prion protein, and we show that mPrP has a redox potential of 0.03 +/- 0.01 V versus the saturated calomel electrode at pH 7. The analysis also indicated that PrP is able to undergo reversible redox cycling with equal oxidative and reductive charges that are largely dependent on the copper bound to the octarepeat. The fifth site provides a small contribution to this redox activity, but only when the octarepeat is present. These results show conclusively that PrP can utilize copper for electron transfer, which would be expected for a radical detoxifying enzyme, and that the octarepeat region is the functional domain.

Comparison of Free Light Chain Assays: Freelite and N Latex in Diagnosis, Monitoring, and Predicting Survival in Light Chain Amyloidosis.

OBJECTIVES: Measurement of serum free light chains (FLCs) is critical in diagnosis, prognosis, and monitoring treatment responses in light chain (AL) amyloidosis. We compare the Freelite assay (polyclonal antibodies to hidden light chain epitopes), which is the current gold standard, with a new assay: a mixture of monoclonal antibodies to light chain epitopes (N Latex). METHODS: We collected 240 serum samples from 94 consecutive patients with newly diagnosed AL amyloidosis (at least three serial serum samples during the first 6 months) analyzed at the National Amyloidosis Centre, London, from January 2011 to April 2012. Concordance in detecting abnormal light chain components and hematologic response was assessed at 2, 4, and 6 months. RESULTS: The κ and λ clonal light chain involvement was 21% and 79%, respectively, with an abnormal κ/λ ratio or detectable protein in 78.7%. Median κ, λ, and difference in involved and uninvolved FLCs by Freelite and N Latex assays were 17.3 vs 16 mg/L (R(2 ) = 0.91), 48.8 vs 52.6 mg/L (R(2) = 0.52), and 43.2 vs 39.1 mg/L, respectively. Discordant κ/λ ratios at presentation were as follows: 10 of 90 abnormal by Freelite/normal by N Latex and 11 of 90 abnormal by N Latex/normal by Freelite. CONCLUSIONS: Both FLC assays show good correlation in detecting the abnormal light chain subtype with discordance in absolute values and thus are not interchangeable.

Analysis of 17 α-hydroxyprogesterone in bloodspots by liquid chromatography tandem mass spectrometry.

BACKGROUND: Monitoring of treatment for patients diagnosed with congenital adrenal hyperplasia (CAH) can be performed by measuring the concentration of 17α-hydroxyprogesterone (17OHP) in bloodspots collected on filter papers. A method is described here for measuring 17OHP by liquid chromatography tandem mass spectrometry (LCMSMS). METHODS: 17OHP was extracted by liquid-liquid extraction and analysed by LCMSMS. The method was validated for sensitivity, specificity, linearity, recovery, ion suppression, precision and bias. RESULTS: The standard curve was linear from 0 to 400 nmol/L. Intra-assay %CVs were <10 and inter-assay %CVs were <15 over the range 10-200 nmol/L. Limit of quantitation was 6 nmol/L. No ion suppression was detected. The only interfering compound detected was deoxycorticosterone, an intermediate steroid with the same molecular weight as 17α-hydroxyprogesterone. The method was more accurate and precise than an existing radioimmunoassay. There was poor correlation between the two assays. CONCLUSIONS: We have developed a sensitive and specific assay suitable for quantitation of 17OHP in bloodspots. This method performs better than radioimmunoassay and allows smaller samples to be used.