Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins.

BACKGROUND: Given the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions. METHODS: By parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of α- and ß-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/ß-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared. RESULTS: The α/ß-ratio is a poor marker for thalassemia patients, yet deviant α/ß-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval. CONCLUSIONS: Although subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the α/ß-ratio as screening parameters.

Therapeutic drug monitoring of infliximab: performance evaluation of three commercial ELISA kits.

BACKGROUND: Therapeutic drug monitoring (TDM) of infliximab (IFX, Remicade®) can aid to optimize therapy efficacy. Many assays are available for this purpose. However, a reference standard is lacking. Therefore, we evaluated the analytical performance, agreement and clinically relevant differences of three commercially available IFX ELISA kits on an automated processing system. METHODS: The kits of Theradiag (Lisa Tracker Infliximab), Progenika (Promonitor IFX) and apDia (Infliximab ELISA) were implemented on an automated processing system. Imprecision was determined by triplicate measurements of patient samples on five days. Agreement was evaluated by analysis of 30 patient samples and four spiked samples by the selected ELISA kits and the in-house IFX ELISA of Sanquin Diagnostics (Amsterdam, The Netherlands). Therapeutic consequences were evaluated by dividing patients into four treatment groups using cut-off levels of 1, 3 and 7 µg/mL and determining assay concordance. RESULTS: Within-run and between-run imprecision were acceptable (≤12% and ≤17%, respectively) within the quantification range of the selected ELISA kits. The apDia assay had the best precision and agreement to target values. Statistically significant differences were found between all assays except between Sanquin Diagnostics and the Lisa Tracker assay. The Promonitor assay measured the lowest IFX concentrations, the apDia assay the highest. When patients were classified in four treatment categories, 70% concordance was achieved. CONCLUSIONS: Although all assays are suitable for TDM, significant differences were observed in both imprecision and agreement. Therapeutic consequences were acceptable when patients were divided in treatment categories, but this could be improved by assay standardization.

Two cases of q-Fever in hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare B-cell lymphoproliferative disorder accounting for about 2% of all leukemias. The clinical course is indolent, however HCL patients are particularly susceptible to infections. Here we report two cases of Q-fever as first manifestation of disease in two patients affected by HCL. Both patients described in this report showed an unusually sluggish clinical response to the antibiotic treatment with ciprofloxacin probably because of the marked immunodeficiency. However, treatment of HCL with cladribine administered soon after the resolution of QF pneumonitis was uneventful and led to a complete remission in both cases. Most probably the association of Coxiella burnetii (CB) infection and HCL that we observed in two patients is due to chance. However, a hairy cell resembling transformation of freshly isolated human peripheral blood lymphocytes upon CB has been showed. We think that the possibility of CB infection in febrile HCL patient should be always taken in mind, especially in endemic areas. In addition the potential for such infections to become chronic in HCL patients should not be overlooked and the reporting of further cases should be encouraged.

The effect of paraproteins on the erythrocyte sedimentation rate: a comparison between the StarrSed and TEST 1.

BACKGROUND: The principle of the erythrocyte sedimentation rate (ESR) as assessed by TEST 1 is different from that of Westergren-based methods. This could result in different influences on the tests by paraproteins. METHODS: We investigated the effect of paraproteins on ESR readings by TEST 1 (y) and the StarrSed (x), a Westergren-based method, in 142 patients with paraproteinaemia. Agreement (Passing-Bablok) and bias (Bland-Altman) between methods was investigated and compared with that of a control population. RESULTS: A poor agreement between the two methods was found in patients with a paraprotein (y = 0.67x + 3.3) in comparison with that of the control population (y = 0.96x + 0.2). Large differences between methods were present when ESR readings were >40 mm/hour, but clinical interpretation was similar in 90% of cases. Linear regression showed a concentration dependent influence of paraproteins on ESR readings by the StarrSed, especially for immunoglobulin class IgM. CONCLUSION: ESR readings by TEST 1 result in similar clinical interpretation for most subjects, but readings are less influenced by the presence of a paraprotein than those of a Westergren-based method.