Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method

Aluminum (Al) components in vaccines are known to act as adsorbents that interfere with accurate protein quantification by the Lowry method. Therefore, certain modifications based on the characteristics and compositions of the vaccine are required for determination of protein contents.We investigated the effects of an additional centrifugal separation and found that protein contents were overestimated by up to 238% without centrifugation through a collaborative study performed with hepatitis B vaccines containing Al. However, addition of a centrifugation step yielded protein concentrations that were similar to the actual values, with small coefficients of variation (CVs). Proficiency testing performed in 11 laboratories showed that four laboratories did not have satisfactory results for vaccines containing aluminum hydroxide, although all laboratories were proficient in protein analysis when samples did not contain aluminum hydroxide. Incomplete resuspension of aluminum hydroxide solution with alkaline copper solution was the major cause of insufficient proficiency in these laboratories.     

The development and validation of methods for evaluating the immune system in preweaning piglets

The preweaning piglet has been found to be a valuable research model for testing ingredients used in infant formula. As part of the safety assessment, the neonates' immune system is an important component that has to be evaluated. In this study three concurrent strategies were developed to assess immune system status. The methods included (1) immunophenotying to assess circulating innate immune cell populations, (2) monitoring of circulating cytokines, particularly in response to a positive control agent, and (3) monitoring of localized gastrointestinal tissue cytokines using immunohistochemistry (IHC), particularly in response to a positive control agent. All assays were validated using white papers and regulatory guidance within a GLP environment. To validate the assays precision, accuracy and sample stability were evaluated as needed using a fit for purpose approach. In addition animals were treated with proinflammtory substances to detect a positive versus negative signal. In conclusion, these three methods were confirmed to be robust assays to evaluate the immune system and GIT-specific immune responses of preweaning piglets.     

Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia

The use of peripheral blood rather than marrow has potential advantages for monitoring minimal residual disease during the treatment of leukaemia. To determine the feasibility of using blood, we used a sensitive polymerase chain reaction method to quantify leukaemia in the blood and marrow in 35 paired samples from 15 children during induction treatment. Leukaemic cells in the blood ranged from 1.1 × 10⁻² to < 9.4 × 10⁻⁷ leukaemic cells/total cells, corresponding to 1.3 × 10⁷ to < 2 × 10³ leukaemic cells/l. In 15 paired samples, leukaemia could be quantified in both tissues and in 20 paired samples, leukaemia was not detected in one or both tissues so that only upper level limits could be set. In the former 15 pairs, the level of leukaemia in peripheral blood was directly proportional to that in marrow but was a mean of 11.7-fold lower. Leukaemia in blood was detected in 10/12 pairs in which the level in marrow was > 10⁻⁴, but in only two of 13 pairs in which the level in marrow was < 10⁻⁵. Patients studied at multiple time-points showed parallel declines in the number of leukaemic cells in both tissues. The results showed that leukaemia could be monitored in peripheral blood during induction therapy, and quantitative considerations based on the results suggest that monitoring of blood during post-induction therapy may be of value in detecting molecular relapse.     

Imaging Risk in Multisystem Inflammatory Diseases

Rheumatic diseases are immune-mediated inflammatory multisystem diseases with frequent cardiovascular manifestations including perimyocarditis, valvular disease, coronary artery disease, heart failure with or without preserved ejection fraction, pulmonary hypertension, aneurysms, and thrombosis. Echocardiography, carotid ultrasonography, cardiac computed tomography, cardiac magnetic resonance imaging, and positron emission tomography are valid diagnostic tools for the detection of the cardiovascular complications of the multisystem diseases that frequently determine prognosis. Furthermore, the findings of these methods may offer additive risk stratification in asymptomatic patients over the conventional risk scores used to assess cardiovascular risk in the primary prevention setting. Finally, the imaging methods offer a unique opportunity to monitor the effects of treatment on atherosclerotic lesions, coronary microcirculatory dysfunction, myocardial inflammation and fibrosis. However, studies are needed to investigate whether improvement of imaging markers by treatment or selection of treatment according to its effects on surrogate imaging markers is linked to improved prognosis.     

Direct Measurement of ATP7B Peptides Is Highly Effective in the Diagnosis of Wilson Disease

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