Studies on the factors influencing the hydrogen peroxide hemolysis test.

There seems to be a greater variation between laboratories in hydrogen peroxide hemolysis techniques than in other clinical laboratory procedures. In this report, factors influencing hemolytic values were examined and the effect of the combination of these factors on the results was analysed statistically by using the orthogonal array. Factors influencing hemolysis induced by hydrogen peroxide were as follows; the concentration of hydrogen peroxide, temperature in the peroxide reagent when added to the red cell suspension, the red cell concentration in the cell suspension and the addition of charcoal to the reaction mixture. However, addition of charcoal may not be essential to stabilize the hemolytic values and other factors such as keeping blood for 4 hours at room temperature before testing, the difference between investigators, a reaction time 2 or 3 hours and the technique of adding peroxide reagent to the cell suspension, had little effect on hemolysis. The most important factor was the temperature in hydrogen peroxide solution. The estimated hemolytic values by the orthogonal array linearly correlated with the plasma tocopherol levels.

Propagation of hepatitis A virus in a renal cell line JTC-12.P3 of cynomolgus monkey origin.

Human hepatitis A virus (HAV) derived from 10% HAV infected marmoset liver homogenate and faeces from acute hepatitis A was successfully propagated in vitro in a new cell line, JTC-12.P3. The cell line originated from the renal cortex of cynomolgus monkey which was adapted to growth in a serum free, protein free, chemically defined synthetic medium. Replication of the virus was followed by solid phase RIA, immunofluorescent staining, and immunoelectron microscopy. The propagation of HAV occurred over several passages, with the 1st and 2nd passages requiring at least 8 weeks each. However, with the increasing serial passage of virus, the period needed to detect it was shortened, suggesting the adaptation of HAV to the cells. The identity of the newly synthetized virus particles with HAV was established by immunoelectron microscopy and immunofluorescent blocking effect with human convalescent serum. The HAV propagated in JTC-12.P3 cells banded predominantly at a density of 1.32 g/cm3 in CsC1 gradient. The infected cells showed no specific signs of CPE. Ultrastructurally, clusters of virus particles 27 nm in diameter were observed mainly in the lysosomal vesicles and freely in crystalline array in the cytoplasm, too. Addition of 0.1% of various anti-HAV negative sera or of prostaglandin E1 to the culture medium caused accelerated propagation of HAV.

Rapid and accurate measurement of anti-A/B IgG antibody in ABO-unmatched living donor liver transplantation by surface plasmon resonance.

High anti-blood group A or B (anti-A/B) immunoglobulin G (IgG) haemagglutination titres are associated with poor graft survival in ABO-unmatched liver transplantation. We have previously reported that the surface plasmon resonance (SPR) method can be used to measure anti-A/B IgG levels in the plasma very quickly and quantitatively. The aim of this study was to brush up this SPR method. The anti-A/B IgG antibodies (Abs) were purified from the plasma of healthy volunteers by affinity chromatography and used to establish standard curves for the SPR and flow cytometry (FCM) methods. The haemagglutination test tube (TT), FCM and SPR methods were then used to measure the changes over time in the anti-A/B IgG titres of 25 ABO-unmatched living donor liver transplantation (LDLT) recipients. The standard curve permitted the SPR values for the anti-A/B IgG titres to be expressed in microg mL(-1) units. The SPR measurements of the anti-A/B IgG levels in the LDLT recipients correlated very well with the FCM values, whereas the TT values correlated poorly with either method. Furthermore, the SPR method accurately detected the effects of plasma exchange. In conclusion, the SPR method is an accurate, time- and labour-saving method for measuring anti-A/B IgG titres that can be easily standardized.

Effect of a synthetic adjuvant for inducing anti-tumour immunity.

An acylated derivative of muramyl dipeptide (MDP), 6-O-(2-tetradecyl-hexadecanoyl)-muramyl-dipeptide (B30-MDP) is a strong adjuvant effective in inducing cell-mediated immunity. We used B30-MDP as an adjuvant for induction of anti-tumour immunity. Guinea-pigs which were injected repeatedly with a mixture of X-ray-treated leukaemic cells and B30-MDP dissolved in phosphate buffered saline resisted a challenge of leukaemia cells and showed no sign of leukocytosis. The immunity induced was tumour-specific and retained for more than 100 days. These results suggest that B30-MDP is useful as a simple but potent immunotherapeutic tool.