Immunofixation detected both light chains down to a concentration of at least 10 mg/L, whereas the detection limits on acid violet-stained HRAGE gels were 20 mg/L for the [kappa] chain and 10 mg/L for the [lambda] chain.
QUANTIFICATION OF BJP IN UNCONCENTRATED URINE BY DENSITOMETRIC SCANNING OF HRAGE GELS
Unconcentrated urine was then subjected to HRAGE and acid violet staining (Fig.
We compared the BJP concentrations obtained with HRAGE and acid violet staining, using on-gel human albumin calibrators with the BJP concentrations obtained with a comparison method, i.e., multiplication of the BJP fraction of the total urine proteins (determined by agarose gel electrophoresis of concentrated urine) by the total urine protein concentration (determined on the Hitachi 917 automated analyzer; Fig.
Among the 230 samples analyzed, discrepancies in the interpretation occurred among the three biologists in only eight cases: seven cases analyzed by HRAGE + IFE and one analyzed by IFPOD.
The sample that was found positive by HRAGE + IFE only consisted of a monoclonal free [kappa] chain, which could therefore not be detected with the IFPOD method in the conditions used.
Statistical analysis showed that for CDMS vs all others patient groups, the sensitivities (95% confidence interval) of the IFPOD and HRAGE + IFE methods were identical at 83% (51-97%).
As currently performed in the laboratory, IFE allows for the detection of isotypes other than IgG by use of anti-[kappa] and anti-[lambda] antisera in addition to the HRAGE. In such cases, identification is pursued by a new IFE performed with anti-IgA and anti-IgM antisera.
A discriminator value of 0.0150 was chosen for the sum of the slopes of included inflection points in the [gamma]-region according to optimal sensitivity and specificity compared with HRAGE evaluations.
The sequential use of all six algorithms for MCs on the total 711 samples detected a total of 94 of the 95 samples judged to contain MCs by HRAGE, corresponding to a sensitivity of 98.9%.
All five contained identifiable peaks attributable to circulating light chains, despite a lack of visible bands on HRAGE in two cases.
The sensitivity of this algorithm for oligoclonality as a whole was low, 37% (16 of 43) compared with the subjective evaluation of HRAGE gels with an optimal discriminator value of 0.0150, but agreement increased from 11% (2 of 19) for mild oligoclonality (+) to 47% (8 of 17) for moderate (++) and 85% (6 of 7) for severe (+++) oligoclonality, confirming a correlation between the visual impression of oligoclonality in the gel and irregularity in the CZE-absorbance curve.