Individualized PCR strategies hamper comparability of molecular results between different laboratories in several fields of medicine. To harmonize BCR-ABL mRNA quantification an international multicenter trial involving 37 laboratories in 14 countries was initiated using 10 samples, each containing various dilutions (10, 2, 1 and 0.1%) of b3a2 or b2a2 BCR-ABL positive in normal leukocytes and negative controls. A novel control plasmid (pME-2) was designed for external calibration containing BCR-ABL and glucuronidase-b (GUS) sequences. Median BCR-ABL/ABL ratios were 9.1, 1.8, 0.85 and 0.11% in b3a2 samples and 9.5, 1.6, 0.84 and 0.11% in b2a2 samples. Median BCR-ABL/GUS ratios were 3.4, 0.77, 0.37 and 0.042% in b3a2 samples and 2.8, 0.48, 0.29 and 0.031% in b2a2 samples. The coefficients of variation were 0.62 for ratios BCR-ABL/ABL and 1.03 for ratios BCR-ABL/GUS. Five of 37 evaluable participating laboratories (13%) detected low BCR-ABL copy numbers in negative control samples; one laboratory failed to detect BCR-ABL in a low-level sample. We conclude that the use of a common control plasmid does indeed improve comparability of BCR-ABL mRNA quantification results. However, further standardizing efforts like introducing a calibrator and regular control rounds are needed.

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The weakness in myasthenia gravis (MG) is mediated by T helper cell (Th)-dependent autoantibodies against neuromuscular epitopes. So far, analyzing Th phenotypes or antigen specificities has yielded very few clues to pathogenesis. Here we adopt an alternative antigen-independent approach, analyzing T cell receptor (TCR) Vb usage/ expansions in blood from 118 MG patients. We found major expansions (! five standard deviations above the mean of 118 healthy, individually age-and sex-matched controls) in diverse Vb in 21 patients (17.6%, p<0.001) among CD4 + T cells, and in 45 patients (38.1%, p<0.001) among CD8 + T cells. In informative probands, the expanded CD4+ cells consistently showed a Th cell phenotype (CD57 + CXCR5 + ) and expressed Th1 cytokines. Furthermore, their expression of markers for activation, lymphocyte trafficking and B cell-activating ability persisted for ! 3 years. Surprisingly, we noted a selective decline in the expansions/their CD57 positivity while the probands' MG was improving. CDR3 spectratyping suggested mono-or oligoclonal origins, which were confirmed by the prevalent TCR Vb CDR3 sequences of Th cells cloned from repeat bleeds. Thus, our data provide evidence for persistent clonally expanded CD4 + B helper T cell populations in the blood of MG patients. These unexpected CD4 + expansions might hold valuable clues to MG immunopathogenesis.