No abstract

Automatic scanning was more efficient and consistent than manual scanning for detection of rare FCs and required considerably less operator time. Automatic scanning using FISH is more sensitive than that using PRINS. The study validates automatic scanning retrieval of FCs from maternal blood.

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1–10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Summary. Persistent B-cell lymphocytosis (PPBL) is a haematological disorder diagnosed primarily in adult female smokers that is characterized by a polyclonal increase in peripheral blood B lymphocytes and a moderate elevation of serum IgM. B lymphocyte-associated cellular abnormalities, such as the occurrence of multi-lobed nuclei, increased bcl2/ Ig gene rearrangements and the identification of an extra long-arm chromosome (i3)(q10) in the B-cell population, indicate that PPBL could be part of a multi-step process leading to the emergence of a malignant B lymphoproliferation. However, the resulting impact on cellular functional properties remains to be elucidated. Our goal was to address that aspect via the study of B-cell activity following stimulation through CD40, a key molecule of the tumour necrosis factor receptor superfamily involved in B lymphocyte development. In contrast to normal B cells, PPBL B lymphocytes were unable to respond to the proliferative signal delivered in vitro by CD40, indicating a defect in the CD40 activation pathway. Polymerase chain reaction amplification and sequencing of the receptor as well as FACScan analysis of patient B lymphocytes dismissed the possibility of a defect in either CD40 structure or expression. Moreover, Western blot analysis of tyrosine phosphorylation, an early event in the CD40-signalling cascade, was similar in patients and controls, leading to the conclusion that the defect affecting B lymphocytes in PPBL patients is probably located downstream of that signalling cascade.

Although intravenous immunoglobulins (IVIg) are widely used in the treatment of many autoimmune and inflammatory diseases, the mechanisms of action are still unclear in most cases. We have recently reported the presence of soluble autoimmune complexes (auto-IC) in human serum after the addition of a dose of IVIg similar to the one used in therapy. Here, we report the isolation and characterization of the responsible auto-IgG present in IVIg. The auto-IgG were purified by affinity chromatography on serum proteins immobilized on Sepharose. The purified auto-IgG constituted approximately 3% of the IgG present in IVIg and recognized a wide variety of structures in ELISA as well as many serum proteins on western blots. Auto-IC were formed in human serum following the addition of an amount of purified auto-IgG sufficient to over-saturate the auto-IgG inhibitory mechanisms known to be present in normal serum. These results indicate that most of the IgG present in IVIg are not involved in the formation of the soluble auto-IC, raising the possibility of preparing from IVIg a novel product which could be used for the treatment of the autoimmune diseases in which IC are thought to play an important role.