Elution mode of Pneumocystis carinii cysts in gravitational field-flow fractionation.

The simplest field-flow fractionation technique, i.e. gravitational, was used in an attempt to purify a Pneumocystis carinii cyst suspension. This parasite is an opportunistic invader in immunocompromised patients, especially those suffering from AIDS. The cyst stage is spherical and 5 microns in diameter. Unexpected retention times, not systematically related to the size and the density of the parasite, were obtained under various experimental conditions. When silicone-coated walls were used, Pneumocystis carinii cysts were eluted in the void volume, whereas when uncoated walls were used with a sodium dodecyl sulphate-enriched carrier phase, retention was observed. These phenomena are probably related to the high degree of hydrophobicity of these micrometre-sized biological particles; this degree can be easily determined. The use of the gravitational field-flow fractionation technique can be of a great interest for the development of new methods for diagnostic purposes. Particle-wall interactions and their modifications due to the carrier phase or to the wall treatment can be employed in the search for new bronchoalveolar lavage solutions.

Monitoring of an experimental red blood cell pathology with gravitational field-flow fractionation.

Gravitational field-flow fractionation is a simple method suitable for the separation of micrometre-size particles, for example the red blood cells (RBC). The variation in the composition of the RBC population in a rabbit during an experimental reversible anaemia induced by phenylhydrazine was studied. Blood samples taken at different stages of the anemia showed differences in the retention and shape of the elution profiles. Microscopic observations of original RBC samples and fractions collected at the outlet of the fractionation channel make a description of the RBC population possible. The three species observed were the normal RBC, the newly produced reticulocytes, and the blood cells containing Heinz bodies (intracellular haemoglobin precipitates). A decrease of the normal RBC from 96 to 1% was observed over five days. The production of reticulocytes in bone marrow is stimulated by the anaemia and increases in percentage after the second day of the anaemia (from 1 to 16%). RBC with Heinz bodies, which appear on the third day, were also studied. Granulometric studies were performed on the RBC sampled from the rabbit each day as well as on some fractions eluted by field-flow fractionation. Reinjection procedures of some cell subpopulations of known size distribution were also performed. The relaxation process of these cells was then studied to approximate their density properties. It was observed that RBC of different density but of the same average size were selectively eluted, as were cells of equivalent density but of different size. Injection of the cells at different stop-flow times enabled the study of the relaxation process on the elution profiles. The results, compared with systematic microscopic observation and size analysis, permit the description of modifications in the RBC composition as well as the purification of subpopulations at each stage of the anaemia. The correlation observed between the fractionation profiles and the progress or the regression of the anaemia opens a new field in the analytical monitoring of this type of pathology.