Cytometric detection of DNA amplified with fluorescent primers: applications to analysis of clonal bcl-2 and IgH gene rearrangements in malignant lymphomas

Follicular lymphomas comprise almost two thirds of the US adult non- Hodgkin's lymphomas (NHL) and are the most common malignancy of B- lineage lymphocytes. Polymerase chain reaction (PCR) protocols have been developed to detect the t(14;18) translocation, which juxtaposes the bcl-2 proto-oncogene to the Ig heavy-chain (IgH) gene in 85% of follicular lymphomas and monoclonal rearrangements of the IgH gene in B- cell NHL that lack bcl-2 rearrangements. We used PCR to amplify bcl-2 and IgH rearrangements in DNA from patients with lymphoproliferative disorders and analyzed the products in parallel by gel electrophoresis and flow cytometry, which detected PCR products incorporating fluoresceinated oligonucleotide primers by sequence-specific capture to oligonucleotide-coated magnetic beads. Overall, flow cytometry was superior to electrophoresis of ethidium-bromide-stained agarose gels for detection of products of nested PCR to detect intergenic rearrangements involving bcl-2 and single primer-pair amplification of clonal rearrangement of IgH. Flow cytometric analysis detected bcl-2 translocations in 12 of 13 CD10+ B-cell lymphomas and clonal IgH rearrangements in 14 of 17 monoclonal B-cell populations. In contrast, analysis by gel electrophoresis detected bcl-2 translocations in only 10 of 13 CD10+ and clonal IgH gene rearrangements in only 9 of 17 monoclonal B-cell populations. Flow cytometric analysis was more sensitive than gel electrophoresis and could detect a 16-fold greater dilution of a bcl-2-amplified product than gel electrophoresis. Similarly, flow cytometry could detect an amplification product when template DNA was diluted 10,000-fold, whereas gel electrophoresis only detected amplification products when template was subjected to dilution between 100- and 1,000-fold. This shows the utility of flow cytometry for the analysis of DNA amplification products incorporating fluorochrome-labeled primers as a rapid, objective alternative to conventional strategies. Because current-generation clinical laboratories emphasize automation, flow cytometric analysis of PCR- amplified products shows increased analytic sensitivity and offers a vehicle for automation of DNA amplification tests.     

Reduced invasion and growth of Plasmodium falciparum into elliptocytic red blood cells with a combined deficiency of protein 4.1, glycophorin C, and p55

In this investigation, we have measured the invasion and growth of the malaria parasite Plasmodium falciparum into elliptocytic red blood cells (RBCs) obtained from subjects with homozygous hereditary elliptocytosis. These elliptocytic RBCs have been previously characterized to possess molecular defects in protein 4.1 and glycophorin C. Our results show that the invasion of Plasmodium falciparum into these protein 4.1 (-) RBCs is significantly reduced. Glycophorin C (-) Leach RBCs were similarly resistant to parasite invasion in vitro. The intracellular development of parasites that invaded protein 4.1 (-) RBCs was also dramatically reduced. In contrast, no such reduction of intracellular parasite growth was observed in the glycophorin C (-) Leach RBCs. In conjunction with our recent finding that a third protein termed p55 is also deficient in protein 4.1 (-) and glycophorin C (-) RBCs, the present data underscore the importance of the membrane-associated ternary complex between protein 4.1, glycophorin C, and p55 during the invasion and growth of malaria parasites into human RBCs.     

Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.