Thirteenth Daniel C. Baker, Jr, memorial lecture. Monoclonal antibodies: the coming revolution in diagnosis and treatment of human disease.

Hybridoma technology has revolutionized the diagnosis of human disease. Owing to the unique specificity of monoclonal antibodies (MABs) of murine origin, hundreds of highly sensitive, accurate in vitro diagnostic tests have been developed for clinical laboratory and at-home use. Major research efforts are now underway to exploit this specificity for direct therapeutic purposes: MABs are being used by themselves in treating lymphomas, and in conjugated form with radionuclides, toxins, and chemotherapeutic drugs to treat a variety of tumors. Human MABs now are being tested in tumor imaging and for treatment of infectious diseases. New technologies have been developed to alter the genetic structure of MABs (eg, chimeric antibodies) to increase their treatment efficacy. As development of therapeutic uses for these reagents continues and, especially, as they receive approval for general clinical use, hundreds of kilograms per year will have to be produced. This paper reviews some of the recent developments in this area and describes two novel technologies that have been developed for large-scale cultivation of hybridoma cells.

Erythrocyte deformability and size measured in a multiparameter system that includes impedance sizing.

A Coulter Counter was modified to allow the photographing of erythrocytes as they exist the sizing orifice. These photographs provide the necessary information to determine the shape of individual cells. This data was combined on a cell-by-cell basis with the corresponding impedance signal to determine accurately both the cell size and deformed state of each cell. The particle shape is required if the size is to be determined accurately. Normal, diabetic and sickle cell samples were used in this study. The cell size was corrected according to theory that relates the impedance signal with cell shape. Although there was no objective test to determine the efficacy of this calculation in terms of improving the size measured, the reduction in the associated distribution width is taken as indicative of an improvement in the measuring process. The data presented represent the first attempt to relate cell size with deformed state on a cell-by-cell basis and indicates the potential usefulness of multiparameter deformability and size measurements.

Differentiation of HL-60 cells: cell volume and cell cycle changes.

HL-60 promyelocytic leukemic cells can differentiate into more mature myeloid cells with the addition of dimethylsulfoxide, butyric acid or retinoic acid and can differentiate into macrophages with the addition of phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). After the addition of an inducer, the HL-60 cell volume shows a daily decrease while the cell number increases at a rate similar to the untreated control cells. Flow cytometry measurements show an increase in G1 cells and a decrease in S cells after day 1. Since the generation time is constant, the data suggest that the length of time spent in the different cell cycle stages has changed during differentiation. Within 3 hours after the addition of TPA to HL-60 cells, selective adhesion of G1 cells occurs. Smaller sized cells are recovered from the flask bottom and larger sized cells are recovered from the supernate. Flow cytometric analysis reveals a G1 and S block in cells obtained from both the supernatant and from the flask bottom. After 1 day of TPA incubation, there is preferential adhesion of G1 and G2 cells with the nonadherent cells being primarily in the S and G2 cell cycle stages and undergoing a cell cycle traverse.

Collodion membrane secures cells sorted by flow cytofluorometry onto microscope slides.

Quantitative microscopic cytology of cells previously sorted by flow cytofluorometry has been hindered by the loss of cells from the microscope slide during staining procedures. The simple application of a semi-permeable membrane of collodion over fixed or unfixed cells sorted directly onto a microscope slide secured virtually 100% of the cells onto the slide. Cells covered with the collodion membrane studied with Papanicolaou's stain as well as routine clinical cervical cytologic preparations. In contrast, fewer than one half of the cells sorted onto uncoated or albumin coated slides were retained after staining.

Multiangle light-scattering analysis of murine teratocarcinoma cells.

Stem cells of the mouse testicular teratocarcinoma are capable of giving rise in vivo and in vitro to a wide variety of cell and tissue types representative of each embryonic germ layer. Multiangle light-scattering measurements in a flow system have been made on these stem cells and on a variety of their differentiated derivatives. This technique is capable of distinguishing the stem cells from parietal yolk sac cells, visceral yolk sac cells, neuronal cells and squamous cells. However, multipotential stem cells cannot be distinguished from stem cells that are restricted in their development to a single pathway.

Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.

Light-scatter analysis of microalgae. Correlation of scatter patterns from pure and mixed asynchronous cultures.

A method is described for the first time for rapid and accurate discrimination among several algal types by their light-scattering properties alone. Using a multiangle light-scattering flow system, we obtained light-scatter patterns for individual cells in asynchronous cultures of Chlorella, Chlamydomonas, and Anacystis. The patterns are consistent and distinct for each species. By these signatures, each algal type can be recognized within mixtures.