ABSTRACT
The use of automated microscopy has reached the maturity necessary for its routine use in the clinical pathology laboratory. In the following study we compared the performance of an automated microscope system (MDS) with manual method for the detection and analysis of disseminated tumor cells present in bone marrow preparations from breast carcinoma patients. The MDS System detected rare disseminated tumor cells among bone marrow mononuclear cells with higher sensitivity than standard manual microscopy. Automated microscopy also proved to be a method of high reproducibility and precision, the advantage of which was clearly illustrated by problems of variability in manual screening. Accumulated results from two pathologists who had screened 120 clinical slides from breast cancer patients both by manual microscopy and by use of the MDS System revealed only two (3.8%) missed by the automatic procedure, whereas as many as 20 out of 52 positive samples (38%) were missed by manual screening.
Subject(s)
Bone Marrow Examination/methods , Image Processing, Computer-Assisted/methods , Neoplasm Metastasis/diagnosis , Neoplastic Cells, Circulating , Bone Marrow Examination/instrumentation , Breast Neoplasms/diagnosis , Carcinoma/diagnosis , Carcinoma/secondary , Female , Humans , Image Processing, Computer-Assisted/instrumentation , Immunohistochemistry , Mass Screening/instrumentation , Mass Screening/methods , Reproducibility of Results , Sensitivity and Specificity , Single-Blind Method , Tumor Cells, CulturedABSTRACT
In this study we evaluated the performance of a system for the enrichment, identification and analysis of fetal cells in maternal peripheral blood. Blood samples were collected from women after chorionic villus sampling and enriched for the presence of nucleated erythrocytes using a three-step procedure, namely: (a) centrifugation to separate nucleated red blood cells (NRBCs) from the majority of red blood cells (RBCs) and white blood cells (WBCs); (b) selective lysis of the remaining maternal RBCs; (c) separating the NRBCs from the remaining WBCs in a three-layer density gradient. Fetal cells were identified by using a monoclonal antibody against the gamma-chain of fetal haemoglobin (anti-HbF) and a nuclear stain (DAPI). Additionally, to further increase the specificity of the identification, and to eliminate some of the undesired staining by maternal leukocytes, a fluorescent antibody (CD45) was added. The sex chromosome complement of the cells was determined by fluorescence in situ hybridization (FISH) with X and Y-specific probes and the results were compared with the karyotypes obtained after analysis of chorionic villi. Using the described method, in all cases where the woman was carrying a male fetus (n=18) at least one XY cell was found, while no male cells were found in women carrying a female fetus. However, in the majority of cases with a male fetus (n=11) female HbF positive cells were found indicating the presence of maternal nucleated erythrocytes. The study demonstrates that the combination of anti-HbF and CD45 is a useful, but not fully specific, marker for fetal NRBCs and that additional markers are needed.
Subject(s)
Cell Separation/methods , Erythrocytes , Fetal Blood/cytology , Prenatal Diagnosis/methods , Antibodies, Monoclonal , Cell Nucleus , Centrifugation , Centrifugation, Density Gradient , Erythrocytes/ultrastructure , Female , Fetal Hemoglobin/analysis , Hemolysis , Humans , In Situ Hybridization, Fluorescence , Leukocyte Common Antigens/analysis , Male , Pregnancy , Sensitivity and Specificity , Sex ChromosomesABSTRACT
The preimplantation-embryo-development (Ped) gene influences the rate of cleavage division of preimplantation mouse embryos. The location of the Ped gene in the mouse major histocompatibility complex (MHC), the H-2 complex, has been inferred from the analysis of cleavage rates of embryos from mouse strains congenic at the H-2 complex. In this paper, formal genetic linkage studies were undertaken to evaluate linkage of the Ped gene to the H-2 complex. Co-segregation of Ped gene phenotype and H-2 haplotype was found in back-cross embryos. These data support the hypothesis that the Ped gene is linked to the H-2 complex.
Subject(s)
Blastocyst/immunology , Embryonic and Fetal Development , Genetic Linkage , Major Histocompatibility Complex , Animals , Blastocyst/cytology , Embryo Transfer , Female , H-2 Antigens/genetics , Haplotypes , Mice , Mice, Inbred C57BL , Phenotype , PregnancyABSTRACT
The preimplantation-embryo-development (Ped) gene, a gene that controls the cleavage rate of preimplantation mouse embryos, maps to the Qa-2 subregion of the mouse major histocompatibility complex (MHC). A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure was used to detect Qa-2 antigens on mouse embryos. The use of a monoclonal antibody specific for Qa-2 antigens showed that Qa-2 antigens were present on oocytes, 2-cell, 8-cell, and blastocyst-stage embryos, with the greatest expression found on blastocysts. Expression of Qa-2 antigens by the embryos correlated completely with Ped gene phenotype. Those embryos expressing the fast Ped allele showed the presence of Qa-2 antigens (Qa-2a mice), whereas those embryos expressing the slow Ped allele showed the absence of Qa-2 antigens (Qa-2b mice). It is hypothesized that the Qa-2 antigen may be the Ped gene product.
Subject(s)
Antigens, Surface/genetics , Blastocyst/immunology , Embryonic and Fetal Development , Histocompatibility Antigens Class I , Alleles , Animals , Blastocyst/cytology , Chromosome Mapping , Gene Expression Regulation , Mice , PhenotypeABSTRACT
An enzyme-linked immunosorbent assay (ELISA), which utilized anti-H-2 monoclonal antibody, was used to detect H-2 antigens on preimplantation mouse embryos. All embryonic stages studied, including unfertilized eggs and 1-cell, 2-cell, 8-cell, and blastocyst-stage embryos, showed the presence of H-2 antigens. To prove that the H-2 antigens were not cytophilically adsorbed to the embryos, blastocysts were treated with papain to strip off the H-2 antigens, and then the embryos were further incubated to allow the H-2 antigens to regenerate. After a 3-h incubation time, 60% of the H-2 antigens on the embryos had reappeared, proving that the H-2 antigens were synthesized by the embryos themselves.
Subject(s)
Embryo, Mammalian/metabolism , H-2 Antigens/analysis , Animals , Embryo, Mammalian/drug effects , Enzyme-Linked Immunosorbent Assay , Mice , Mice, Inbred Strains , Papain/pharmacology , Pronase/pharmacologyABSTRACT
An enzyme-linked immunosorbent assay (ELISA) was developed to detect H-2 antigens on preimplantation mouse embryos. The presence of these antigens was shown on early blastocysts from the outbred strain CF1. The assay consists of treating the embryos with purified anti-H-2 monoclonal antibodies followed by treatment with rabbit anti-IgG and protein A-beta-galactosidase conjugate. Removal of the zonae pellucidae by pronase did not change the sensitivity of the assay. However, longer treatment of the embryo surface with pronase eliminated the binding of the anti-H-2 antibodies. The embryo ELISA was shown to be more sensitive than an embryo cytotoxicity assay also described in this paper. The ELISA technique should be useful for future studies of the surface of early embryos.
Subject(s)
Antigens, Surface/immunology , Embryo, Mammalian/immunology , Embryonic Development , Enzyme-Linked Immunosorbent Assay , H-2 Antigens/immunology , Immunoenzyme Techniques , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Binding Sites, Antibody , Cytotoxicity Tests, Immunologic/methods , Female , Mice , Mice, Inbred C57BL , Pregnancy , RabbitsABSTRACT
The rate of preimplantation mouse embryo development was found to be influenced by genes both within and outside the H-2 complex. An H-2 associated gene, called Ped, was shown to govern either fast or slow development. Analysis of congenic strains on a C57BL/10Sn background showed that fast development is associated with the b, d, q, s, and u haplotypes, whereas slow development is associated with the k and r haplotypes. Analysis of the recombinant haplotype, a suggested that the fast Ped gene maps to the D end of the H-2 complex. The study of congenic strains on a BALB/c background has shown that background genes, as well as the H-2 associated Ped gene, play a role in the control of the rate of early embryonic cleavage.
Subject(s)
Blastocyst/physiology , Genes , Mice, Inbred Strains/genetics , Animals , Growth , H-2 Antigens/genetics , Kinetics , Mice , Mice, Inbred BALB C/genetics , Mice, Inbred C57BL/geneticsABSTRACT
The H-2 complex was found to influence early mouse embryo development. Slow development was shown to be associated with the H-2k haplotype. By the use of congenic strains, it was clearly demonstrated that gene(s) in the H-2 complex affect the time of the first cleavage division and the rate of subsequent preimplantation development
Subject(s)
Blastocyst/physiology , H-2 Antigens/genetics , Mice/embryology , Animals , Blastocyst/cytology , Cell Count , Cleavage Stage, Ovum , Growth , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBA , Mice, Inbred Strains , Time FactorsABSTRACT
The number of cells in preimplantation mouse embryos of different H-2 haplotypes was analysed. It was found that embryos of the H-2k haplotype have fewer cells per embryo than those of the H-2b haplotype. By analysing reciprocal congenic pairs of mice it was demonstrated unequivocally that slow development is linked to the H-2k haplotype and fast development to the H-2b haplotype. The gene (s) in the H-2 complex which influence early mouse embryo development have been named Ped: preimplantation embryo development. Analysis of F1 hybrid embryos showed that fast development is dominant. Reciprocal F1 crosses yielded identical results, which indicated there was no apparent effect of the maternal egg cytoplasm on Ped gene expression. Analysis of F2, and back-cross embryos was consistent with the interpretation that there is a major gene located in the H-2 complex (Ped), which is modified by environment and genetic background, that influences early mouse embryo development.
