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The special situation of multiple pregnancies following IVF has led to a growing interest in the assessment of embryonal development by means of molecular genetics. We report a case of congenital blood chimerism in dizygotic triplets (two boys, one girl) present in erythrocytes and leukocytes in both sexes. Routine pre-operative blood serology of the 6 year old female triplet revealed chimerism of the red cells. Flow cytometry of the erythrocytes and DNA analysis of the leukocytes demonstrated that all three children had the same proportions of male and female cells. Fluorescent in-situ hybridization (FISH) analyses revealed Y chromosomes in 84% of the girl's leukocytes and in 89/92% of the two boys' leukocytes. The true genetic lines were determined by analysing polymorphism of serum groups (glycoprotein, transferrin, protease inhibitor and plasminogen) secreted by non-haematopoetic tissue, by blood group typing of hair roots and by DNA analysis of endothelial cells. Evidently placental anastomoses allowed a reciprocal intra-uterine transfusion of blood stem cells in the triplets.

Trisomy 3, trisomy 5, and an X additional chromosome are the most frequent chromosome aberrations in angioimmunoblastic lymphadenopathy with proteinemia (AILD)-type T-cell lymphomas. To evaluate the frequency of +3 and +X clones, fluorescence in situ hybridization studies with centromere-specific probes for chromosome 3 and X were done in 41 patients with peripheral T-cell lymphomas (PTL). With this interphase cytogenetic approach, 32 of 41 patients (78%) showed +3 clones, and 14 patients (34%) +X clones. These frequencies far exceeded those observed with metaphase cytogenetics (+3, 41%; +X, 20%). Summing up the results of metaphase and interphase cytogenetics, aberrant clones were found in 37 of 41 patients with PTL (90%) and 32 of 36 patients with AILD-type T-cell lymphoma (89%). Although AILD-type T- cell lymphoma is considered a neoplastic disease, it is an exception in that it shows a high frequency of cytogenetically unrelated clones and single cells that cannot be derived from a common cell of origin because of their completely different karyotypes. In five patients, double hybridization with centromere-specific probes for chromosomes 3 and X showed that these aberrations occurred in different cells. When the results of metaphase and interphase cytogenetics were combined, 17 of 36 patients with AILD-type T-cell lymphoma (47%) had unrelated clones. This high frequency of oligoclonal proliferations may be caused by increased genetic instability and an immune defect resulting in impaired elimination of aberrant cells.

A new damping mechanism of multi-bunch oscillations in a storage ring with help of a narrow-band feedback system has been experimentally tested in PETRA. The oscillations of a multi-bunch filling were sucessfully damped. The bandwidth of the active loop was 1.5 kHz instead of 5 MHz needed for standard damper systems.

In Hodgkin's disease, cytogenetically aberrant clones have been demonstrated in a minority of cases studied. In the remaining cases, only normal metaphases have been found, but it is questionable whether normal karyotypes actually correspond to the pathognomonic Hodgkin and Reed-Sternberg (HRS) cells. Numerical aberrations could be studied by fluorescence in situ hybridization (FISH). However, in Hodgkin's disease, the percentage of tumor cells is mostly below the detection limit of FISH, which is near 1%. With the technique of simultaneous fluorescence immunophenotyping and interphase cytogenetic analysis (FICTION), this problem can be overcome. By FICTION, hybridization signals can selectively be evaluated within the CD30a+ cell population. We have studied 30 cytogenetically analyzed cases of Hodgkin's disease by means of FICTION. In all cases, we found numerical chromosome aberrations within the majority of CD30+ HRS cells. In cases with complex and hyperdiploid karyotypes, the cytogenetic results agreed with the FICTION data. There was considerable variability in the chromosome numbers, demonstrating that karyotype instability is an in vivo phenomenon of HRS cells. Lymphocytes never displayed numerical chromosome changes. Our results indicate that HRS cells regularly exhibit numerical chromosome aberrations and that the chromosome numbers are always in the hyperploid range.

Chronic myeloid leukaemia (CML) is believed to represent a stem cell disorder involving all three cell lineages. The typical chromosomal aberration, the Philadelphia chromosome, is the translocation (9;22)(q34;q11). Several studies with cytogenetics, fluorescence in situ hybridization (FISH), or polymerase chain reaction have investigated the presence of the t(9;22) in different cell compartments. However, questions still remain. In six cases of CML we combined the standard May-Grunwald-Giemsa staining with FISH at the single-cell level and were able to demonstrate that not only all maturation stages of granulopoiesis, erythropoiesis, and megakaryocytes, but also plasma cells, eosinophils, basophils and monocytes carried the Philadelphia chromosome in 53-98% of samples. Using immunological identification of single cells we were able to demonstrate that the t(9;22) is detectable in 34% of CD3-positive T lymphocytes, in 32% of CD19-positive B lymphocytes, and in 82% of CD34-positive precursor cells. The results give new insight into the biology of CML and may have implications for future therapeutic strategies.

Combined immunophenotyping and karyotyping was performed in seven cases of peripheral T cell lymphoma with complex aberrant clones. Various lymphocytic cell populations entered mitosis, whereas all aberrant cells belonged to the T helper/inducer cell population. Lymphomas with the same recurrent chromosome aberrations, i.e. inversion inv(14)(q11q32.1) and isochromosome i(8)(q10), had a very similar immunophenotype. The aberrant cells in these cases expressed CD3 +, CD4+, CD7+, CD45RO+. The immunophenotypic similarity is underlined in one case of T prolymphocytic leukemia, in whom the aberrant cells lost the CD8 antigen originally present, during cultivation with PHA.In one case of Sézary's syndrome, two or possibly even three different clones as well as nonclonal aberrations were identified within the T (helper/inducer) cell population, providing further evidence that chromosomal instability is a characteristic feature of cutaneous T cell lymphoma.

In Hodgkin's disease, cytogenetically aberrant clones have been demonstrated in a minority of cases studied. In the remaining cases, only normal metaphases have been found, but it is questionable whether normal karyotypes actually correspond to the pathognomonic Hodgkin and Reed-Sternberg (HRS) cells. Numerical aberrations could be studied by fluorescence in situ hybridization (FISH). However, in Hodgkin's disease, the percentage of tumor cells is mostly below the detection limit of FISH, which is near 1%. With the technique of simultaneous fluorescence immunophenotyping and interphase cytogenetic analysis (FICTION), this problem can be overcome. By FICTION, hybridization signals can selectively be evaluated within the CD30a+cell population. We have studied 30 cytogenetically analyzed cases of Hodgkin's disease by means of FICTION. In all cases, we found numerical chromosome aberrations within the majority of CD30+HRS cells. In cases with complex and hyperdiploid karyotypes, the cytogenetic results agreed with the FICTION data. There was considerable variability in the chromosome numbers, demonstrating that karyotype instability is an in vivo phenomenon of HRS cells. Lymphocytes never displayed numerical chromosome changes. Our results indicate that HRS cells regularly exhibit numerical chromosome aberrations and that the chromosome numbers are always in the hyper-ploid range.

The French-American-British subtype acute myelomonocytic leukemia with abnormal eosinophils (FAB AML M4Eo) with pericentric inversion of chromosome 16 is cytomorphologically defined by a myelomonoblastic blast population and abnormal eosinophils. Until now, it remained an open question whether these abnormal eosinophils are part of the malignant clone or an epiphenomenon. We analyzed five cases of AML M4Eo with inv(16) and combined May-Grunwald-Giemsa staining with fluorescence in situ hybridization using yeast artificial chromosome clone 854E2, which spans the inv(16) breakpoint on 16p. In the case of inv(16), three instead of the normal two hybridization signals can be observed both on metaphase spreads and in interphase cells. With this approach, we were able to show inversion 16 in abnormal eosinophils and, therefore, identified them as a part of the leukemic cell population.

In immunocytochemical studies, the phenotypic evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypical variability, such as bone marrow. A method that identifies mitotic tumor cells by chromosomal aberrations and permits the subsequent immunophenotypical analysis was a first progress, demonstrated by Teerenhovi et al. However, the results are usually hampered by the low number of analyzable mitoses. We demonstrate here a method that simultaneously combines immunophenotyping and in situ hybridization with centromere-specific probes. Using our method, numerically aberrant tumor cells can be identified by interphase cytogenetics and subsequently analyzed immunophenotypically. Since all interphase cells can be analyzed, we are not limited by the number and banding quality of analyzable mitoses.

For about 20 years we have known from cytogenetic studies that there is a clonal cell population in Hodgkin's disease. Most karyotypes are complexly aberrant and chromosome numbers typically lie in the hyperploid range. Some chromosome regions seem to be preferentially involved, but a chromosome aberration specific for Hodgkin's disease has not yet been determined. Although the existence of a clonal cell population is evident from these cytogenetic studies, there is still an ongoing debate, whether in all cases the pathognomonic Hodgkin and Reed-Stemberg cells belong to one single aberrant clone and thus represent a monoclonal proliferation. This article reviews the current knowledge on cytogenetics in Hodgkin's disease. Moreover, our recent data from simultaneous immunophenotyping and interphase cytogenetics (FICTION) are introduced into the passionate discussion on the monoclonality of the Hodgkin and Reed-Sternberg cells.

We describe a method that enables detection and immunophenotypical characterization of distinct subpopulations within a cytogenetically defined tumor clone. Coexisting normal cells do not hinder microscopic evaluation because they can be distinguished from cytogenetically aberrant tumor cells. This is also true when normal and neoplastic cells cannot be clearly distinguished by cytology or immunohistochemistry, i.e., if both constituents have similar immunophenotypes and morphology. The method is based on fluorescence double staining for two different antigens combined with interphase cytogenetic analysis. It is referred to as "Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)." In a case of follicular lymphoma we demonstrate that FICTION can differentiate bcl-2-positive malignant and non-malignant cells and can verify the presence of bcl-2-positive but cytogenetically inconspicuous T-lymphocytes.

Recently, we have presented a new technique for immunophenotyping cells that have numerical chromosome aberrations. We referred to this method as “Fluorescence-Immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms” (FICTION). We present here an advanced FICTION method with three-color staining and improved sensitivity.

Cytogenetic studies on Hodgkin’s disease (HD) typically reveal very complex karyotypes with a variety of numerical and structural abnormalities. The confusing thing is that about 10% of cases contain relatively discrete chromosome aberrations, for example a simple trisomy or loss of one single chromosome. Whether these karyotypes really correspond to Hodgkin and Reed-Sternberg (HRS) cells is uncertain. They could, for example, represent early stages in the evolution of the karyotype of the pathognomonic HRS cells. On the other hand, they could be artificial events that occur during the cytogenetic procedure. In our experience, isolated loss of the Y chromosome is the most frequent finding of this type. This aberration is usually considered to be a preparation artifact. However, if one takes into account that in HD up to 50% of male cases with complex karyotypes also lack the Y chromosome, a possible relation to HRS cells must be considered. The technique of simultaneous fluorescence immunophenotyping and interphase cytogenetic analysis (referred to as FICTION) is a powerful tool for studying the nature of cytogenetically abnormal cells. With the FICTION technique we studied four cases of HD in which the chromosome analysis had shown only the loss of the Y chromosome. Our aim was to clarify whether these karyotypes corresponded to the CD30-positive HRS cells. In two cases we found that HRS cells actually lacked the Y chromosome. There was strong evidence, however, that the HRS cells additionally had other chromosome aberrations and thus could not correspond to the cytogenetically determined karyotypes.

The recently developed MAC (morphology-antibody-chromosome) method allows simultaneous immunophenotype and karyotype analysis in the same cell. To date, application of this new method has been hampered by the poor quality of chromosome banding. In this paper, we describe a modified simultaneous immunofluorescence and Q-banding technique, as well as a new combination of immunohistochemical and fluorescent R-banding methods. By further modifying the MAC method, we were able not only to achieve unequivocal results with weakly expressed antigens but also to improve the quality of the banding techniques, so that even structural chromosome abnormalities were well defined.