Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Purification and biochemical characterization of a virus-specific reverse transcriptase from human osteosarcoma tissue.

A RNA-dependent DNA polymerase (RTase) was purified from human osteosarcoma tissue by successive column chromatography of the microsomal fraction on DEAE-cellulose (DE-23 and DE-52) and phosphocellulose. The purified enzyme has a molecular weight of about 68,000, a pH optimum of 8.1, a Mg2+ optimum of 0.8 mM, Mn2+ optimum of 1.0 mM and a KCl optimum of 60 mM. The enzyme transcribes (rA)n . (dT)12, (rC)n . (dG)12-18 and (2-O-methyl C)n . (dG)18, but is unable to transcribe (dA)n . (dT)10. The enzyme has no catalytic activity in the presence of oligodeoxynucleotide initiators alone, indicating the absence of terminal deoxynucleotidyl transferase. The purified enzyme is able to transcribe the heteropolymeric regions of a 70S RNA from R(Mu)LV. The presented data support the presence of a RNA-dependent DNA polymerase in human osteosarcoma tissue with biochemical properties, resembling those of C-type RNA tumor viruses.

Serological characterization of a purified reverse transcriptase from osteosarcoma of a child.

Serological analysis of the reverse transcriptase (RTase), purified from human osteosarcoma tissue, has shown that it is antigenically related to DNA polymerases from BEV and from RD-114. No cross-reactivity of the osteosarcoma RTase was observed with RTases purified from AMV, RLV, SiSV, GaLV and from human spleen of a patient with myelofibrosis.

Direct molecular genetic diagnosis and heterozygote identification in X-linked Emery-Dreifuss muscular dystrophy by heteroduplex analysis.

X-linked Emery-Dreifuss muscular dystrophy (EMD) is a very rare, relatively benign muscle disorder. The disease is associated with potentially lethal cardiac arrhythmias in affected males and some heterozygous females. X-linked EMD can be genetically distinguished from phenotypically similar autosomal EMD. Heterogenic mutations are identified as the cause of X-linked EMD. We introduced heteroduplex analysis to follow the segregation of heterogenic emerin gene mutations in the families of six unrelated EMD patients. Heteroduplex analysis was proved to be a simple, fast and reliable tool for direct molecular genetic diagnosis of EMD in male patients and identification of heterozygotes even in families where affected males are not available as index cases.

Uptake and release of iodine-labelled m-iodobenzylguanidine in a neuroblastoma cell culture system and its importance in neuroblastoma therapy.

A neuroblastoma cell line was established from bone marrow of a patient in stage IV of the disease and used as a model system in order to elaborate experimental data of importance in neuroblastoma therapy, such as cell-drug interactions, the mode of uptake and conditions for storage and release. m-Iodo benzylguanidine (MIBG) is rapidly taken up from culture medium, giving high concentrations of cell-bound radioactivity reaching a maximum level 4 h after the addition of the compound. A removal of the radiopharmacon from the culture medium causes a dramatic loss of cell-associated radioactivity, suggesting that neuroblastoma cells are not able to retain MIBG in a drug-free environment. Replacement of labelled by unlabelled MIBG prevents a similar release and maintains high levels of cellular radioactivity. Variations of cell culture conditions only result in minor changes of uptake rates, whereas a pretreatment with drugs used in neuroblastoma chemotherapy harms the cells extensively: even after a short-term exposure the cells lose the capacity for MIGB uptake and fail to recover within a long period of incubation in growth medium. The importance of our results is discussed, leading to the following suggestions: 1. The performance of radiotherapy with labelled MIBG is recommended prior to the chemotherapy protocols. 2. Therapeutically effective radioactivity in tumor tissues may be maintained by the additional infusion of unlabelled MIGB.

Minimal residual disease in leukemia in children.

Many chromosomal translocations involved in leukemia have been defined at the molecular level in recent years. In addition to advancing the understanding of pathological mechanisms underlying the transformation process, the cloning and sequencing of the genes altered by the translocations have provided new tools for diagnosis and monitoring of patients. In particular, the polymerase chain reaction (PCR) method yields sensitive and accurate diagnostic and prognostic information. Minimal residual disease (MRD) is not clearly defined. In ALL we define MRD as fewer than 5% blast cells in the bone marrow by conventional cytology and proof of leukemic cells with more sensitive methods. The techniques for detecting MRD are imaging for detection of single leukemic cells in the blood, bone marrow, or other tissues by means of immunocytology or PCR/RT-PCR. Highly sensitive PCR, immunocytology, FACS analysis, or conventional cytology are important tools to use in the process of deciding on appropriate therapy. Detection limits at present are 10(-2) for cytology and FISH, up to 10(-4) for immunological procedures, and 10(-5) to 10(-6) for PCR. But multiple methods also imply the possibility of mistakes (e.g., PCR). The question must be raised what method should be decisive in assessing MRD for evaluating autologous peripheral blood stem cells (PBSC) or autologous bone marrow transplants? Prospective studies will have to answer the question whether MRD should be treated or not and whether purging of bone marrow or PBSC is useful or damaging. When applied, should a positive or a negative immunopurging or a chemotherapeutic purging be used? MRD refers to the organism of the patient as well as to the peripheral blood stem cells and autologous bone marrow that had been taken before myeloablative therapy and kept for retransfusion.

Polynucleotides containing 5-mercapto-substituted pyrimidines: inhibition of viral DNA polymerases and the biological implication.

Partially thiolated polycytidylic acids MPC I-III, containing 1.7%, 3.5% and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA polymerase of Friend leukemia virus (FLV) in the endogenic reaction as well as in the presence of poly(A)-(dT)14 or poly[d(a-T)] templates; the inhibitory activities were directly related to the percent of thiolation. Various partially thiolated RNA and DNA isolates from Ehrlich ascites cells (containing one 5-mercaptopyrimidine nucleotide/50-100 nucleotide units) also inhibited the DNA polymerases of FLV in the endogenic reaction, and also in the presence of the synthetic templates. The thiolated DNA was the most active, but the thiolated tRNA also showed substantial inhibitory effects, while the thiolated ribosomal RNA was less effective. In a bacterial DNA polymerase (E. coli-K12, using denatured DNA as template), MPC I-III showed no activity. By contrast, MPC III and several partially thiolated nucleic acid isolates significantly inhibited a regenerating rat liver DNA polymerase (I) system; among those tested, the thiolated DNA from Ehrlich ascites cells showed the highest activity. Kinetic analysis of the inhibitory action of this thiolated DNA in the rat liver enzyme system, using as template the corresponding unmodified DNA, demonstrated that the thiolated DNA acts as a competitive inhibitor of the template, with a Ki/Km ratio of 0.5.

Rapid immunodiagnosis of childhood leukemia using microwave-stimulated APAAP-complex system.

Important insights into leukocyte differentiation and the cellular origin of leukemias have been achieved by the use of monoclonal antibodies for the detection of cellular antigens with impact on the diagnosis and classification of hematological malignancies. A successful rapid immunoenzymatic technique using application of microwave irradiation (MIWI) on bone marrow cells of various children with ALL is described. The MIWI-stimulated immunotyping of acute leukemia cells with a panel of monoclonal antibodies against differentiation antigens (i.e. CD2, CD7, CD10, CD19, CD20, CD24, HLA-DR and TdT) has been compared with the conventional APAAP procedure developed by Mason et al 1983. The commercial microwave oven we used operates at 2.45 GHz. Fifteen sec irradiation at 350 W during all incubation steps produced excellent color reactions with Fast Red TR and Fast Blue BB similar to the conventional immunoenzymatic method. The results so far have demonstrated that the application of the MIWI-technique eliminates the need for long incubation periods without loss of sensitivity. With this technique an immunological diagnosis of childhood leukemia cells is possible using air dried smears in an microwave oven within 30 minutes.

Expression of markers shared between human haematopoietic cells and neuroblastoma cells.

Neuroblastoma (NB) is a solid tumor of childhood with a relatively bad prognosis, with the exception of young infants (less than 1 year), in whom spontaneous regression of tumor burden occurs. The reasons for this are still unknown but immune mechanisms may be involved. In this study, we have examined the ability of several monoclonal antibodies (MoAbs), which recognize markers predominantly expressed on human haematopoietic cells, to react with four human neuroblastoma cell lines (UKF-NB 1-4) and SK-N-SH as control cell line. In order to define the phenotype of NB cells, we used a large panel of MoAbs consisting of 2 major groups: a) well characterized MoAbs raised against antigens of neuroectodermal origin from the Kemshead-serie (e.g. UJ 13A, UJ 127.II, UJ 167.11, UJ 181.4, UJ 223.8, A2B5), b) monoclonal antibodies which have been considered to react with haematopoietic cells (HLA-DR and anti-CD-molecules CD1, CD7, CD9, CD10, CD13, CD16, CD19, CD20, CD24, CD57). The phenotypic analyses were performed at various times of culture by an immunoenzymatic procedure (APAAP-technique). Most of the MoAbs used against neuroblastoma cells showed a strong reactivity pattern with the NB cell lines. None of the antibodies against T-lymphocytes bound to any of the NB cells assayed in our study, with the exception of anti-CD 1. On the contrary, B-cell markers BA-2 (CD9) and BA-1 (CD24) cross-reacted with the NB cells just as well as the marker for NK-cells (CD57), but they did not express reactivity with Leu-11b (CD16), anti-CALLA (CD10) and anti-HLA-DR.

[Microwave stimulated cell marker analysis. Possibilities for more rapid immune diagnosis]. / Mikrowellen-stimulierte Zellmarkeranalysen. Möglichkeiten einer beschleunigten Immundiagnostik.

We describe a successful rapid APAAP-complex technique using innovative application of microwave irradiation (MIWI) on Ficoll separated peripheral blood mononuclear cell smears of healthy donors. The typing with several monoclonal antibodies (MoAbs) against different cell surface antigens is compared with the conventional APAAP procedure. The commercial domestic microwave oven was operated at 2.45 GHz. Fifteen second irradiation at 350 W during all incubation steps, e.g. primary antibody, bridging antibody and APAAP-complexes produced excellent color reactions with Fast Red TR, Fast Blue BB, New Fuchsin or NBT similar with the conventional immunoenzyme procedure. The routinely usage of a Silicon-Chamber-System developed by us is applicable without limitation under microwave conditions. The results till now have shown that the application of microwave-technique (MIWI) eliminated the need for much longer incubation periods without lost of sensitivity. All immunological markers could be detected in the same degree as observed with the conventional method. We could demonstrate that an immunological diagnosis is possible within 30 minutes using air dried smears in an microwave oven.

[Use of a new silicon chamber system for carrying out immunologic technics with blood and bone marrow samples and histologic specimens]. / Anwendung eines neuen Silikon-Kammersystems (SCS) zur Durchführung immunologischer Detektionssysteme an Blut-/KM-Ausstrichen und an histologischen Präparaten.

We are reporting for the first time on the successful application of the newly developed method of Silicon-Chamber-System (SCS). For this purpose we are utilizing a commonly manufactured silicon sealant by which we are able to obtain reaction fields of any size and number. We are applying the sealant on cell- and on tissue-slides without any special preparation. These reaction fields on the slides allow us to immunohistochemically analyse cells with multiple different monoclonal antibodies without running great risk of a cross-reaction between the different immunoreagents. It is a simple inexpensive and convenient method, giving us the opportunity to analyze elaborate cell material.

[Retrospective marker analyses performed with blood and bone marrow smears using an immunoenzyme procedure (alkaline phosphatase-anti-alkaline phosphatase technic)]. / Retrospektive Marker-Analysen durchgeführt an Blut- und Knochenmark-Ausstrichen mittels eines immunenzymatischen Verfahrens (APAAP-Technik).

In the present study the possibility of immunophenotyping of routinely prepared (air dried) peripheral blood and bone marrow smears is described after storage. The immunoenzymatical alkaline-phosphatase-anti-alkaline-phosphatase (APAAP)-method was carried out in differently stored blood smears (+4 degrees C and -80 degrees C). Last results were compared with originals made from freshly prepared mononuclear cells at time of diagnosis. The results showed no remarkable decrease of antigenicity, neither under the procedure refrigeration and thawing of the frozen smears (-80 degrees C), nor under storage conditions of more than 22 months. In our opinion this highly sensitive method enables us to get additional results from unexplained haematological disorders using retrospective analysis.

[Immunologic identification of undetectable neuroblastoma cells by current cytohistological studies of bone marrow samples]. / Immunologische Identifizierung von durch herkömmliche zytohistologische Untersuchungen an Knochenmarksproben nicht erkennbare Neuroblastomzellen.

Immuno-alkaline phosphatase staining (APAAP technique) has been used to identify neuroblastoma cells on bone marrow samples from 12 children at various stages of the disease. On 72 occasions immunological analyses were performed using a panel of monoclonal antibodies which selectively bind to cells of neuroectodermal origin. In 57 of these procedures, tumor cells were detected, whereas histological and cytological analyses revealed pathological cells in 45 and 37 cases respectively. Reactivity of minimal residual tumor cells--mainly with three MAbs (UJ13A, UJ167.11, A2B5)--points to the fact that these cells belong to a resistant neuroblastoma clone, which remains in bone marrow despite intense therapy. Our study demonstrates that immunological staining may identify and define a small number of neuroblastoma cells which are not yet detectable by traditional histological and cytological criteria.

Immunological detection and definition of minimal residual neuroblastoma disease in bone marrow samples obtained during or after therapy.

Immunological staining by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique has been used to recognize low levels of neuroblastoma cells in bone marrow mononuclear cells. Immunological phenotyping with 11 well characterized monoclonal antibodies was performed on 16 children with neuroblastoma and BM involvement during or after therapy. Neuroblasts were detected in 11 of 16 patients (0.1-5%), whereas BM biopsies on six of these patients were classified as normal. Aspirates, stained conventionally, were positive for pathological cells in three patients only. The comparison of the phenotype of the neuroblastoma cells at the time of diagnosis to the phenotype of the residual cells within one patient revealed differences. The phenotype of residual disease in different patients on the other hand showed a unique pattern. The above mentioned results lead to the conclusion that the immunological procedure is particularly suitable for the analysis of minimal residual neuroblastoma since the technique allows very minor cell populations to be identified in BM samples.

[Expression of AC133 vs. CD34 in acute childhood leukemias]. / Expression von AC133 vs. CD34 bei akuten Leukämien im Kindesalter.

AC133, a newly discovered antigen on human progenitor cells, demonstrating 5-transmembranous domains is expressed by 30-60% out of all CD34+ cells. Our aim therefore was to investigate the extent of human stem-/progenitor cells expressing AC133 antigen in umbilical cord blood, peripheral blood without or following an application of granulocyte-colony stimulating factor (rhG-CSF). The main task was the investigation of bone marrow aspirates derived from children suffering from newly diagnosed acute leukemias, as well as from patients with a relapse or during a complete remission. The determination of antigen expression was done by application of flow cytometry (FACScan analysis) and the usage of newly developed monoclonal antibodies (AC133/1 and AC133/2; Miltenyi Biotec GmbH) in combination with monoclonal antibody directed against CD34-antigens (HPCA-2; BD). Our studies till now show average percentages in umbilical cord blood derived from 43 newborns about 0.294 +/- 0.165% AC133+ vs. 0.327 +/- 0.156% CD34+ hematopoietic stem-/progenitor cells (HSPC). In peripheral blood from 11 healthy donors we verified up to 0.15% CD34+ as well as AC133+ HSPC's. The concentration of progenitor cells was found to be obviously higher in peripheral blood from children with various diseases (neuroblastoma, rhabdomyosarcoma, ALL/AML) and undergoing application with rhG-CSF in order to be prepared for PBSC-transplantation. In those cases we found up to 3.51% AC133+ cells as well as slightly higher values (3.94%) for CD34 antigens. Additionally we quantified 128 bone marrow (BM) samples for AC133+ and CD34+ cells. In 10 BM samples, derived from patients without any neoplasia, the CD34+ cells were about 0.03% and 1.49%, whereas AC133 values were up to 0.64%. Bone marrow aspirates from 53 children with acute leukemias at time of diagnosis (ALL: n = 41/AML: n = 12) have been immunophenotyped and leukemic blast cells have been proved for AC133- and CD34 antigen expression. 32/41 (78%) of lymphoblastic leukemic cells showed to be positive for CD34 antigen and 24/41 (58%) demonstrated AC133 antigens. Interestingly there were 2 ALL-patients with pathological blast cells positive for AC133 but lacking of any CD34 antigens. 42% (5/12) of investigated AML patients showed CD34+ phenotype, on the other hand there were only 25% (3/12) with AC133+ phenotype. Similar values were found in relapsed patients (n = 18). In BM samples from patients during complete remission (n = 47) we could detect percentages up to 5.55% for CD34 and up to 1.25% for AC133 positive stem-/progenitor cells. Such quite high data may be explained by occasionally application of rhG-CSF therapy. Our results till now lead to the conclusion, that it seems to be useful, to recruit quantification of CD34+ HPSC by additionally detecting AC133 antigens. This new stem cell marker (AC133) may be of great value in case of autologous peripheral blood stem cell transplantation (PBSCT) because it could be an alternative to the usual CD34+ MACS selection system.

Increased HIV-1 production in chronically infected H9 cells grown in protein-free medium.

Human T cell line H9 was established in a protein-free 1:1 mixture of Ham's F-12 and IMDM. After 230 passages (3 years) in protein-free medium, the cells designated H9-PF were infected with HIV-1. The infectivity titers of HIV-1 in cell culture medium were monitored by determining the median tissue culture infectious doses (TCID50). Additionally, the production of viral antigen in cells was measured by an immunoenzymatical alkaline phosphatase anti-alkaline phosphatase (APAAP) method using a monoclonal antibody against HIV-1-p24 antigen. In acutely infected H9-PF and H9 cultures similar TCID50 values and percentage of cells positive for p24 antigen were found. In contrast, both TCID50 values and percentage of cells positive for p24 antigen were by far greater in chronically infected H9-PF than in H9 cultures.