Differential expression of miR-17~92 identifies BCL2 as a therapeutic target in BCR-ABL-positive B-lineage acute lymphoblastic leukemia.

Despite advances in allogeneic stem cell transplantation, BCR-ABL-positive acute lymphoblastic leukaemia (ALL) remains a high-risk disease, necessitating the development of novel treatment strategies. As the known oncomir, miR-17~92, is regulated by BCR-ABL fusion in chronic myeloid leukaemia, we investigated its role in BCR-ABL translocated ALL. miR-17~92-encoded miRNAs were significantly less abundant in BCR-ABL-positive as compared to -negative ALL-cells and overexpression of miR-17~19b triggered apoptosis in a BCR-ABL-dependent manner. Stable isotope labelling of amino acids in culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) identified several apoptosis-related proteins including Bcl2 as potential targets of miR-17~19b. We validated Bcl2 as a direct target of this miRNA cluster in mice and humans, and, similar to miR-17~19b overexpression, Bcl2-specific RNAi strongly induced apoptosis in BCR-ABL-positive cells. Furthermore, BCR-ABL-positive human ALL cell lines were more sensitive to pharmacological BCL2 inhibition than negative ones. Finally, in a xenograft model using patient-derived leukaemic blasts, real-time, in vivo imaging confirmed pharmacological inhibition of BCL2 as a new therapeutic strategy in BCR-ABL-positive ALL. These data demonstrate the role of miR-17~92 in regulation of apoptosis, and identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL.

Inhibition of Abl tyrosine kinase enhances nerve growth factor-mediated signaling in Bcr-Abl transformed cells via the alteration of signaling complex and the receptor turnover.

Receptor tyrosine kinase-mediated signaling is tightly regulated by a number of cytoplasmic signaling molecules. In this report, we show that Bcr-Abl transformed chronic myelogenous leukemia (CML) cell lines, K562 and Meg-01, express the receptor for nerve growth factor (NGF), TrkA, on the cell surface; however, the NGF-mediated signal is not particularly strong. Treatment with imatinib, a potent inhibitor of Bcr-Abl tyrosine kinase, downmodulates phosphorylation of downstream molecules. Upon stimulation with NGF, Erk and Akt are phosphorylated to a much greater degree in imatinib-treated cells than in untreated cells. Knockdown of expression of Bcr-Abl using small interfering RNA technique also enhanced NGF-mediated Akt phosphorylation, indicating that Bcr-Abl kinase modifies NGF signaling directly. Imatinib treatment also enhanced NGF signaling in rat adrenal pheochromocytoma cell line PC12 that expresses TrkA and c-Abl, suggesting that it is not only restoration of responsiveness to NGF after blocking oncoprotein activity, but also c-Abl tyrosine kinase per se may be a negative regulator of growth factor signaling. Furthermore, inhibition of Abl tyrosine kinase enhanced clearance of surface TrkA after NGF treatment and simultaneously enhanced NGF-mediated signaling, suggesting that as in neuronal cells 'signaling endosomes' are formed in hematopoietic cells. To examine the role of TrkA in CML cells, we studied cell growth or colony formation in the presence or absence of imatinib with or without NGF. We found that NGF treatment induces cell survival in imatinib-treated CML cell lines, as well as colony formation of primary CD34+ CML cells, strongly suggesting that NGF/TrkA signaling contributes to aberrant signaling in CML.

Stable RNA interference (RNAi) as an option for anti-bcr-abl therapy.

RNA interference (RNAi) has recently been used for sequence-specific gene silencing of disease-related genes including oncogenes in hematopoietic cells. To characterize its potential therapeutic value, we analyzed different modes to activate RNAi as well as some pharmacokinetic aspects of gene silencing in bcr-abl+ cells. Using lentiviral gene transfer of transcription cassettes for anti-bcr-abl shRNAs and red fluorescence protein (RFP) as a quantitative reporter, we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+ K562 and murine TonB cells from suspension cultures. Importantly, depletion of bcr-abl+ cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of transduced target cells: RFP-high K562 cells are eradicated, whereas RFP-low or -intermediate cells may recover after prolonged cell culture. Interestingly, these cells still show reduced bcr-abl mRNA levels, aberrant proliferation kinetics, and enhanced sensitivity to the Bcr-Abl-kinase inhibitor STI571. Quantitative PCR from genomic DNA suggests that more than three lentiviral integrations are required for effective depletion of K562 cells. Finally, we demonstrate that lentivirus-mediated anti-bcr-abl RNAi can inhibit colony formation of primary CD34+ cells from chronic myeloid leukemia patients. These data demonstrate dose-dependent gene silencing by lentivirus-mediated RNAi in bcr-abl+ cells and suggest that stable RNAi may indeed be therapeutically useful in primary hematopoietic cells.

Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction.

Autologous transplantation of gene-modified hematopoietic stem cells may provide a therapeutic strategy for several monogeneic disorders. In previous studies, retroviral gene transfer of coagulation factor VIII (FVIII) into FVIII(-/-) mouse bone marrow (BM) cells did not result in detectable plasma FVIII levels. However, specific immune tolerance was achieved against neo-antigenic FVIII. Here, we used lentiviral vectors to study the ability of various hematopoietic cell types to synthesize and secrete recombinant FVIII. Several myeloid, monocytic and megakaryocytic cell lines (K-562, TF-1, Monomac-1, Mutz-3, Meg-01) expressed FVIII at 2-12 mU/10(4) cells. In contrast, two lymphatic cell lines, BV-173 and Molt-4, were less-efficiently transduced and did not express detectable FVIII. Similarly, peripheral blood-derived primary monocytes were transduced efficiently and expressed up to 20 mU/10(4) cells, whereas primary lymphocytes did not express FVIII. Although human and canine CD34(+) cells were transduced efficiently, the cells expressed very low levels of FVIII (up to 0.8 mU/10(4) cells). Following xenotransplantation of transduced CD34(+) into NOD/SCID mice, ELISA failed to detect FVIII in the plasma of engrafted mice. However, NOD/SCID repopulating cell (SRC)-derived human monocytes isolated from BM of these mice secreted functional recombinant FVIII after culture ex vivo. Again, SRC-derived human lymphocytes did not secrete FVIII. Therefore, certain hematopoietic cell types are able to synthesize and secrete functional recombinant FVIII. Our results show for the first time that transplantation of transduced CD34(+) progenitors may give rise to differentiated hematopoietic cells secreting a nonhematopoietic recombinant protein.

Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography.

Lentiviral vectors have been shown to stably transduce dividing and non-dividing target cells in vitro and in vivo. However, in vivo gene transfer applications with viral vectors in the central nervous system require highly efficient vector preparations, because only very small volumes can be injected stereotactically without damage to the brain tissue. Since lentiviral vectors are generated in transient co-transfection systems, viral preparations need to be purified and efficiently concentrated before injection into the brain. We describe an alternative procedure to concentrate lentiviral preparations by binding viral particles to an anion exchange column. Viral particles are eluted with sodium chloride, desalted and further concentrated by ultrafiltration. These vector preparations allowed high levels of gene transfer into terminally differentiated neuronal and glial cells and long-term transgene expression without any signs of acute and long-term toxicity or inflammation. The purification of lentiviral vectors from large-scale preparations by anion exchange chromatography allowed us to concentrate the virus to small volumes and to use these preparations to genetically modified target cells in vivo without signs of acute inflammatory responses.

Donor cell-derived acute myeloid leukemia developing 14 months after matched unrelated bone marrow transplantation for chronic myeloid leukemia.

We report a patient with Ph-positive CML who developed a Ph-negative AML in donor cells 14 months after BMT from an HLA-identical male unrelated donor. The Ph translocation could not be detected by either conventional cytogenetics, FISH or RT-PCR analysis excluding relapse of CML in myeloid blast crisis. Chimerism studies were performed by variable number of tandem repeats (VNTR) analysis. These revealed donor-type hematopoiesis in both unseparated mononuclear cells and CD34+ selected blasts proving the leukemia to be of donor origin. The patient received three cycles of polychemotherapy with mitoxantrone, topotecan and ara-c resulting in CR after the first treatment cycle and reconstitution with donor hematopoiesis. A second transplant from a female alternative matched unrelated donor was performed after conditioning with fludarabine and 200 cGy TBI and was well tolerated. Nine months after the second transplant the patient is at home and in CR. T cell chimerism was studied by sex chromosome analysis and revealed complete female donor chimerism.

Quantitative determination of lentiviral vector particle numbers by real-time PCR.

Here, we describe a quantitative, DNA-based, real-time PCR approach to determine the number of lentivirus particles that are present in vector preparations. In this approach, the minus strong-stop cDNA fragment that is present in viral capsids serves as template for PCR. Using this technology, we found that only 0.1%-1% of the virus particles that are present in vector preparations are infectious. The approach described here is rapid, reliable, and simple in concept and can be used to estimate both vector particles in supernatants and the number of infectious particles. Also, this approach can easily be adapted to a high-throughput system by using 96-well plates and a 2-h running time.

Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation.

To analyze the value of real time RT-PCR for monitoring of bcr-abl expression in CML patients after allogeneic or autologous stem cell transplantation (SCT), we generated pairs of PCR-primers and TaqMan probes specific for either the b2a2- or the b3a2-variant of bcr-abl. Either variant could be detected specifically from cDNA from a single K562 (b3a2) and BV173 (b2a2) cell with the respective TaqMan probe. Bcr-abl expression was normalized by comparison with GAPDH expression, and samples were quantitated using standard cDNA dilutions from K562 or BV173 cells. In a retrospective analysis 13 patients with CML after allogeneic (n = 10) or autologous (n = 3) SCT including patients with relapsed or persistent CML were analyzed by both real-time and conventional nested RT-PCR. In addition chimerism was monitored by FISH analysis of sex chromosomes in three patients with relapsed disease. The bcr-abl/GAPDH ratio dropped at least 1000-fold in all seven patients evaluable prior to and after allogeneic SCT as estimated by real-time RT-PCR, and conventional RT-PCR became negative in 6/7 patients. In five patients with relapsed or persistent disease after allogeneic SCT the bcr-abl/GAPDH ratio eventually increased again, and real-time RT-PCR was as sensitive as conventional RT-PCR for detection of bcr-abl. Donor lymphocyte infusions (DLI) were given to all five patients, and the bcr-abl/GAPDH ratio dropped to undetectable levels in two patients both remaining in continuing molecular remission. In contrast, in three other patients the bcr-abl/GAPDH ratio decreased only or did not change significantly after DLI. In three patients undergoing autologous SCT the bcr-abl/GAPDH ratio dropped only 1.1 to 30-fold, and the patients were tested positive with real-time RT-PCR at all time points. These data demonstrate that real-time RT-PCR is valuable to quantitate bcr-abl expression in CML patients after transplantation.

Technical and safety aspects of blood and marrow transplantation using G-CSF mobilized family donors.

An allogeneic transplantation programme using immunoselected blood progenitor and bone marrow CD34+ cells has been established. Thirteen healthy HLA-matched, MLC negative sibling donors received two doses of 5 micrograms kg-1 G-CSF (s.c. daily) for 5 days. On days 4 and 5, large-volume mononuclear cell aphereses were performed (COBE Spectra) and on day 5 one unit of autologous blood was obtained. Mononuclear cells were pooled and cryopreserved after CD34+ cell-immunoselection on day 5. Bone marrow (BM) of the same donors was procured under routine conditions 10-45 days later (median: 27 days). The final graft consisted of blood CD34+ cells with either complete BM (n = 5) or immunoselected BM CD34+ cells (n = 8). The present paper describes the progenitor cell mobilization and apheresis protocol and analyzes the cell loss by BM and peripheral blood progenitor cell (PBPC) donation. Considerably larger amounts of mononuclear cells (CD45+), T-lymphocytes (CD3+) and platelets were lost by the apheresis as compared to bone marrow without apparent immediate clinical consequences for the donors. Owing to cross-cellular contamination of the apheresis concentrate, blood platelet count (PC) significantly decreased (mean PC after the second apheresis 116 x 10 microL-1); furthermore on average 3.04 x 10(10) CD3+ cells were removed by two apheresis sessions. This loss did not lead to long-term total lymphocyte count changes (2370 microL-1 versus 1889 microL-1) as observed during the long-term follow-up of 7/13 donors (mean 290 days). Subjectively, the PBPC collections were better accepted than BM donations in all but one family donor.

[Ex vivo increase in hematopoietic progenitor cells for clinical use]. / Ex-vivo-Vermehrung von hmatopoetischen Progenitorzellen für die klinische Anwendung.

BACKGROUND: The clinical use of ex vivo expanded hematopoietic progenitor cells is currently explored. MATERIAL AND METHODS: In this study the culturing of G-CSF mobilized and purified CD34+ blood cells was investigated. The interleukins IL-1 beta, IL-3 and IL-6 (each at a dose of 300 U/ml) and stem cell factor (25 ng/ml) without or with erythropoietin (1 U/ml) were used. Cells of 10 healthy sibling donors and 10 patients with solid tumors were incubated under small-scale (n = 15, 2 ml) and large-scale (n = 7, 50 ml) culture conditions at 37 degrees C for 5 and 4 weeks, respectively. The cell density was adjusted to about 1 x 10(5) cells/ml. RESULTS: The nucleated cell counts increased approximately 7-fold and 10- to 70-fold after 1 and 2 weeks of incubation. Numbers of CD34+ cells doubled to triplicated within this time interval, without any significant changes in their clonogenicity (CFU-GM and BFU-E output). Thereafter a depletion of the CD34+ cell pool was noticed. However the numbers of CD34+/CD38(-)- or CD34+/ HLA-DR(-)- cells were reduced to a lesser extent. The expanded cells generated predominantly myeloid and almost no lymphoid cells. More glycophorin-A+ cells were produced when erythropoietin was added. Replacement of non-human additives with heat-inactivated autologous plasma had no influence on cell growth. Almost no proliferation was obtained with a 10-fold higher cell density (1.7 x 10(6)/ml in 100 ml), but the cells maintained their viability for 13 to 16 days. CONCLUSIONS: This study suggests, that the chosen culture conditions might be feasible for a large-scale ex vivo expansion of hematopoietic progenitor cells for clinical application. The impact of the ex vivo generated cells on hematopoietic regeneration after chemotherapy is currently under clinical investigation.

Combined transplantation of allogeneic bone marrow and CD34+ blood cells.

Allogeneic peripheral blood progenitor cells (PBPCs) were transplanted after immunoselection of CD34+ cells. Two patient groups were studied: group I patients received immunoselected blood CD34+ cells and unmanipulated marrow cells from the same donor. Group II patients were given immunoselected blood and bone marrow (BM) CD34+ cells. One to 6 weeks before bone marrow transplantation (BMT), PBPCs from HLA-identical and MLC- sibling donors were mobilized with granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg twice daily subcutaneously) for 5 days. Aphereses were performed at days 4 and 5 of G-CSF application. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption onto avidin with the biotinylated anti-CD34 monoclonal antibody 12.8 and then stored in liquid nitrogen. BM was procured on the day of transplantation. Patients were conditioned with either busulfan (16 mg/kg) or total body irradiation (12 Gy) followed by cyclophosphamide (120 mg/kg). Cyclosporin A and short methotrexate were used for graft-versus-host disease (GVHD) prophylaxis. After transplantation, all patients received 5 micrograms G-CSF/kg/d from day 1 until greater than 500 neutrophils/microL were reached and 150 U erythropoietin/kg/d from day 7 until erythrocyte transfusion independence for 7 days. Group I consisted of patients with acute myeloid leukemia (AML) (n = 2), chronic myeloid leukemia (CML) (n = 2), and T-gamma-lymphoproliferative syndrome and BM aplasia (n = 1). The patients received a mean of 3.3 x 10(6) CD34+ and 3.7 x 10(5) CD3+ cells/kg body weight of PBPC origin and 4.5 x 10(6) CD34+ and 172 x 10(5) cells/kg body weight of BM origin. Group II consisted of five patients (two AML, two CML, one non-Hodgkin's lymphoma). They received a mean of 3.3 x 10(6) CD34+ and 3.2 x 10(5) CD3+ cells/kg from PBPC and 1.4 x 10(6) CD34+ and 0.6 x 10(5) CD3+ cells from BM. A matched historical control group (n = 12) transplanted with a mean of 5.2 x 10(6) CD34+ and 156 x 10(5) CD3+ cells/kg from BM alone was assembled for comparison. In group I, the median time to neutrophil recovery to > 100, > 500, and > 1,000/microL was 12, 15, and 17 days, respectively. Patients from group II reached these neutrophil levels at days 13, 15 and 17 post BMT. Neutrophil recovery in the control patient group occurred at days 17, 18, and 20 respectively. Group I patients were given platelet transfusions within 18 days and red blood cells within 10 days, whereas for group II patients, these time points were 26 and 17 days, respectively. These same transfusions could be ceased within 38 and 24 days, respectively, in control patients. The addition of about 2% more peripheral blood CD3+ cells (group I patients) did not result in higher grades of acute GVHD (median grade II) as compared with the controls (median grade II). Four of five group II patients showed no signs of acute GVHD. These data suggest that the addition of immunoselected allogeneic CD34+ progenitor cells to BM cells may accelerate hematopoietic recovery.

Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells.

BACKGROUND: Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells. MATERIALS AND METHODS: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out. RESULTS: The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation. CONCLUSIONS: The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.

DNA fingerprint analysis in acute leukemias.

Restriction fragment length polymorphism (RFLP) analysis by Southern blotting or direct in-gel hybridization is a routine procedure in any genetic laboratory. Minisatellites and simple repeat probes for RFLP analysis have proved to be highly informative genetic markers, depending on their degree of homology and index of heterozygosity. Several of these probes have considerable individualization potential, thus yielding 'fingerprint' pattern. In the setting of acute leukemia DNA fingerprint (DNA-F) analysis is able to provide considerable information concerning the genetic instability of the leukemic clone. DNA-F is capable of detecting randomly occurring genetic alterations of unknown localization and to identify new hotspots of malignant transformation. As DNA-F analysis is not likely to be hampered by the effects of chemotherapy or DNA methylation, altered fingerprints may be regarded as characteristic of the leukemic clone. With the introduction of polymerase chain reaction (PCR) and increasing sensitivity, DNA-F analysis is likely to be of significant importance in monitoring minimal residual disease in human leukemia.

Treatment of poor marrow graft function with allogeneic CD34+ cells immunoselected from G-CSF-mobilized peripheral blood progenitor cells of the marrow donor.

The treatment effect of immunoselected allogeneic CD34+ blood cells was evaluated in two patients with poor graft function following BMT without evidence for immune-mediated rejection. Patient A had no signs of hematopoietic recovery up to day +34 post-BMT and patient B had normal leukocyte counts only with G-CSF support and remained platelet transfusion-dependent for > 200 days post-BMT. PBPC from the HLA-identical sibling BM donors were mobilized with G-CSF (2 x 5 micrograms/kg sc daily) for 5 days. Aphereses were performed on days 4 and 5 of G-CSF administration. CD34+ cells were separated from the pooled PBPC concentrates by immunoadsorption with the anti-CD34 moAb 12.8 in a biotin-avidin system. Patient A received 0.4 x 10(6) CD34+ and 4.3 x 10(5) CD3+ cells/kg body weight and patient B 3.4 x 10(6) CD34+ and 1.4 x 10(5) CD3+ cells/kg body weight. The trilineage repopulation of BM and the rapid improvement of peripheral blood parameters correlated with CD34+ cell infusion. Patients' blood and BM cell analyses proved the donor origin. Patient A died from CMV pneumonitis and multiorgan failure 27 days after CD34+ cell infusion (day +61 post-BMT). Patient B is still stable and in remission 260 days after CD34+ cell infusion (day +478 post-BMT). Neither patient suffered further exacerbation of GVHD). Thus, immunoselected allogeneic CD34+ blood cells might be appropriate for treatment of post-BMT graft failure.

Cytomegalovirus infection in leucocytes after bone marrow transplantation demonstrated by mRNA in situ hybridization.

Cytomegalovirus (CMV) infection in leucocytes after bone marrow transplantation (BMT) was identified using a 35S-labelled antisense RNA probe specific for CMV immediate early (IE) gene mRNA. 54 patients were examined regularly up to 14 weeks after BMT. 36 after allogeneic BMT and 18 after autologous BMT. Only mononuclear cells with monocyte morphology were CMV IE mRNA positive. The number of CMV positive leucocytes was higher after allogeneic BMT than after autologous BMT (P = 0.006), and in patients with acute graft-versus-host disease (GvHD) II-IV than with GvHD O-I (P = 0.06). In patients who later developed chronic GvHD, the mean value of CMV infected leucocytes during the first week after BMT was higher than in patients without chronic GvHD (P = 0.034). Patients with symptomatic CMV infection had greater numbers of CMV infected leucocytes during the fourth and fifth week after BMT than patients without CMV disease, but the difference did not reach statistical significance. CMV infection of monocytes may be an important factor in the early onset of CMV infection and of GvHD.

Monitoring of relapse and remission in acute leukaemias by DNA-fingerprint analysis.

DNA-fingerprint (DNA-F) analysis was successfully performed with DNA from 22 adult patients with acute leukaemia, including 13 patients with acute myeloid leukaemia (AML) and nine patients with acute lymphoblastic leukaemia (ALL). The purpose of this study was to detect differences between the leukaemic phase (at diagnosis or relapse) and remission-phase DNA. We applied one simple repeat probe (GTG)5 and one minisatellite (M13) after DNA-digestion with different restriction endonucleases (HinfI and HaeIII) and agarose gel electrophoresis. In 7/13 patients with AML and 5/9 patients with ALL it was possible to detect loss of bands, additional bands or band shift with at least one of the probes. Together the probes M13 and (GTG)5 unveiled deviating fingerprint patterns in 54.6% of patients between leukaemic cells and remission-phase leucocytes. Allogeneic bone marrow transplantation was performed on six patients. In each case the DNA-F pattern of the donor was different from the relapse and the remission-phase pattern. We conclude from our studies that the probes M13 and (GTG)5 are useful in the detection of relapse and remission in acute leukaemias after chemotherapy and bone marrow transplantation.

Influence of the freezing-thawing-washing procedure on the cytomegalovirus DNA content of cellular blood products.

There is a proven relation between cytomegalovirus (CMV) infection in seronegative immunocompromised patients and transfusion of random CMV unscreened cellular blood products (BP), where the leukocytes are thought to be the vector of transmission. We examined whether freezing, thawing, and washing, in attempt to reduce the quantity of leukocytes, also reduces the CMV-DNA-carrying cells of BP of CMV-seropositive donors and their infectivity for fibroblasts, using the polymerase chain reaction (PCR). The leukocyte contamination of the thawed-washed erythrocyte concentrates was < 1 x 10(8)/l and of the thawed-washed platelet concentrates, approximately 2.5 x 10(8)/l. All plasma samples and most samples of BPs of the CMV-seronegative controls were PCR-CMV-DNA-negative. The differences of the PCR results with the samples of CMV-seropositive donors before and after freezing were not significant. Thus we concluded that freezing-thawing-washing of cellular BP could not eliminate all CMV-DNA-bearing leukocytes. Plasma carries low risk for CMV infection, if at all.

Detection of cytomegalovirus-infected cells by flow cytometry and fluorescence in suspension hybridisation (FLASH) using DNA probes labeled with biotin by polymerase chain reaction.

A biotin-labeled DNA probe specific for the immediate early gene of the cytomegalovirus (CMV) strain AD 169 was generated with the polymerase chain reaction. Nucleic acid hybridisation was carried out with CMV-infected T-lymphoblastoid cells (MOLT-4) after 4 hr to 6 days of culture. Biotin molecules were made visible with streptavidin coupled with fluorescein. The fluorescence signal of the hybridised probe was measured by flow cytometry in the cell suspension. The number of CMV-positive cells was 7% at 4 hr, 8% after 28 hr, 18% after 2 days, 26% after 3 days, 91% after 4 days, 97% after 5 days, and 98% after 6 days. The first detection of CMV antigen (pp65) was possible with immunoenzymatic labeling by day 4, whereas CMV-DNA was detected by PCR after 4 hr. CMV-specific RNA could be detected in a similar way. The analysis of mononuclear peripheral blood leukocytes in a patient with active CMV infection showed 14.7% CMV DNA-positive cells at day 1 and 7% at day 8, as compared to 0.9% and 0.0% cells which were positive for CMV antigen (pp65) by immunoenzymatic labeling at day 1 and day 8, respectively. We conclude that flow cytometry and fluorescence in suspension hybridisation (FLASH) offers a new tool for analysing exactly and quantifying large numbers of cells for specific DNA or RNA and may be useful for other laboratory and clinical applications.