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We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8(+)-recipient cells decreased rapidly, while CD4+ remained stable (17%). This work demonstrates the feasibility of FISH after cell sorting, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.

We retrospectively analysed the factors that influenced rate of haemopoietic recovery (HR) in 243 patients after transplantation with chemotherapy-mobilised autologous peripheral blood progenitor cells (PBPC). Approximately half the patients also received haemopoietic growth factors (HGF) for mobilisation. Conditioning for transplantation was with either chemotherapy alone or chemotherapy plus total body irradiation (TBI). Median time to recovery of granulocytes > or = 0.5 x 10(9)/l was 13 days (range 7-93 days) and of platelets > or = 50 x 10(9)/l 14 days (7-440). Speed of HR was greater, both for neutrophils and platelets for patients who received more rather than less CFU-GM than our median value of 18.9 x 10(4)/kg (P < 0.0001 in both instances) and more rather than less CD34-positive cells than our median value of 8.8 x 10(6)/kg (P < 0.0001 and P < 0.0005, respectively). For granulocyte recovery, in the multivariate analysis the dose of infused CFU-GM (P = 0.05) and the use of HGF for both mobilisation and post-transplantation (P < 0.0014) were significant positive factors. For platelet recovery in the multivariate analysis the dose of infused CFU-GM (P < 0.0016) was a positive factor. The use of busulphan and of TBI were significant adverse factors for rate of platelet recovery (P = 0.005 and 0.0004, respectively). When compared with non-HGF-mobilised PBPC, HGF-mobilised PBPC reduced the number of days of hospitalisation (28 vs 24, P = 0.0001) and of treatment with intravenous antibiotics (15 vs 11, P = 0.0004). These findings emphasise the importance of cell dose in accelerating haemopoietic recovery after autologous blood stem cell transplantation.

Expression of the multidrug resistance (MDR) phenotype is responsible for chemotherapy failure in numerous cancers. This phenotype is generally due to the expression of the mdr1 gene-encoded P-gp. Modulation of P-gp activity by chemotherapy has limited possibilities because of toxicity and poor specificity. In contrast, specific transcription blockage of the mdr1 gene can be obtained by oligonucleotides forming a triple helix structure at the DNA level. We used here immunofluorescence and both flow cytometry and image analysis to evaluate surface and total P-gp content in K562 MDR cells. The mdr1 mRNA content was measured by RT-PCR. We confirm the capacity of a 27-mer oligodeoxynucleotide, targeted to an mdr1 DNA fragment, to cause a 10-fold decrease in mdr1 mRNA level. However, this specific genetic inhibition was functionally limited because cellular growth was not modified in a cytotoxic environment. We found that total P-gp content was reduced in resistant cells treated with the mdr1-targeted oligonucleotide, while it remained in high levels on the cell surface, suggesting the existence of a large cytoplasmic pool of P-gp (approximately 50% of the total cellular P-gp). Moreover, when cycloheximide was used for 72 h to suppress protein synthesis, surface P-gp expression showed no decrease, whereas total P-gp was considerably lowered. A rapid 35% decrease in surface P-gp level was reached when resistant cells were treated for 24 h with brefeldin A, an inhibitor of intracellular protein trafficking. Simultaneously, the total P-gp level remained stable, thus indicating a probable accumulation of cytoplasmic P-gp, in agreement with the interruption of protein migration. We propose that the cytoplasmic P-gp pool could be a storage pool consumed for maintaining a steady-state level of surface P-gp. Cytometry could be a useful tool to study such a mechanism of P-gp trafficking and cellular distribution, which could explain the difficulties encountered in achieving stable and rapid effects of MDR reversal with oligonucleotides.

An increasing number of methods are being described to detect apoptotic cells. However, attempts to detect apoptotic cells in clinical samples are rarely successful. A hypothesis is that apoptotic cells are cleared from the circulation by phagocytosis before they become detectable by conventional morphological or cytometric methods. Using LR73 adhering cells as phagocytes in a model of in vitro phagocytosis, we found that phagocytosis of daunorubicin (DNR)-treated U937, HL60, or K562 leukemia cell lines occurred prior to phosphatidylserine externalization, DNA hydrolysis, chromatin condensation, nuclear fragmentation, or mitochondrial potential alteration. Moreover DNR-treated K562 cells were eliminated by phagocytes while apoptosis was never observed by any of the above methods. By contrast, using a fluorometric batch analysis assay to detect caspase activity in ceramide- or DNR-treated cells (fluorogenic substrate for caspase), we found that caspase activity increased in apoptosis-committed cells before they were detected by flow cytometry or recognized by phagocytes. Similarly a caspase activity increase was detected in circulating mononuclear cells of luekemic patients 15 h after the beginning of anthracyclin treatment. We suggest that recent findings on enzymatic events (caspase activation) occurring in the early events of apoptosis must now allow the development of new markers for apoptosis, irrespective of the morphological features or internucleosomal fragmentation which are late events in apoptosis.

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

The aim of this study was to compare the effectiveness of two methods of antibiotic susceptibility testing performed on Chlamydia trachomatis-infected cells: a flow cytometric detection method and the standard method, which consists of a microscopic reading of minimal inhibitory concentration (MIC). The L2 reference strain and 13 clinical strains isolated from six patients presenting recurrent infections were tested. McCoy cells infected with an inoculum of 10(5) inclusion forming units (IFU)/ml were incubated with serial dilutions of doxycycline, ofloxacin, and erythromycin. Mean fluorescence intensity (MFI) of cells was determined by flow cytometry after staining of chlamydial inclusions with an anti-Chlamydia fluorescent monoclonal antibody. The end-point values determined by flow cytometry and microscopic reading were equivalent but presented the same imprecision. Calculation of the inhibitory concentration 50 (IC50) by flow cytometry, defined as the antibiotic concentration required to reduce the drug-free control MFI by 50%, allowed a more objective and precise evaluation of antibiotic activity than MIC. Moreover, IC50 values were reproducible, independent of the antibiotic dilution series tested, and could be used to compare the in vitro efficiency of various drugs on C. trachomatis. No resistant strain was found among the 13 clinical isolates of C. trachomatis tested.

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT-PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.

A flow cytometry method has been introduced into the routine investigation of whole bone marrow samples following red blood cell lysis on the basis of a primary CD45/side scatter (SSC) gating procedure. Blast cells were first identified by CD45/SSC gating in 74 cases of acute myeloid leukemia (AML) and the results were compared to a conventional FSC/SSC gating procedure and to MGG-staining smears. The percentages of blast cells in these samples as defined by the morphological analysis of MGG smears correlated better with the values determined by CD45/SSC gating (r = 0.94) than with the blast cell counts recorded with FSC/SSC gating (r = 0.76). These findings were not surprising because while CD45 expression was regularly lower on leukemic blasts than on normal lymphoid and monocytic cells, the FCS/SSC characteristics of these populations were overlapping. In 53 samples, the blast cell populations were also analyzed with a panel of FITC-conjugated monoclonal antibodies that were utilized in double labeling with CD45-PE. We show that the CD45/SSC gating procedure improved phenotypic determination of the blast cells in three ways: (1) by discriminating between leukemic blast cells and residual normal cells; (2) by excluding normal cells from the phenotypic analysis of leukemic blast cells; and (3) by identifying blast cell heterogeneity in many cases of leukemia on the basis of different CD45 display. Moreover, this immunophenotyping procedure on whole bone marrow samples also allowed an efficient discrimination between the various cell lineages and facilitated the analysis of leukemic blasts present in low proportions.

Splenic lymphoma with villous lymphocytes (SLVL) is a chronic monoclonal B-cell lymphoproliferative disorder characterized by massive splenomegaly and typical villous lymphocytes in the peripheral blood (PB). The diagnosis of SLVL relies on blood smear examination, phenotypic features, and marginal zone involvement of the spleen. The histologic pattern of bone marrow (BM) involvement has not been well characterized. We report four cases associated with a peculiar intrasinusoidal BM involvement. This intrasinusoidal pattern was highlighted by immunostaining that also showed the cytoplasmic projections of villous lymphocytes within routinely fixed and decalcified BM biopsy specimens. Therefore, a simple immunohistochemical analysis of BM involvement would help to identify SLVL. Combined with cytologic and immunophenotypic evaluation of marrow and blood smears, this intravascular pattern might be helpful in differentiating SLVL from hairy cell leukemia and its variant. Whether this peculiar intravascular pattern combined with cytologic evaluation represents a practical alternative to the diagnostic splenectomy must be confirmed by extensive studies focusing on this immunohistochemical criterion.

A flow cytometric method for simultaneous apoptotic cell detection and cell cycle analysis was applied on the U937 cell line. Four antitumoral drugs currently used in the treatment of acute myeloid leukaemia were studied in vitro: DNR, IDR, MITO and Ara-C. Our results show a dissociation between the cytostatic effect (the block in the cell cycle observed for low drug concentrations) and the cytotoxic effect (the induction of apoptosis induced by higher concentrations) for all the tested molecules. Low concentrations of Ara-C induced a block in the S phase while higher concentrations (>10(-7) M) induced apoptosis at the G1-S boundary. Low concentrations of anthracyclines (<40 nM DNR and <20nM IDR) induced a block in G2 without apoptosis. Apoptosis was induced in G1 and/or early S phases by higher concentrations of anthracyclines. The concentration inducing 50% apoptosis (IC50) was found to be, respectively, 200 and 40 nM for DNR and IDR. Analysis of MITO-treated cells showed a parallel increase in the percentages of S phase and apoptotic cells. However, the bivariate analysis showed that apoptosis did occur in a population with G1 DNA content. For two other drugs (CAM and COLC), apoptosis occurred for the same concentrations and in the same phase as the block (in S and G2M, respectively). The IC50 of MITO was found to be 100 nM. Cotreatment of the cells with colchicin and either Ara-C or IDR showed that the passage through mitosis was not necessary for the completion of apoptosis at the G1-S boundary. Short incubations of U937 cells with high concentrations of anthracyclines were found to be efficient in inducing further apoptosis. We conclude that, for all the assayed molecules, the cytotoxic and/or cytostatic effects of the antitumoral drugs tested greatly depend on the concentrations used and that, depending on their in vivo pharmacokinetics, the induction of apoptosis could be an important mechanism of action for some of these drugs.

The response to therapy of leukemic cells is largely determined by their capacity of proliferation and apoptosis in presence of the administered drugs. We describe here the main markers used in flow cytometry (FCM) and involved in the assessment of cell cycle parameters: single labeling by Propidium Iodide (PI) and double labeling anti-Bromodeoxyuridine (BrdUrd)/PI which, both in vitro and in vivo, gives cell percentages in the different cell cycle phases. The markers of cell cycle progression can be divided into proliferation markers such as PCNA (proliferating cell nuclear antigen) or Ki-67 and cell cycle progression markers. The latter, which are the core of the cell cycle machinery, are molecules recently characterized (Cyclins, CDKs (cell dependent kinases), CDIs (cyclin-dependent kinase inhibitors)) and their cell expression can be analyzed using FCM. FCM is also one of the best means to detect and quantitate apoptotic cells. Several techniques are described: Nuclear labeling using Hoechst 33342: mitochondrial labeling using DiOC6(3): detection of DNA fragmentation using 1) labeling of fixed and permeabilized cells with a DNA marker or 2) labeling of the free 3' DNA ends using incorporation of labeled deoxynucleotides; detection in apoptotic cells (Bcl-2, Fas, phospholipids...). At last, we analyzed flow cytometry methods to study the cell resistance to Ara-C and anthracyclins. In combination with cell kinetic studies and detection of apoptotic cells, they should increase the efficiency of the acute leukemia treatment.

Recruitment of quiescent, clonogenic blasts from patients with acute myeloid leukemia (AML) by hematopoietic growth factors (HGFs) may improve the cytotoxic effects of cell-cycle-specific drugs like cytosine-arabinoside (Ara-C). Using the culture methods described by Nara and McCulloch and making a distinction between self-renewing and post-deterministic mitoses, we analyzed the effects of stem cell factor (SCF), a growth factor acting on early hematopoietic progenitor and stem cells. First, we demonstrated that SCF, used in combination with other HGFs included in fetal calf serum (FCS) and/or in 5637 cell line supernatant (5637-CM), stimulated both colony formation and self-renewal of blast progenitors from 10 patients, unlike SCF alone. We tested the effects of SCF on the recruitment of cells in the S-phase by using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM). We showed that SCF stimulated proliferation of AML cells significantly in 9/18 patients with AML. Second, we tested the influence of SCF on the sensitivity to Ara-C of self-renewing leukemic cells from 18 patients with AML. We showed that SCF was efficient in increasing the toxicity of Ara-C on the self-renewing blast progenitors, especially with high concentrations of Ara-C. However, a large patient-to-patient heterogeneity was found and the activity of SCF was not correlated with its effect on the cell cycle. These data indicate that SCF can enhance sensitivity to Ara-C of some leukemic cells with self-renewing capacity.

Background: Converting enzyme inhibitors meet most of the criteria required to be used in acute pulmonary edema. However, they could also induce deleterious effects on renal function and electrolytes. The purpose of this study was to evaluate the efficacy and safety of a single intravenous 2-hour infusion of enalaprilat (1 mg) after an acute pulmonary edema., Methods and Results: This was a placebo-controlled, randomized, double-blind study performed in 20 congestive heart failure patients (New York Heart Association class III or IV). Systemic and regional hemodynamic parameters, biological parameters, and blood gases were measured before and repeatedly after the onset of infusion. Compared with placebo, enalaprilat decreased pulmonary capillary wedge pressure (-37% versus -10%, P = .001), diastolic and mean systemic blood pressures (-21% versus 0%, P = .009, and -18% versus -1%, P = .026, respectively), diastolic and mean pulmonary blood pressures (-21% versus -8%, P = .040; -18% versus -9%, P = .046), and brachial and renal resistances (-44% versus -14%, P = .017, and -22% versus -2%, P = .014, respectively); increased brachial and renal blood flows (+77% versus +8%, P = .036, and +12% versus 0%, P = .043, respectively), arterial oxygen tension (+2% versus -16%, P = .041), and arterial oxygen saturation (+1% versus -2%, P = .045); and tended to decrease rate-pressure product (-19% versus -7%, P = .076), increase brachial artery diameter (+13% versus 0%, P = .081), and improve intrapulmonary shunt (-18% versus +16%, P = .080). Enalaprilat did not affect cardiac output or carotid or hepatosplanchnic hemodynamics., Conclusions: Early administration of enalaprilat is effective and well tolerated in acute pulmonary edema.

Twenty-three patients with acute myelogenous leukemia (AML) in first relapse were treated with high-dose cytosine-arabinoside (Ara-C) and amsacrine or idarubicin. To prime the cells, the patients were given rhGM-CSF. We studied the influence of 48-h infusion of rhGM-CSF on proliferation and Ara-C sensitivity of leukemic cells both ex vivo and in vitro. We found that a 48-h infusion of rhGM-CSF increased both white blood cell counts and peripheral blood blast cell percentages. Using a Bromodeoxyuridine/DNA (BrdUrd/DNA) staining in flow cytometry, we found an non-constant increase in cells in the S-phase. Ex vivo 48-h culture of leukemic cells with or without rhGM-CSF, with or without other hematopoietic growth factors (HGFs), showed a greater increase of the cells in the S-phase with GF but no correlation with the ex vivo results. We used a method of quantitation of the DNA synthesis previously described (Lacombe F., et al. (1992) Cytometry 13, 730) to monitor the Ara-C sensitivity of the cells in S-phase before and after 48-h infusion with rhGM-CSF. We observed a great variation in the Ara-C sensitivity of the leukemic cells before and after infusion with rhGM-CSF from one patient to another. The BrdUrd/DNA method seems a convenient method to study the influence of HGFs on Ara-C sensitivity of the patients.

The treatment of acute myeloid leukaemia in adults is based on chemotherapy which is divided in two phases: induction (to achieve a complete remission) and post-induction treatment (to maintain the complete remission). Induction chemotherapy usually combines cytosine arabinoside and one anthracycline. The complete remission rate which varies from 50 to 90% is mainly influenced by the age of patients, the presence of certain cytogenetic abnormalities or the presence of a preleukaemic phase. The efficacy of post-induction treatments remains controversial. Maintenance treatment (with low-dose chemotherapy) has probably a marginal efficacy. More intensive (as intensive as induction chemotherapy) chemotherapies seems capable to prolong survival but can only be applied to the youngest patients. The rate of 5-years survivors is about 30-45% and influenced by a very low numbers of factors. The treatment of relapses is very difficult and seems to prolong survival only when the duration of the first remission has been longer than 18 months. In other cases, it can be justified to restrict the treatment to transfusions and oral chemotherapy.

Antisense oligonucleotides were used to determine the role of the BCR-ABL gene in the proliferation of chronic myeloid leukaemia (CML) clonogenic cells. Peripheral blood Philadelphia chromosome positive cells were obtained from eight CML patients at diagnosis (chronic phase = 7; accelerated phase = 1). Mononuclear cells were incubated with synthetic antisense 18-mer oligonucleotides complementary to the two different junctions b2a2 or b3a2. The type of junction (b2a2 or b3a2) was previously determined by RT-PCR techniques. Cells incubated for 12 to 14 hours with or without sense oligonucleotides served as controls. After incubation with oligonucleotides, the cell DNA synthesis was analysed by flow cytometry using the BrdUrd/DNA method and the cell plating efficiency in methylcellulose was determined. In six of the seven patients in chronic phase, there was a significant inhibition of CFU-GM production which was only 68.4 +/- 19%; (p < .01) of that found in controls. The S phase index, which depends upon the percentage of S phase cells as well as the fluorescence intensity, was 48 +/- 29% (p < .01) of the control values for the seven patients in chronic phase. Interestingly, for the only CML patient in accelerated phase, antisense oligomers had no inhibitory effect on either the production of CFU-GM or the number of S phase cells. In improving the specificity of oligomers, it might be useful for gene-targeted anti-leukemic therapy and/or bone marrow purging.

To evaluate the leukocyte differential flags of the Cobas Argos 5 Diff., the authors performed a comparative study between their current analyzer, the Technicon H2, and the manual leukocyte differential. Samples (n = 1,600) were collected from the Blood Disease Department of their hospital and were tested on both Cobas Argos 5 Diff. (ABX/Roche Hematology Division, Montpellier, France) and Technicon H2. Abnormalities of the manual leukocyte differential (immature granulocytes, blast cells, atypical lymphocytes, hyperbasophil cells, erythroblasts, and hairy cells) were found in 597 samples. The authors determined the best cut-off of the quantitative flags--atypical lymphocytes (ALYs) and large immature cells (LICs)--using the likelihood ratio method, and the capability of the 5 Diff. qualitative flags to determine abnormal subpopulations by the predictive value of a positive result. The presence of particular combinations of flags was associated with band cells and blast cells of acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML). The sensitivity, specificity, and efficiency of the two analyzers were 92.1, 53.9, and 68.2, respectively, for the Argos 5 Diff., and 93.5, 36.7 and 57.9, respectively, for Technicon H2. In conclusion, the Cobas Argos 5 Diff. is well adapted for detecting leukocyte abnormalities.

Haematopoietic colony-stimulating factors are a group of molecules that can stimulate haemopoiesis. With the advent of recombinant DNA technology and the cloning of their genes, they can be utilized against malignant blood diseases. They might be useful in therapy of acute myelogenous leukaemia either by increasing the efficacy of antileukaemic drugs or by shortening the duration of granulocytopoenia following chemotherapy or bone marrow transplantation. Encouraging results have been obtained, but stimulation of leukaemic cells is not ruled out. Further clinical and biological investigations are required to provide insight into the role of cytokines in treatment of acute myelogenous leukaemia.

We evaluated the expansion capacity of untreated and 5-fluorouracil (5-FU)-resistant peripheral blood stem cells (PBSC) after 7-day incubation with interleukin-1 (IL-1) plus IL-3 plus stem cell factor (SCF) or with medium alone. We found a significant increase in the proportion of CD34+ cells in the PBSC fraction resistant to 25 micrograms/mL 5-FU after 7-day incubation with IL-1 plus IL-3 plus SCF as compared with the untreated fraction (p = 0.011). We also showed that 5-FU-resistant PBSC have a greater capacity for expansion of IL-1/IL-3/SCF-responsive immature progenitors (p = 0.05), amplification of IL-3 plus GM-CSF responsive progenitors (p = 0.01), and production of committed single growth factor-responsive (granulocyte-macrophage colony-stimulating factor [GM-CSF]) precursors (p = 0.01) than the untreated PBSC. The expansion of all types of progenitors and CD34+ cells was only observed after 7-day incubation with IL-1 plus IL-3 plus SCF. These results suggest that PBSC contain a primitive stem cell population with an enhanced expansion capacity that is identified by 5-FU resistance. As these cells can be expanded in vitro, they may then be suitable for a number of clinical applications.

Ara-C is currently used in the treatment of adult acute myeloid leukemia (AML). The cytotoxicity of Ara-C derives from an inhibition of DNA synthesis which can be determined using flow cytometry from the amount of bromodeoxyuridine (BrdUrd) incorporated into cells after a short exposure to BrdUrd. We developed a computer program to quantify inhibition of the rate of DNA synthesis by analysis of the distribution of BrdUrd/DNA. A resistance index (RI) was expressed as the ratio of the amount of BrdUrd incorporated into S phase cells incubated with Ara-C to that incorporated in the absence of Ara-C. In Ara-C sensitive and resistant HL60 cell lines, a linear relationship between RI and log Ara-C concentration was observed. This technique was applied to 96 bone marrow samples from patients with de novo AML treated by a regimen containing Ara-C. A first group of nine patients with high RI values included only drug resistant (DR) patients; a second group of 63 patients with low RI values included 62 patients who achieved a complete remission (CR); a third group of 24 patients with intermediate RI values included 19 CR and five DR patients. In view of these results, we think that it is possible to detect a majority of DR patients treated by Ara-C.

The techniques of flow cytometry are becoming more and more important for the clinical hematology laboratory. No longer a novelty confined to a few specialized institutions as it was 10 years ago, flow cytometry has blossomed into a mature discipline. The methodology is well-known, the mechanical apparatus is readily available, and the role it plays in clinical hematology is increasingly appreciated. The burgeoning number of scientific articles devoted to this topic attests to the interest it has aroused as a tool for both medical research and patient care. In fact, more than a thousand such papers are now published each year and it would be impossible to deal with all the methodologies and applications of FCM currently utilized or under development. Throughout this paper four relevant hematologic fields are briefly discussed, in which FCM appears to be of great help at present: the immunophenotyping of leukemias and lymphomas, the measurement of proliferative activity and DNA ploidy in hematological malignancies, the detection of drug resistant leukemic cells and the use of FCM in the study of platelets.

Apoptosis or programmed cell death (PCD) was measured in two human cell models by flow cytometric analysis. Blood neutrophils underwent spontaneous apoptosis in short-term culture. Pentoxifylline (PTX) inhibited spontaneous neutrophil PCD. We confirmed that granulocyte/macrophage colony-stimulating factor (GM-CSF) inhibited apoptosis of polymorphonuclear neutrophils. Treatment with both GM-CSF and PTX did not increase the inhibition of PCD by either GM-CSF or PTX alone. Because apoptosis could be due to the accumulation of H2O2 in the culture medium, and because PTX has been described to reduce peroxide production, we studied the effect of adding catalase to the medium. Catalase reduced the neutrophil apoptosis and this effect was cumulative with the effect of PTX. Camptothecin, an inhibitor of topoisomerase I, induces a block in the S-phase of the cell cycle followed by apoptosis of the U937 cell line. This drug-induced apoptosis was partially inhibited by PTX, whereas the S-phase cell block was not affected. In conclusion, PTX was found to inhibit apoptosis in two different human cell types. In neutrophils, this effect appears to occur regardless of the inhibition of phosphodiesterase activity and inhibition of H2O2 release.

A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells.

Cytarabine (Ara-C) is currently used in the treatment of adult acute myeloid leukemia (AML). To predict the results of induction chemotherapy, it could be useful to detect leukemic cells that are resistant to Ara-C in patients with AML. Using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM), we have developed a cell resistance index to Ara-C (RI). The technique has been applied to 121 bone marrow (BM) samples from patients with de novo AML treated by a regimen containing Ara-C and daunorubicin (DNR). Ninety-seven patients achieved a complete remission (CR), and 24 patients did not and were considered drug-resistant (DR). The BM cells collected at diagnosis were cultured for 48 hours and underwent BrdUrd/DNA analysis. Among 25 patients with no or very low proliferative activity (<3% of cells in S-phase), the proportion of DR patients (nine of 25) was significantly higher than in a second group of 96 patients with detectable proliferative activity (15 of 96) (P < .025). Within this second group, there was a first group of nine patients with high RI values, which included only DR patients; a second group of 63 patients with low RI values, which included 62 CR patients; and a third group of 24 patients with intermediate RI values, which included 19 CR and five DR patients. In view of this series, our results show that it is possible to detect a majority of DR patients treated by Ara-C.

To further characterize the primitive stem cell subpopulation present in chemotherapy mobilized peripheral blood stem cells (PBSC), we evaluated the functional characteristics of 5-FU-resistant PBSC and normal bone marrow (BM) cells after 7 days incubation with IL-1 + IL-3+SCF. The resulting 5-FU-resistant cells were evaluated for (1) the production of GM-CSF-responsive clonogenic elements (CE), (2) the production of IL-3+GM-CSF-responsive CE, and (3) their self-renewal capacity (production of IL-1+IL-3+SCF-responsive CE). We also evaluated the percentage of CD34+ cells, the percentage of cells in S phase of the cell cycle, and the number of nucleated cells before and after cytokine-mediated expansion. We demonstrated an overall loss in nucleated cells after cytokine-mediated expansion in all cell fractions. We demonstrated a significantly greater increase in the percentage of CD34+ cells in the 5-FU-resistant PBSC fraction as compared to 5-FU-resistant BM cells (p = 0.012). We also showed that 5-FU-resistant PBSC have a greater capacity for self-renewal, amplification of IL-3+GM-CSF-responsive progenitors, and the production of committed GM-CSF-responsive progenitors as compared with BM cells, but this did not reach statistical significant. These results suggest that PBSC contain a truly primitive stem cell with an enhanced self-renewal and differentiative capacity that is recruited by 5-FU resistance and IL-1+IL-3+CSF-mediated expansion.(ABSTRACT TRUNCATED AT 250 WORDS)

By using flow cytometry, the intracellular accumulation (Acc) of idarubicin (IDA) and daunorubicin (DNR) and the effect of verapamil (VRP) on both anthracycline accumulation (VRP index) were studied in leukemic cell lines (K562 and HL60 and their two DNR-resistant subclones) and fresh leukemic cells. IDA accumulated more than DNR in both parental (K562: p < 0.03 and HL60: 0.09) and resistant cell lines (p < 0.01 for both cell lines) irrespective of whether or not they were treated with VRP. VRP index was higher for DNR than for IDA (p < 0.05). Similar results were observed in fresh leukemic blasts from 25 patients with ANLL (IDA Acc superior to DNR Acc: p < 0.0001; higher VRP index for DNR than for IDA: p < 0.01). The higher Acc of IDA than DNR seen in fresh leukemic cells could explain the better clinical efficacy of IDA reported in patients with ANLL.

The DNR accumulation (DNR Acc) and the verapamil (VRP) index (percent increase of VRP on DNR accumulation) was studied by using flow cytometry. Fresh leukemic mononuclear bone marrow blasts from 80 unselected ANLL patients' samples were incubated with DNR in the presence or absence of VRP. The DNR accumulation was determined by flow cytometry. The median DNR Acc was 28 (range: 4-101) and the median VRP index was 4% (range 0-53). VRP significantly enhanced DNR Acc in 42 of the ANLL samples (52.5%). DNR Acc or VRP index were not influenced by age, sex, or WBC counts. Only the FAB subclassification and the blast immunophenotyping were found to influence the parameters studied here. The lowest DNR Acc was found in M0 and M6 blast cells (15 range 0-46 and 10.5 range 8-13 respectively). M4 and M5 ANLL samples accumulated significantly more DNR than M0 and M6 blast cells. The VRP index was significantly higher in M0 compared with M1 and M2 samples, as well as in M4 compared with M1 samples. A slightly positive correlation was found between the percentage of CD34-positive cells in the CD34-positive samples and DNR Acc. In this study, DNR Acc and the VRP index were not significantly correlated with the response to chemotherapy or survival. In conclusion, this study shows that ANLL leukemic cells differ in anthracyclin accumulation and response to VRP in vitro.

The antigen Ki-67 Ag, regarded as a marker for proliferating cells, was identified as a protein(s) (pKi-67) which can exist free or associated with DNA as evidenced by DNA digestion of cells before or after immunolabeling with Ki-67. The dual nature of this antigen was also supported by reconstitution of Ki-67 Ag from purified DNA and nuclear proteins extracted from the K562 cell line. The immunoreactivity of the resulting complexes was examined in solution using Ki-67 and MIB-1 antibodies. The interaction between Ki-67 or MIB-1 antibodies and pKi-67 was enhanced in the presence of undegraded ds DNA, indicating that ds DNA modulates the conformation of pKi-67 and that the altered conformation of pKi-67 is more reactive than the pure protein to both Ki-67 and MIB-1 antibodies.

Cell proliferation of blast cells in acute myeloid leukemia (AML) plays a major role in determining response to therapy. Therefore, studies of leukemic cell growth and related factors have been very numerous involving many techniques. They have used a wide panel of proliferation markers but despite their close correlation, we consider here markers assessing cell cycle parameters and those related to cell proliferation.

Over the past decade, Hematopoietic Growth Factors (HGFs) have been shown to improve the treatment of malignant blood diseases. They reduce the duration of leucopenia after chemotherapy and the associated infectious morbidity, without affecting remission duration, or percentage of patients resistant to chemotherapy. Moreover, HGFs are able to increase the percentage of leukemic cells in the S phase. In consequence, HGFs have been used in acute myeloblastic leukemia (AML) to recruit quiescent clonogenic blasts to improve the cytotoxic effects of cell cycle specific drugs. The clinical results are divergent and do not allow definite conclusions. However, in vitro, the efficacy of HGFs to enhance cytosine arabinoside (ara-C)-mediated cytotoxicity has been reported. Whether the HGFs-mediated increase in the antileukemic activity of ara-C is caused by changes in the cell cycle status of AML blasts or by other mechanism(s) is currently debated.

Ten patients with acute myeloblastic leukemia (AML) in first relapse were treated with high-dose cytosine-arabinoside (ara-C, 3 g/m2/12 hours x 4) and amsacrine (150 mg/m2/day x 5). In order to prime the cells, the patients were given rh-GM-CSF (3 micrograms/kg/d) for four days, the first infusion starting 48 hours before chemotherapy. Two patients died during the aplastic phase, seven patients achieved a second complete remission (CR2) and one patient remained leukemic. The median duration of aplasia was 17 days (14-21). These results were comparable to those obtained in our previous series of 27 patients treated for AML in first relapse with the same chemotherapy but without GM-CSF (66% CR2). After 48 hours of GM-CSF infusion, (before chemotherapy was started), seven patients had an increase in the white blood cell count with an increase in the absolute number of blast cells in five of these cases. Marrow blast cells percentages increased in 3 of the 8 patients analysed. Six of seven patients tested showed an increase in the percentage of cells in S-phase (studied by flow cytometry using the bromodeoxyuridine (BrdU/DNA) labelling technique and BrdU incorporation). GM-CSF used to prime leukemic cells may be safely administered but its clinical usefulness needs to be further evaluated.

Peripheral blood stem cells (PBSC) contain a mixture of mature and immature hematopoietic progenitors. Resistance to 5-Fluorouracil (5-FU) has been used to identify and characterize primitive quiescent stem cells among bone marrow (BM) cells. To see if the same technique could be used to isolate a similar population of cells among PBSC, low-density peripheral blood mononuclear cells (PBMNC) were collected by cytapheresis in the regenerative phase after high-dose chemotherapy from patients with hematological malignancies. These PBMNC were incubated with increasing concentrations of 5-FU for 24 h. The viable 5-FU resistant cells were then cultured in semi-solid media in the presence of either single cytokines: TCM 5637, Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), or a combination of cytokines: interleukin 1 (IL-1) IL-1 + IL-3 + 5637, IL-1 + IL-3 + Stem Cell Factor (SCF). Low concentrations (5-10 micrograms/ml 5-FU) eliminated mature day 7 Colony Forming Units-Granulocyte Macrophage (CFU-GM) and spared day 7 clusters while enriching for day 14 CFU-GM, irrespective of the growth factors used. Higher concentrations of 5-FU (15, 20, 25 micrograms/ml) selected for later forming clonogenic elements. A combination of synergistic growth factors was required for the development of morphologically identifiable clonogenic elements resistant to 25 micrograms/ml 5-FU at day 21 of culture. Further experimentation demonstrated that SCF could effectively replace TCM 5637 in the cytokine combination for the detection of primitive late forming clonogenic elements. The presence of SCF potentiated colony formation by 5-FU resistant PBMNC. It was confirmed that GM-CSF alone was unable to support colony formation by PBMNC resistant to 25 micrograms/ml. These observations demonstrate that PBSC contain a heterogenous mixture of hematopoietic progenitors and that incubation with 25 micrograms/ml 5-FU permits access to a quiescent primitive stem cell population that requires a combination of synergistic growth factors for the development of morphologically identifiable clonogenic elements at day 21. Taken together, these results suggest that PBSC have similar characteristics to BM derived stem cells.

Two different molecular techniques were used to monitor chimerism following 17 non-T cell-depleted BMTs from female donors to male recipients: pHY10, a Y chromosome-specific probe (Southern or slot blots), and a set of primers for Y chromosome sequence-specific amplification by the polymerase chain reaction (PCR). On Southern blots, male DNA was detectable at a level less than 1% of 10 micrograms DNA while cross-reactivity with autosomal sequences was avoided. On slot blots, male DNA was reliably detectable at levels less than 0.5%, even in small sample (0.5 microgram DNA). With the PCR technique, male DNA was detectable at levels of 1:10(6) to 1:10(7) of 0.5 microgram DNA. Slot blot and PCR results were concordant in 19 of 23 samples. Both techniques demonstrated a constant small mixed chimerism during the first year after BMT and in four of nine patients, this chimerism persisted even longer (up to 29 months after BMT).

A method for the detection of poly(A)+ RNA in cell suspensions by in situ hybridization and flow cytometry is described. The hybridizing properties of oligothymidylate o(dT) was well preserved after coupling to fluorescein. This fluorescent oligonucleotide was used as a probe to determine the poly(A)+ RNA content of fixed HL60 leukemic cells by flow cytometry. Labeling was considerably reduced by treating the cells with RNAse, and by competitive hybridization with either poly(U) or free poly(A). Labeling was also decreased in a time-dependent fashion by incubating the cells with actinomycin D prior to fixation. This method represents an improvement on the methods measuring total RNA and could be of value in investigations on the effect of drugs on RNA metabolism.

Bone marrow blast cell antigen expression from 86 patients with de novo acute myeloid leukemias (AML) was studied and correlated with FAB classification and clinical outcome. Among a panel of 14 monoclonal antibodies routinely used for the diagnosis of acute leukemias we studied the expression of six antibodies (CD13, CD15, VIM2, CD33, CD14, CD34) of the granulomonocytic lineage and found that some of them were useful for diagnosis and/or prognosis. For FAB subclassification of AML, the CD13 or VIM2 antigen expression was of no benefit. Monocytic leukemias (M4 + M5PD + M5WD) more frequently expressed CD34 antigen (28/31) than granulocytic (M1 + M2 + M3) subtypes (33/53) (P < 0.01). Finally, the most striking differences were found with CD14 antigen expression: CD14 antigen was more frequently expressed in M4 + M5 leukemias (21/31) than in M1 + M2 + M3 subtypes (12/33) (P < 0.01). The mean percentage of CD14 positive blast cells was accordingly higher in monocytic leukemias than in granulocytic leukemias and the difference was highly significant (P < 0.0001). The CD15 antigen was more frequently expressed in differentiated leukemias (M2 + M3 + M4 + M5WD) (35/44) than in poorly differentiated forms (M1 + M5PD) (17/37) (P < 0.001). The statistical difference was higher when the mean percentage of CD15 positive blast cells were compared (P < 0.0003). Moreover these latter percentages were different in M1 and M2 subtypes (P < 0.003). The blast cell expression of CD13, CD14, CD15 or CD33 was not predictive of the length of CR or survival. Moreover, our results support previously published findings suggesting a longer overall survival duration for patients whose leukemic cells do not express the CD34 antigen (P < 0.01). We also confirm that patients with the more differentiated subtypes of AML (CD13-, CD34+) tend to survive longer than patients with the less differentiated subtypes of AML (CD13-, CD34+) (P < 0.001).

Non-invasive assessment of mechanical properties of the aorta may prove useful in the early detection of atheroma. We have evaluated several of the available echocardiographic indices using ability to detect age-related changes in putatively disease-free vessels as a measure of sensitivity to changes in aortic mechanical properties. Suprasternal imaging was used in 49 healthy non-smoking volunteers to measure minimum and maximum aortic arch diameters. Maximal flow velocities, with corresponding acceleration times and heart periods, were determined in the descending aorta in 24 of these subjects. Blood pressure was recorded non-invasively immediately after the echocardiographic study. Doppler derived measurements of aortic flow acceleration did not relate to age (P greater than 0.05). Three different 2D echo assessments of aortic distensibility, however, all showed a close relationship to age. Ep elastic modulus and Beta index (derived from different stress-strain mechanical relationships) were significantly related to age with r = 0.69 and 0.65 respectively. There were no significant effects of gender or left ventricular systolic function on these relationships. There was a tendency for the relationship between these distensibility indices and age more closely to fit an exponential than a linear relationship. We conclude that 2D echocardiographic assessment of aortic distensibility is able to detect sensitively changes in aortic mechanical properties. Even in the absence of risk factors for cardiovascular disease there is a marked reduction in aortic distensibility with increasing age.

Serial marrow karyotyping was performed in 31 chronic myeloid leukemia (CML) patients treated with allogeneic bone marrow transplantation (BMT). Of 11 hematological relapses, seven were heralded for up to 20 months by a cytogenetic relapse (characterized by increasing percentages of Philadelphia (Ph)-positive metaphases, seen on serial karyotypes). Chromosomal abnormalities additional to the Ph, seen before BMT, were not found again at relapse. Relapses were characterized by clonal evolutions of the Ph-positive cells, likely corresponding to cytogenetic patterns of treatment-induced leukemia [del(5q), del(7q), complex karyotypes] and were different from those generally found in CML evolution. Involvement of chromosome 1 was also frequent. Sporadic Ph-positive metaphases (not seen in repeated karyotypes) were seen only during the first 8 months after BMT.

An eight-month-old was admitted for acute congestive heart failure with fever. The respective parts played by hypocalcemia (due to vitamin-D deficiency rickets) and acute Epstein-Barr virus infection are discussed. Hypocalcemia was sufficiently marked to induce heart failure per se but replenishment of calcium stores was followed by only partial improvement in cardiac manifestations. Initial management was difficult because of the risks associated with concomitant administration of calcium and digitalis. After eighteen months during which the patient's status remained stable, evaluation showed that clinical features were consistent with sequelae of acute viral myocarditis. The possibility of primary hypokinetic dilated cardiomyopathy was then considered. Esterified carnitine levels were found to be increased leading to further investigations which outruled mitochondrial cytopathy.

Long term and semi-solid culture techniques were used to evaluate the quality of stroma produced by bone marrow from 33 normal subjects and 57 patients (46 allogeneic bone marrow and 11 autologous blood stem cell transplant recipients). Bone marrow from transplant recipients was capable of sustained CFU-GM and nucleated cell production in long term culture. However, only 13% of the marrow investigated developed a complete, confluent stromal layer. These stromal abnormalities were observed in spite of complete hematopoietic reconstitution after transplantation and rarely improved with time. Our results suggest that the hematopoietic microenvironment is very fragile and susceptible to long term damage as a result of chemotherapy and the conditioning regimes used prior to transplantation.

The cytotoxicity of ara-C derives from an inhibition of DNA synthesis after incorporation of ara-CTP into DNA. The rate of DNA synthesis can be determined from the amount of bromodeoxy-uridine (BrdUrd) incorporated into cells after a short exposure to BrdUrd. We developed a computer program to quantify the inhibition of the rate of DNA synthesis by analysis of the distribution of BrdUrd/DNA. Inhibition was evaluated in ara-C-sensitive and resistant cells after incubation with different doses of ara-C. An index of resistance to ara-C (RI) was expressed as the ratio of the amount of BrdUrd incorporated into S phase cells incubated with ara-C to that incorporated in the absence of ara-C. In the ara-C-sensitive and resistant HL60 cells, a linear relationship between RI and log ara-C concentration was observed. Small numbers of slightly resistant cells in mixtures of ara-C-sensitive and resistant cells could be determined using this method, making it suitable for clinical use to test the resistance of leukemic cells to ara-C.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.

The relationship between heparin and fibrinolysis is strongly suggested. We have studied the influence on fibrinolysis of standard heparin (SH), Calciparin and low molecular weight heparin (LMWH), Fraxiparin, given preventively in an elderly population. Patients were randomized into two groups (SH, LMWH). We investigated fibrinolytic parameters (ECLT, t-PA antigen, PAI-1 activity and antigen, t-PA/PAI-1 complexes) before treatment (D0) and at D30 and D60, before and after venous occlusion (VO). Values at D0, D30 and D60 were compared within each group. A significant and marked increase in t-PA and t-PA/PAI-1 complexes at D30 and D60 before and after VO was noted only in the SH group. The mechanism and clinical relevance of this increase remains to be established.

The stiffness of the thoracic aorta can be assessed non-invasively. If aortic stiffness can be shown to be related to coronary heart disease, perhaps it can be used to identify which patients with hypercholesterolaemia are most likely to have atheromatous changes and thus to be selected for intensive cholesterol-lowering treatment. Hence the distensibility of the transverse aortic arch was measured by echocardiography of the aortic arch in four groups of patients--symptom-free patients with normal serum cholesterol; symptom-free patients with raised serum cholesterol; patients with coronary heart disease (all with raised serum cholesterol), and post-heart-transplant patients. In all groups distensibility fell with age. The regression slope was steeper (p less than 0.05) for patients with known coronary disease than for either of the disease-free groups, and among cardiac transplant recipients there was also a segregation of distensibility values between those with and without atheroma in their native hearts. The results indicate that aortic distensibility might be an indicator of coronary heart disease and that it might be useful in identifying which symptom-free subjects with modest hypercholesterolaemia should be treated aggressively.

Forty-seven patients with chromosome Philadelphia-positive (Ph1) chronic granulocytic leukaemia (CGL) in transformation underwent autologous transplantation of peripheral blood stem cells (ABSCT) collected at the original diagnosis before any treatment. They were treated with three consecutive strategies: single transplant (group I = 17 patients), double transplant (group II = 13 patients), double transplant followed by recombinant alpha interferon (group III = 17 patients). Forty-three patients were restored to a second chronic phase with a cytogenetic conversion (more than 10% Ph1-negative marrow metaphases) occurring in 14 of the 29 evaluable patients. Most patients had a recurrent transformation occurring 2-43 months after ABSCT and finally eight patients are still alive in second chronic phase 4-49 months after ABSCT (median = 24 months). The actuarial median duration of second chronic phase was 3 months, 10 months and 18 months for group I, group II and group III patients (P less than 0.0001). The encouraging results observed for group III patients prompt us to propose ABSCT for patients in chronic phase with initial prognostic factors, suggesting that recombinant alpha interferon will not be effective if administered as front-line therapy.

The antibody Ki67 is currently used to evaluate the proliferative fraction of solid tumors and some hematological malignancies. We have used phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes as a model to study the entry of quiescent cells into cell cycle and to follow their progress to the next cycle. Flow cytometric analysis of lymphocyte samples stained with the antibody Ki67 and a DNA marker has allowed us to follow the expression of Ki67 antigen (Ki67 Ag) as a function of the position of the cells in the cell cycle. The use of drugs blocking the stimulated lymphocytes in different phases of the cell cycle permitted us to demonstrate that Ki67 Ag expression started from the beginning of the first S phase. The level of Ki67 Ag increased during S phase until mitosis, when its expression was maximal. After division, the cells in G1 phase showed a decrease in Ki67 Ag expression (possibly corresponding to degradation) until they reentered S phase, when the level of Ki67 Ag increased again. The results confirm that the expression of Ki67 Ag is related to the proliferative state of the cells and suggest that it may be used to determine the proliferative cell fraction in hematopoietic tissues.

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.