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Background: The Flow-Self Organizing Maps (FlowSOM) artificial intelligence (AI) program, available within the Bioconductor open-source R-project, allows for an unsupervised visualization and interpretation of multiparameter flow cytometry (MFC) data., Methods: Applied to a reference merged file from 11 normal bone marrows (BM) analyzed with an MFC panel targeting erythropoiesis, FlowSOM allowed to identify six subpopulations of erythropoietic precursors (EPs). In order to find out how this program would help in the characterization of abnormalities in erythropoiesis, MFC data from list-mode files of 16 patients (5 with non-clonal anemia and 11 with myelodysplastic syndrome [MDS] at diagnosis) were analyzed., Results: Unsupervised FlowSOM analysis identified 18 additional subsets of EPs not present in the merged normal BM samples. Most of them involved subtle unexpected and previously unreported modifications in CD36 and/or CD71 antigen expression and in side scatter characteristics. Three patterns were observed in MDS patient samples: i) EPs with decreased proliferation and abnormal proliferating precursors, ii) EPs with a normal proliferating fraction and maturation defects in late precursors, and iii) EPs with a reduced erythropoietic fraction but mostly normal patterns suggesting that erythropoiesis was less affected. Additionally, analysis of sequential samples from an MDS patient under treatment showed a decrease of abnormal subsets after azacytidine treatment and near normalization after allogeneic hematopoietic stem-cell transplantation., Conclusion: Unsupervised clustering analysis of MFC data discloses subtle alterations in erythropoiesis not detectable by cytology nor FCM supervised analysis. This novel AI analytical approach sheds some new light on the pathophysiology of these conditions., (© 2022 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Multiparameter flow cytometry (MFC) is a powerful and versatile tool to accurately analyze cell subsets, notably to explore normal and pathological hematopoiesis. Yet, mostly supervised subjective strategies are used to identify cell subsets in this complex tissue. In the past few years, the implementation of mass cytometry and the big data generated have led to a blossoming of new software solutions. Their application to classical MFC in hematology is however still seldom reported. Here, we show how one of these new tools, the FlowSOM R solution, can be applied, together with the Kaluza® software, to a new delineation of hematopoietic subsets in normal human bone marrow (BM). We thus combined the unsupervised discrimination of cell subsets provided by FlowSOM and their expert-driven node-by-node assignment to known or new hematopoietic subsets. We also show how this new tool could modify the MFC exploration of hematological malignancies both at diagnosis (Dg) and follow-up (FU). This can be achieved by direct comparison of merged listmodes of reference normal BM, Dg, and FU samples of a representative acute myeloblastic case tested with the same immunophenotyping panel. This provides an immediate unsupervised evaluation of minimal residual disease. © 2019 International Society for Advancement of Cytometry., (© 2019 International Society for Advancement of Cytometry.)

The complete blood cell count and white blood cell differential are the first step in the biological diagnosis of hematological diseases. Both are currently performed by automated instruments which control data and produce alerts. If such flags are activated, the automated differential cannot be validated and the operator must activate a visual blood smear review. Microscopic examination is still today the reference method despite its lack of sensitivity and reproducibility. The HematoFlow™ (Beckman Coulter) system is the first flow cytometry commercialized method designed for the routine differential. Using six markers in five colors and an automated gating strategy, it provides differentials proven to be reliable for 17 leukocyte subpopulations detection. Relying first on a retrospective analysis of 6,462 blood samples processed by HematoFlow™, thresholds were determined to detect the presence of immature granulocytes and/or blast cells. All possible gating strategy misclassifications of leukocyte subpopulations were then summarized in a systematic nomenclature leading to the development of an original flag system based on the detection of aberrant localization of cell events in specific new bivariate histograms. Ultimately, more than 50% of the results could be automatically validated using the HematoFlow™ system, without any false negative, thereby dramatically contributing to an important decrease of technicians' workload. Moreover a noticeable help was given for smear review interpretation and new immunological flags led to the confirmation of blood disease after classical immunophenotyping. These results were confirmed in a second prospective study including 15,335 cases, where more than 50% of the results were automatically validated by this new flag system. MFC stands as being more and more essential for analyzing differentials in routine and this new flag system could greatly improve its implementation., (© 2015 International Clinical Cytometry Society.)

Flow cytometry (FC) instruments settings classically rely on local establishment of photomultipliers (PMT) voltages adapted to the measurements expected to be performed. In the era of multiparameter FC (MFC), it appears more and more desirable that comparable patterns of fluorescence are obtained in different settings. This relies on a harmonization of settings between instruments. Although this has been shown to be feasible within a given brand of flow cytometers, little information is available about broader comparisons in a given center or in a multicenter fashion. Here, we report a two-phase series of experiments first performed between a Canto II (BD Biosciences) and a Navios (Beckman Coulter) instruments in the same center. PMT values adjusted on the reference instrument (RI) Canto II were used to establish target values for PMT settings on the paired Navios practice instrument (PI). This allowed to show the good correlation of all but peaks 1 and 2 of Rainbow(®) beads between RI and PI. Using 4- or 8-color stained leukocytes, the similitude of the settings was further confirmed. A complex set of matrices was then established between five centers all equipped with both instruments. Using Bland & Altman difference comparisons for median fluorescence values, it was shown that using either Rainbow beads or CD16 stained polymorphonuclears to set-up target values on the RI CantoII, highly superimposable results could be obtained on all 9 PI. The latter were obtained using Rainbow beads or Compbeads(®) for comparisons. In summary, this two-phase study demonstrates the feasibility of different methods allowing for a robust harmonization of settings for MFC., (© 2013 International Society for Advancement of Cytometry.)

Background: The development of multiparameter flow cytometry (FCM) and increasingly sophisticated analysis software has considerably improved the exploration of hematological disorders. These tools have been widely applied in leukaemias, lymphomas, and myelodysplasias, yet with very heterogeneous approaches. Consequently, there is no extensive reference document reporting on the characteristics of normal human bone marrow (BM) in multiparameter FCM. Here, we report a reference analysis procedure using relevant antibody combinations in normal human BM., Methods: A first panel of 23 antibodies, constructed after literature review, was tested in four-color combinations (including CD45 in each) on 30 samples of BM. After evaluation of the data, a second set of 22 antibodies was further applied to another 35 BM samples. All list-modes from the 65 bone marrow samples were reviewed collectively. A systematised protocol for data analysis was established including biparametric representations and color codes for the three major lineages and undifferentiated cells., Results: This strategy has allowed to obtain a reference atlas of relevant patterns of differentiation antigens expression in normal human BM that is available within the European LeukemiaNet. This manuscript describes how this atlas was constructed., Conclusions: Both the strategy and atlas could prove very useful as a reference of normality, for the determination of leukemia-associated immunophenotypic patterns, analysis of myelodysplasia and, ultimately, investigation of minimal residual disease in the BM.

Electronic white blood cell (WBC) differential by standard cytology (hematology analyzer and visual inspection of blood smears) is limited to five types and identification of abnormal cells is only qualitative, often problematic, poorly reproducible, and labour costing. We present our results on WBC differential by flow cytometry (FCM) with a 6 markers, 5 colors CD36-FITC/CD2-PE+CRTH2-PE/CD19-ECD/CD16-Cy5/CD45-Cy7 combination, on 379 subjects, with detection of 12 different circulating cell types, among them 11 were quantified. Detection of quantitative abnormalities of whole leucocytes, neutrophils, eosinophils, basophils, monocytes, or lymphocytes was comparable by FCM and by standard cytology in terms of sensitivity and specificity. FCM was better than standard cytology in detection and quantification of circulating blast cells or immature granulocytes, with a first lineage orientation in the former case. All cases of lymphocytosis, with lineage assignment, were detected by FCM. FCM identified a group of patients with excess of CD16pos monocytes as those having an inflammatory syndrome. WBC differential by FCM is at least as reliable as by standard cytology. FCM superiority consists in identification and systematic quantification of parameters that cannot be assessed by standard cytology such as lineage orientation of blast cells or lymphocytes, and expression of markers of interest such as CD16 on inflammatory monocytes., (Copyright (c) 2007 International Society for Analytical Cytology.)

Background: The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response., Methods: We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment., Results: We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk-VAD-FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk-VAD-FITC, in the d15 residual bone marrow blast cells, correlated with lower disease-free survival probability., Conclusion: More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction., (Copyright 2006 International Society for Analytical Cytology.)

Background: Multiparametric flow cytometry has become an indispensable but complex tool for the diagnosis of acute leukemias. Interpretation of immunophenotypic data within a six-parameter analytical space relies on the standardization and validation of the instrument, the reagents, and the procedure. To address whether or not residual normal lymphocytes, usually present within leukemic samples, can serve as internal quality control for fluorescence intensity, 116 leukemic and 35 normal samples were analyzed., Methods: Eight laboratories participated in the study and recruited a total of 151 individuals including 29 patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), 77 with acute myeloid leukemia (AML), 10 with T-cell precursor acute lymphoblastic leukemia (T-ALL), and 35 normal bone marrow donors. Lymphocytes were gated according to the CD45hi/SSClo gating strategy, after which median fluorescence intensities (MFI) as well as percentages of positive cells (%positive) for CD19, CD22, CD7, and CD3 were recorded. Nonparametric statistics were used to compare variation within and between laboratories., Results: Normal lymphocytes within leukemic samples do not show substantial differences compared to lymphocytes from normal controls with respect to expression of CD19, CD22, CD7, and CD3. In particular, longitudinal control charts of MFI values for CD3 antigen provide useful information on analytical and instrument performance., Conclusion: Residual normal lymphocytes can serve as internal quality control for studies addressing fluorescence intensity in the setting of immunophenotyping of acute leukemias., (Copyright (c) 2005 Wiley-Liss, Inc.)

CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells that disappears with their maturation. This gene is transcribed in two alternatively spliced mRNAs that encode full length and truncated form of CD34 cell surface antigen. Some publications suggested that CD34 full length plays a role in the maintenance of their self renewal capacity. An examination of CD34 regulation by a low O2 concentration that ensures a better maintenance of stem cells may provide important insights into the molecular control of hematopoiesis. Using human cord blood CD34+ cells, we first compared the effect of short term (24 h) culture in hypoxia (1% O2) and normoxia (20% O2) on the expression of full length and truncated form of cd34 transcripts and on the expression of the CD34 antigen. Hypoxia maintained a larger quantity of cd34 full length transcripts and a higher cd34 full length/cd34 truncated form ratio than normoxia. After 72 h of culture at 1% and 20% O2, sorted CD34low sub-population from 1% O2 primary culture still contained more cd34 full length mRNAs than those from 20% O2, maintained better CD34 antigen expression during secondary culture at 20% O2 and contained more undifferentiated cells. This work provides the first evidence of the regulation of the cd34 gene by hypoxia resulting in a delayed higher and longer antigen expression by cord blood cells. We suggest that this phenomenon is related to the better maintenance of primitive stem cells in hypoxia., (2005 Wiley-Liss, Inc.)

Background: Procaspase 3 is a constitutive proenzyme that is activated by cleavage during apoptosis. The resulting enzyme is able to cleave several target proteins after the second aspartate of a DEVD sequence common to all the substrates of caspases 3 and 7 (DEVDase). Because active caspase 3 is a common effector in several apoptotic pathways, it may be a good marker to detect (pre-)apoptotic cells by flow cytometry (FCM). Materials and Methods Apoptosis was induced in U937 or bone marrow mononuclear cells by daunorubicin (DNR), idarubicin (IDA), or camptothecin (CAM). Viable and apoptotic cells were sorted by FCM on the basis of either fluorescein isothiocyante (FITC)-annexin V binding or DiOC6(3) accumulation. DEVDase activity was measured in sorted populations by spectrofluorometry. Cleaved caspase 3 was labeled in situ with phycoerythrin (PE)-conjugated anti-activated caspase 3 antibodies and analyzed by FCM., Results: DEVDase activity was detected in sorted viable CAM- and DNR-treated U937 cells, demonstrating that a partial caspase activation occurred earlier than phosphatidyl-serine exposure and mitochondrial membrane potential dissipation. The presence of a low amount of active caspase 3 in the treated viable cells was confirmed in situ with PE-conjugated anti-active caspase 3 antibodies. The same antibody was used in combination with FITC-annexin V and CD45-PC5 to study caspase 3 activation in acute leukemia blast cells after in vitro DNR and IDA treatment. Both anthracyclines induced a caspase 3-dependent apoptosis that was more efficient in blast cells than in contaminating lymphocytes., Conclusions: These results demonstrate that anti-active caspase 3 labeling can be an alternative to fluorogenic substrates to efficiently detect early apoptosis by FCM in heterogeneous samples. They also confirm that anthracyclines induce blast cell apoptosis by a caspase 3-dependent pathway., (Copyright 2000 Wiley-Liss, Inc.)

Background: Cell chemosensitivity to cytotoxic drugs has been attributed to their ability to trigger apoptosis. The emergence of resistance in drug-exposed cells is often characterized by the appearance of drug efflux mechanisms including P-gp transport. Nevertheless, mdr1 expression may coexist with other resistance features, in particular those interfering with apoptotic signaling pathways., Methods: Leukemic cell lines cultured in a progressively toxic environment were analyzed for Fas and P-gp expression by immunostaining and flow cytometry. Their mdr1 mRNA expression level was determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and their apoptotic response was microscopically evaluated. Activation of the Fas pathway was obtained by cross-linking the Fas receptor with the 7C11 anti-Fas agonist., Results: We demonstrate a dose-dependent Fas overexpression after short-term (18 h) incubation with daunorubicin. The subsequent sensitization to Fas activators led to a significant increase in the apoptotic response induced by 7C11. After long-term exposure to daunorubicin and acquisition of drug resistance, expression of P-gp was accompanied by a decrease in the number of Fas sites at the cell surface with a correlated desensitization to Fas-induced apoptosis. Additional alterations in the Fas signaling pathway can also be hypothesized in the most resistant Jurkat cell line., Conclusions: The induction of Fas expression could be one of the mechanisms of action of chemotoxic drugs and thus might enhance the cell susceptibility to Fas-mediated apoptosis. On the contrary, the emergence of the multidrug resistance phenotype is associated with a down-regulation of Fas expression and possible defects in the Fas signaling pathway., (Copyright 2000 Wiley-Liss, Inc.)

Background: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes., Methods: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results., Results: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis., Conclusions: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle., (Copyright 2000 Wiley-Liss, Inc.)

Background: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment., Methods: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot., Results: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype., Conclusions: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.

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.

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.

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.

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.

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.

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 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.

Four published methods of DNA-content histogram analysis (those of Fried, Dean and Jett, simplified Dean, and Fox) were compared using a double labeling of different cell populations. Partially synchronized and asynchronous cell populations were incubated with bromodeoxyuridine (BrdUrd) and then stained with an anti-BrdUrd monoclonal antibody and propidium iodide (PI). The fractions of cells in the G1, S, and G2 + M phases were calculated by each method and compared with those derived from G1, S, and G2 + M areas plotted on BrdUrd/DNA bivariate histograms, taken as the "true" values. This procedure enabled an optimal choice of method for a given cell population.