3D-printed scaffold combined to 2D osteoinductive coatings to repair a critical-size mandibular bone defect.

The reconstruction of large bone defects (12 cm3) remains a challenge for clinicians. We developed a new critical-size mandibular bone defect model on a minipig, close to human clinical issues. We analyzed the bone reconstruction obtained by a 3D-printed scaffold made of clinical-grade polylactic acid (PLA), coated with a polyelectrolyte film delivering an osteogenic bioactive molecule (BMP-2). We compared the results (computed tomography scans, microcomputed tomography scans, histology) to the gold standard solution, bone autograft. We demonstrated that the dose of BMP-2 delivered from the scaffold significantly influenced the amount of regenerated bone and the repair kinetics, with a clear BMP-2 dose-dependence. Bone was homogeneously formed inside the scaffold without ectopic bone formation. The bone repair was as good as for the bone autograft. The BMP-2 doses applied in our study were reduced 20- to 75-fold compared to the commercial collagen sponges used in the current clinical applications, without any adverse effects. Three-dimensional printed PLA scaffolds loaded with reduced doses of BMP-2 may be a safe and simple solution for large bone defects faced in the clinic.

Biomechanical behaviour of human bile duct wall and impact of cadaveric preservation processes.

Biliary diseases are the third most common cause of surgical digestive disease. There is a close relationship between the mechanical performance of the bile duct and its physiological function. Data of biomechanical properties of human main bile duct are scarce in literature. Furthermore, mechanical properties of soft tissues are affected by these preservation procedures. The aim of the present work was, on the one hand, to observe the microstructure of the human bile duct by means of histological analysis, on the other hand, to characterize the mechanical behavior and describe the impact of different preservation processes. A mechanical study in a controlled environment consisting of cyclic tests was made. The results of the mechanical tests are discussed and explained using the micro-structural observations. The results show an influence of the loading direction, which is representative of an anisotropic behavior. A strong hysteresis due to the viscoelastic properties of soft tissues was also observed. Embalming and freezing preservation methods had an impact on the biomechanical properties of human main bile duct, with fiber network deterioration. That may further provide a useful quantitative baseline for anatomical and surgical training using embalming and freezing.

Biodistribution and preliminary toxicity studies of nanoparticles made of Biotransesterified ß-cyclodextrins and PEGylated phospholipids.

BACKGROUND: The modification of ß-cyclodextrins (ßCDs) by grafting alkyl chains on the primary and/or secondary face yields derivatives (ßCD-C10) able to self-organize under nanoprecipitating conditions into nanoparticles (ßCD-C10-NP) potentially useful for drug delivery. The co-nanoprecipitation of ßCD-C10 with polyethylene glycol (PEG) chains yields PEGylated NPs (ßCD-C10-PEG-NP) with potentially improved stealthiness. The objectives of the present study were to characterize the in vivo biodistribution of ßCD-C10-PEG-NP with PEG chain length of 2000 and 5000Da using nuclear imaging, and to preliminarily evaluate the in vivo acute and extended acute toxicity of the most suitable system. RESEARCH DESIGN AND METHODS: The in vivo and ex vivo biodistribution features of naked and decorated nanoparticles were investigated over time following intravenous injection of 125I-radiolabeled nanoparticles to mice. The potential toxicity of PEGylated ßCD-C10 nanosuspensions was evaluated in a preliminary in vivo toxicity study involving blood assays and tissue histology following repeated intraperitoneal injections of nanoparticles to healthy mice. RESULTS: The results indicated that ßCD-C10-PEG5000-NP presented increased stealthiness with decreased in vivo elimination and increased blood kinetics without inducing blood, kidney, spleen, and liver acute and extended acute toxicity. CONCLUSIONS: ßCD-C10-PEG5000-NPs are stealth and safe systems with potential for drug delivery.

Guidelines for May-Grünwald-Giemsa staining in haematology and non-gynaecological cytopathology: recommendations of the French Society of Clinical Cytology (SFCC) and of the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP).

OBJECTIVE: Since the guidelines of the International Committee for Standardisation in Haematology (ICSH) in 1984 and those of the European Committee for External Quality Assessment Programmes in Laboratory Medicine (EQALM) in 2004, no leading organisation has published technical recommendations for the preparation of air-dried cytological specimens using May-Grünwald-Giemsa (MGG) staining. DATA SOURCES: Literature data were retrieved using reference books, baseline-published studies, articles extracted from PubMed/Medline and Google Scholar, and online-available industry datasheets. RATIONALE: The present review addresses all pre-analytical issues concerning the use of Romanowsky's stains (including MGG) in haematology and non-gynaecological cytopathology. It aims at serving as actualised, best practice recommendations for the proper handling of air-dried cytological specimens. It, therefore, appears complementary to the staining criteria of the non-gynaecological diagnostic cytology handbook edited by the United Kingdom National External Quality Assessment Service (UK-NEQAS) in February 2015.

Cytotoxicity of chalcone derivatives towards glioblastoma.

BACKGROUND: Among seventeen compounds derived from chalcones investigated as potential anticancer drugs towards LN229 glioblastoma cell line, only two were effective. MATERIALS AND METHODS: Anticancer activity was investigated by evaluating the cell growth, cell cycle, mitotic index and the cell death. RESULTS: Two compounds, namely C2 and C12, inhibited cell proliferation associated with a blockade in the G(2)/M phase of the cell cycle and arrested the growth of tumour spheroid mimicking in vivo tumour. C2 blocked cells in the G(2) phase whereas C12 blocked cells in the M phase of the cell cycle. C12 and C2 killed 40% and 95% of the cells respectively using complex mechanisms. The two compounds increased the fluorescence of rhodamine-123 and N-acetylcysteine inhibited their activity, suggesting a role for reactive oxygen species in cell death mediated by these two compounds. CONCLUSION: C2 and C12 are markedly cytostatic and cytolytic to glioblastoma cells and act through different pathways.

Cathodal iontophoresis of treprostinil and iloprost induces a sustained increase in cutaneous flux in rats.

BACKGROUND AND PURPOSE: The treatment of scleroderma-related digital ulcers is still a therapeutic challenge. The most effective drugs are prostacyclin analogues. However, their usage is limited to an intravenous route of administration and by their frequent side effects. The objective of this study was to test whether treprostinil, iloprost and epoprostenol can induce sustained vasodilatation in rats when delivered locally using cutaneous iontophoresis. EXPERIMENTAL APPROACH: Treprostinil, iloprost and epoprostenol were delivered by cathodal and anodal iontophoresis onto the hindquarters of anaesthesized rats (n= 8 for each group). Skin blood flow was quantified using laser Doppler imaging and cutaneous tolerance was assessed from day 0 to day 3. KEY RESULTS: Cathodal but not anodal iontophoresis of treprostinil (6.4 mM), iloprost (0.2 mM) and epoprostenol (1.4 mM) induced a significant and sustained increase in cutaneous blood flow. The effects of treprostinil and iloprost were significantly different from those of treprostinil vehicle. Only weak effects were observed when both drugs were applied locally without current. Skin resistance was unchanged in areas treated with prostacyclin analogues. Finally, skin tolerance was good, with no evidence of epidermal damage. CONCLUSIONS AND IMPLICATIONS: Cathodal iontophoresis of treprostinil and iloprost increases cutaneous blood flow with a good local tolerance. The effects of cathodal iontophoresis of these drugs should be investigated in humans, as they could have potential as new local therapies for digital ulcers in patients with scleroderma.

[Early detection of leptomeningeal metastasis in patients with metastatic breast carcinoma: validation of CA 15-3 measurement in cerebrospinal fluid]. / Détection précoce des métastases leptoméningées chez les patientes atteintes de cancer du sein métastatique: validation du dosage du CA 15-3 dans le liquide céphalorachidien.

Fifteen per cent of metastatic breast cancer will develop symptomatic leptomeningeal metastases. The introduction of trastuzumab (Herceptin) therapy has improved the response rates of survival of patients with metastatic breast cancer overexpressing HER2. Although previous studies are retrospective and of limited number, involving small study groups and different types of patient management, several authors have reported a 30% incidence of leptomeningeal metastases in patients with metastatic breast cancer overexpressing HER2 who were treated with trastuzumab, while 70 to 80% of cases of the disease were controlled systemically. In order to improve control of the disease at the level of the central nervous system (CNS), routine detection of leptomeningeal metastases in high-risk patients could be offered. CA 15-3 in cerebrospinal fluid (CSF) detection might be useful in helping to diagnose CNS metastases, particularly where cytology results are negative--which applies to 30% of cases--because tumor markers are more sensitive in detecting the tumor process. Our study validate CA 15-3 measurement in CSF and reference values were given.

A pilot study in two French medical schools for teaching histology using virtual microscopy.

PURPOSE: Glass slides and standard microscopes associated to a brief review of the lectures with projection slides were used during practical training in histology and histopathology for many years. Today it is necessary to develop new tools to improve teaching, and to face a lower number of teachers, as well the increase of the microscope maintenance costs. The goal of this study was to evaluate the feasibility of virtual slide implantation in several medical schools, the feedback from students, and to develop the interest in microscopic histology. METHODS: We used virtual slides generated by the Samba 2050 system produced by Samba technologies. A collection of all organs for histology training was realized and overviewed by three MD, PhD. A questionnaire was distributed in middle of the year to evaluate the feedback. RESULTS: The feedback of the students is highly positive. Students works faster, on better resources, interactivity between students is increased, and the fact that this is a new modality of teaching raises the students' interest. CONCLUSION: Today the teaching program in two French medical schools (Lyon and Grenoble) include virtual slides alone or in addition to microscopic glass slide examination to teach histology or pathology.

Optimization of PKH67 labeling conditions for proliferation monitoring in daunorubicin-treated leukemic cells.

Daunorubicin (DNR) is commonly used to treat acute myeloid leukemia (AML). The aim of this study was to determine whether PKH67 dye dilution could be used for differential proliferation monitoring in chemosensitive (K562S) and chemoresistant (K562R) leukemic sublines after drug treatment. Cells were labeled with PKH67 and treated for 2 h with a sublethal dose of DNR or vincristine either immediately or after 3 h in fresh medium. Viability (TOTO-3 exclusion) and DNR uptake (total cellular DNR fluorescence) were assessed by flow cytometry, and nuclear DNR accumulation was determined by confocal laser microspectrofluorimetry. Immediate DNR treatment led to enhanced DNR uptake and decreased viability in PKH67-labeled K562S, whereas no excess toxicity was seen if DNR treatment was delayed for 3 h. Treatment with vehicle control (Diluent C) gave similar results. In contrast, PKH67 labeling had no effect on K562S viability after vincristine treatment. For K562R, DNR uptake measured at 120 min was unaltered by prior exposure to PKH67 or vehicle, but viability was again significantly reduced after immediate DNR treatment. As with K562S, delaying DNR treatment for 3 h normalized viability in K562R. The excess DNR toxicity seen for PKH67-labeled K562S appears to be drug related, since it is not seen with vincristine, and may be due to the daunosamine sugar moiety present in DNR. However, with the addition of a 3-h incubation in fresh medium prior to drug exposure, PKH67 dye dilution can be used for proliferation monitoring of both K562S and K562R cells after treatment with DNR.

Fluorescence-based assessment of LRP activity: a comparative study.

BACKGROUND: Broad resistance to anticancer drugs is a major cause of failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a protein associated with drug resistance, which is involved in nucleo-cytoplasmic transport and is known to predict a poor response to chemotherapy in acute myeloid leukaemia. The only method allowing the detection of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal was to develop a fluorescence-based assay to analyse LRP function. MATERIALS AND METHODS: We used human colon carcinoma cell lines treated with sodium butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in isolated nuclei was measured by flow cytometry, the localization and quantification of Daunorubicin analysed by confocal laser scanning microscopy (CLSM) and the diffusion coefficient of this drug estimated by Fluorescence Correlation Spectrometry (FCS). RESULTS: According to the method using [14C] Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and showed a higher dispersion of Daunorubicin kinetics with cells previously NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify anticancer drug repartition in the cells. LRP activity assessment needs either isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may be analysed.

A flow cytometric assay for simultaneous assessment of drug efflux, proliferation, and apoptosis.

BACKGROUND: Proliferative status and multidrug resistance play a key role in determining cell response to chemotherapy. There is a need to develop multiple labeling method that allows simultaneous assessment of multidrug resistant (MDR) phenotype, proliferative status, apoptosis related changes in mitochondrial potential, in chemosensitive and chemoresistant tumor cell populations. METHODS: A three-color labeling was performed using Hoechst 33342 (DNA), JC1 (mitochondrial potential), and a far red fluorescent membrane intercalating dye: PTIR271 (proliferation). RESULTS: Combined staining of DNA and mitochondrial potential allows identification of subpopulations expressing and MDR phenotype mediated by P-glycoprotein (Pgp), and, in Pgp negative subpopulations, identification of apoptotic cells and evaluation of cell cycle status in viable cells. Addition of a far red fluorescent membrane intercalating dye, PTIR271, allows simultaneous monitoring of cell division status by dye dilution in both drug sensitive and drug resistant populations. CONCLUSION: This triple labeling is an interesting method to study the proliferation status of drug sensitivity and drug resistance in viable tumor cells.

TIMP-1/MMP-9 imbalance in an EBV-immortalized B lymphocyte cellular model: evidence for TIMP-1 multifunctional properties.

Tissue inhibitors of metalloproteinases (TIMPs) were initially described as agents controlling metalloproteinase activity. The purpose of this study was to investigate the expression and the roles of TIMP-1 secreted by Epstein-Barr-virus (EBV)-immortalized B lymphocytes. TIMP-1 was isolated from conditioned medium of interleukin (IL)-1beta stimulated EBV-B lymphocytes; purified TIMP-1 was identified by mass spectrometry and immunochemistry. TIMP-1-free MMP-9 was quantified after purification by zymography and enzyme-linked immunosorbent assay. EBV-B lymphocyte-secreted TIMP-1 inhibited MMP-9 gelatinolytic activity resulting in decreased B-cell transmigration as measured in vitro. The release of huge amounts of TIMP-1 in proportion to MMP-9 from B lymphocytes after EBV transformation was shown to be correlated with secretion of IL-10 and dependent on culture time. In contrast, there was little TIMP-1 and almost no IL-10 released from native B cells, suggesting a possible IL-10 mediated autocrine regulation mechanism of TIMP-1 synthesis. The MMP-9/TIMP-1 imbalance observed in the culture medium of EBV-B lymphocytes (TIMP-1>MMP-9) and of native B cells (MMP-9>TIMP-1) is suggestive of a new function for TIMP-1. We propose that TIMP-1 acts as a survival factor controlling B-cell growth and apoptosis through an autocrine regulation process involving IL-10 secreted by EBV-B lymphocytes.

Nucleus labeling or membrane labeling for studying the proliferation of drug treated cells?

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia (AML). This study compared cell cycle analysis (nuclear labeling) with cell division analysis (membrane labeling, PKH67) for studying the proliferation of cells cultured with daunorubicin (DNR) and/or Cytarabine (Ara-C), drugs commonly used in AML treatment. PKHs are a family of lipophilic, fluorescent membrane intercalating dyes. When labeled cells divide, the resulting daughter cells receive half the label, reducing fluorescence intensity to one-half that of the parent cells. DNR has the disadvantage of overlapping the spectrum of propidium iodide (PI), which is the most commonly used marker of membrane integrity. In this study, necrosis was evaluated using TOTO-3, a marker of nucleic acids emitting fluorescence above 645 nm and which incorporates cells with disrupted membranes. Comparison of cell cycle analysis with cell division for studying proliferation revealed that PKH67 was a marker of choice for analyzing the mitotic process in cells cultured with drugs.

Identification of amylase crystalloids in cystic lesions of the parotid gland.

OBJECTIVE: To identify alpha-amylase crystalloid formations in parotid specimens obtained by fine needle aspiration. STUDY DESIGN: The study concerned three cases of sialadenitis with crystalloid formation observed between 1993 and 1998. In one of these cases, transmission electron microscopy, mass spectrometry and measurement of amylase activity were used to characterize the nature of the crystalloids. RESULTS: Light microscopy revealed the same crystalloid structure in all three cases. In one case, where the material was saved, a biochemical method made it possible to reveal high amylase activity, while protein electrophoresis and mass spectrometry were used to identify salivary alpha-amylase. CONCLUSION: Crystalloids of salivary alpha-amylase can be identified by May-Grünwald-Giemsa and Papanicolaou stain and can be rapidly confirmed through determination of amylase activity.

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis.

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines.

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukaemia (AML). Anthracyclines such as daunorubicin (DNR) are typically used to treat AML and can induce drug resistance. The goal of the studies described here was to select a combination of fluorescent probes that could be used in combination with flow cytometry to monitor cell proliferation vs. cell death/necrosis as a function of anthracycline uptake. Propidium iodide (PI), the most commonly used marker of membrane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap. A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was also selected. The results show that DAPI and Cascade Blue (CB), like PI, were able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI. In the presence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR. However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR accumulation, necrotic cells based on CB incorporation, and proliferating cells based on partitioning of PKH67 fluorescence between daughter cells. It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation.

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

Usefulness of PKHs for studying cell proliferation.

Classical methods for proliferative assessment (such as tritiated thymidine or bromodeoxyuridine (BrdUrd) incorporation) need sample fixation. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine the rate and extent of proliferation in cell lines. Flow cytometric analysis associated with modelling software makes it possible to estimate the number of cells having undergone different numbers of cell divisions by sampling the cell population at varying times post-labelling. Two major questions were addressed in these studies. (i) Does PKH26 give a stable and reproducible labelling? (ii) Does labelling with PKH26 alter cellular proliferation characteristics? We conclude that the methods developed here provide a simpler, more complete means for assessment of cell proliferation in patients with hematological malignancies.

PKH26 probe in the study of the proliferation of chemoresistant leukemic sublines.

Proliferative status and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia. Although classical methods for proliferative assessment such as tritiated thymidine or BrdUrd incorporation, are correlated with treatment outcome, they are time consuming and difficult to standardize. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine rate and extent proliferation in drug sensitive and resistant cell lines. When cells labelled with this fluorescent membrane intercalating dye divide, each resulting daughter cell receives half of the dye. Using flow cytometric analysis, it is possible to estimate the number of cells having undergone different numbers of cell divisions. Four different questions were addressed in these studies: a) does PKH26 give stable and reproducible labelling? b) does labelling with PKH26 alter cellular proliferation characteristics? c) is PKH26 a substrate for PGP and MRP? d) does PKH26 labelling alter PGP expression and/or PGP activity? We found that PKH26 labelling is stable, reproducible and has no effect on cell proliferation. It does not modify PGP activity or expression, nor does it appear to be a substrate for PGP or MRP, since the rate of decrease in fluorescence intensity is similar for sensitive and resistant cells which are proliferating at the same rate. Using the dye dilution method, it is possible to simultaneously assess PGP, proliferative status, and level of PGP expression. We conclude that the methods developed here provide a simpler, more complete means for assessment of the effects of the drug therapy on sensitive and resistant cell populations in patients with hematologic malignancies.

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.