Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy.

Point-source digital in-line holographic microscopy with numerical reconstruction is ideally suited for quantitative phase measurements to determine optical path lengths and to extract changes in refractive index within accuracy close to 0.001 on the submicrometer length scale. This is demonstrated with simulated holograms and with detailed measurements on a number of different micrometer-sized samples such as suspended drops, optical fibers, as well as organisms of biological interest such as E. coli bacteria, HeLa cells, and fibroblast cells.

Fast exact scalar propagation for an in-line holographic microscopy on the diffraction limit.

In lensless digital in-line holographic microscopy, currently applied fast reconstruction techniques use approximations limiting the usable NA for optical resolution. The computational effort for an exact scalar reconstruction with straightforward algorithms depends on the relation between the desired resolution and the given pixel pitch of the detector. So there is a trade-off between achievable image resolution and required computation time. We present an exact reconstruction algorithm that guaranties optimum resolution with affordable computation time. Experimental results show a realized NA of at least 0.62. A 1 megapixel hologram was reconstructed in about 1.5 s.

Reconstruction of high-resolution holographic microscopic images.

In in-line holographic microscopy a pinhole illuminates an object and a CCD-detector directly records the hologram in a pixel-pitch-dependent distance. A rapidly calculating exact reconstruction technique using a reorganized hologram with a low number of pixels, the tile superposition technique, is presented. The algorithm is applied on imaging of a 2 microm bead cluster, and it is compared with other reconstruction techniques. The high-contrast image corresponds to an NA of 0.7. A full 4 megapixel reconstruction with a resolution approaching the diffraction limit is possible in less than a minute. The technique is a base for high-resolution wide-field imaging by multispot illumination.

Phase resolving microscopy by multi-plane diffraction detection.

An easily applicable quantitative phase-contrast microscopy is presented. A bright-field microscope with a coherent illumination is used. Only a series of diffraction images is taken in different distances from the sample. For reconstruction, a conjugate gradient technique as a new variant of phase retrieval technique is developed. The technique is experimentally investigated in detail. A phase accuracy up to 0.07 rad or a height accuracy of 13 nm in transmission for a binary test sample is reached, respectively.

[Development of an incubation system for an inverted microscopy for long-term observation of cell cultures using chamber slides]. / Entwicklung eines Inkubationssystems für ein inverses Mikroskop zur Langzeitbeobachtung von Zellkulturen in gekammerten Objektträgern.

Trifunctional bispecific antibodies open up new immunological possibilities in tumour treatment. Prior to clinical application, comprehensive investigations using animal models and in vitro examinations need to be done. To investigate long-term interactions between various immunologically active blood cells and individual tumour cells in the presence of antibodies, we developed an incubation system for experimental cell cultures on an inverted microscope. The system consists of a perspex box with a central moisture chamber with integrated water reservoir, external air circulation heating, and a CO2 supply. The sterile cell cultures are located in the wells of a slide positioned within a depression in the water reservoir. The newly developed incubation system enables continuous observation over the long term of experiments under optimal cell cultures conditions in combination with modern video techniques.

Lysis of prostate carcinoma cells by trifunctional bispecific antibodies (alpha EpCAM x alpha CD3).

Bispecific monoclonal antibodies (bsAbs) are a promising immunotherapeutic option for treatment of cancer, especially in situations of minimal residual disease. The combination of an anti-CD3 and anti-tumor-associated antigen antibody redirects cytotoxic T-lymphocytes towards malignant cells. Using a trifunctional bispecific antibody against EpCAM x CD3, that additionally activates Fc gamma R(+) accessory cells via its Fc region, we investigated the interaction between three EpCAM(+) prostate carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) of healthy donors and patients with prostate carcinoma (PC). Visualization was performed by double immunocytochemical methods and computerized sequential video microscopy. Tumor cells and PBMCs supplemented with alpha EpCAM x alpha CD3 in 16-well chamber slides resulted in lysis of tumor cells within 1--3 days without any differences between patient and healthy donor PBMCs. The characteristic necrotic way of tumor cell killing (rounding, swelling, disrupting) could be observed in computerized sequences of video frames. Simultaneously, we could not reveal any form of apoptotic signal using three different apoptotic markers (TUNEL, M30 cyto death, anti-active caspase 3). Within the first 48 hr we observed typical PBMC cluster formation with increasing cell proliferation. PBMCs surrounding the tumor cells were not dominated by CD4(+), CD8(+), or CD14(+) cells. Lymphocytes with pore-forming perforin proteins concentrated towards the tumor target cells. Our combination of double immunocytochemical and computerized video microscopic techniques may serve as an important improvement of validity of cell-cell interaction experiments using in vitro models. (J Histochem Cytochem 49:911-917, 2001)

Immunomagnetic cell enrichment detects more disseminated cancer cells than immunocytochemistry in vitro.

PURPOSE: We describe a method to improve tumor cell detection compared to currently available immunocytochemical methods by using immunomagnetic cell enrichment. MATERIALS AND METHODS: Two different methods of immunomagnetic cell enrichment using antibody coated magnetic beads were tested and compared with unenriched immunocytochemistry. One method was positive selection of epithelial cells from mononuclear cells with the antiepithelial antibody BER-EP4 and the other was depletion of mononuclear cells with the antileukocyte antibody CD45. Mononuclear cells were isolated from peripheral blood by density centrifugation and various numbers of tumor cells were added. The 5 different cell lines from urological malignancies used in the study were DU-145, RT-4, CAKI-2, KTCTL-2 and KTCTL-30. Following incubation of cell suspensions with the beads, cell separation was performed in a magnetic field. After centrifugation on glass slides immunocytochemical staining for cytokeratin was performed. A total of 112 experiments were completed and negative controls were obtained. RESULTS: The number of tumor cells detected by positive selection and depletion was significantly higher than by immunocytochemistry (p <0.001). The median enrichment factor and tumor cell recovery rate for positive selection and depletion were 15.3 and 61.2%, and 13.0 and 57.3%, respectively (not significant). With less than 1 tumor cell suspended in 106 mononuclear cells, the probability of tumor cell detection was 23% for immunocytochemistry alone and 93.3% for both enrichment methods (p <0.01). No false-positive results were observed. CONCLUSIONS: Compared to immunocytochemistry, immunomagnetic cell enrichment significantly improves the sensitivity of detection of epithelial cells added to mononuclear cells. Both methods of enrichment were equally effective and may be important for clinical practice in the future.

Placebo-controlled, multicenter study of sertraline treatment for obsessive-compulsive disorder.

The safety and efficacy of sertraline versus placebo were examined in a group of nondepressed outpatients with obsessive-compulsive disorder (OCD). Patients with moderate-to-severe OCD were recruited at 10 sites. After a 1-week placebo lead-in, patients were treated in a double-blind fashion for 12 weeks with sertraline or placebo. Sertraline was administered at a starting dose of 50 mg/day, with flexible titration up to 200 mg/day. The efficacy measures were the Yale-Brown Obsessive Compulsive Scale (Y-BOCS), the National Institute of Mental Health Global Obsessive Compulsive Scale (NIMH), and the Clinical Global Impression Scale (CGI) Severity of Illness and Improvement subscales. One hundred sixty-seven patients were randomly assigned and received at least one dose of double-blind medication: 86 received sertraline and 81 received placebo. All efficacy measures showed significantly greater improvement in the sertraline group from the end of week 8 until the end of week 12. Significantly greater improvement (p < 0.05) in the sertraline group first became apparent by the end of week 3 on the Y-BOCS and the CGI Improvement scale, and by the end of weeks 6 and 8, respectively, on the NIMH and CGI Severity scale. Sertraline was well tolerated, without serious adverse effects. In conclusion, sertraline was safe and effective in the treatment of patients with OCD.

Sertraline in children and adolescents with obsessive-compulsive disorder: a multicenter randomized controlled trial.

CONTEXT: The serotonin reuptake inhibitors are the treatment of choice for patients with obsessive-compulsive disorder; however, empirical support for this assertion has been weaker for children and adolescents than for adults. OBJECTIVE: To evaluate the safety and efficacy of the selective serotonin reuptake inhibitor sertraline hydrochloride in children and adolescents with obsessive-compulsive disorder. DESIGN: Randomized, double-blind, placebo-controlled trial. PATIENTS: One hundred eighty-seven patients: 107 children aged 6 to 12 years and 80 adolescents aged 13 to 17 years randomized to receive either sertraline (53 children, 39 adolescents) or placebo (54 children, 41 adolescents). SETTING: Twelve US academic and community clinics with experience conducting randomized controlled trials. INTERVENTION: Sertraline hydrochloride was titrated to a maximum of 200 mg/d during the first 4 weeks of double-blind therapy, after which patients continued to receive this dosage of medication for 8 more weeks. Control patients received placebo. MAIN OUTCOME MEASURES: The Children's Yale-Brown Obsessive Compulsive Scale (CY-BOCS), the National Institute of Mental Health Global Obsessive Compulsive Scale (NIMH GOCS), and the NIMH Clinical Global Impressions of Severity of Illness (CGI-S) and Improvement (CGI-I) rating scales. RESULTS: In intent-to-treat analyses, patients treated with sertraline showed significantly greater improvement than did placebo-treated patients on the CY-BOCS (adjusted mean, -6.8vs -3.4, respectively; P=.005), the NIMH GOCS (-2.2 vs -1.3, respectively; P=.02), and the CGI-I (2.7 vs 3.3, respectively; P=.002) scales. Significant differences in efficacy between sertraline and placebo emerged at week 3 and persisted for the duration of the study. Based on CGI-I ratings at end point, 42% of patients receiving sertraline and 26% of patients receiving placebo were very much or much improved. Neither age nor sex predicted response to treatment. The incidence of insomnia, nausea, agitation, and tremor were significantly greater in patients receiving sertraline; 12 (13%) of 92 sertraline-treated patients and 3 (3.2%) of 95 placebo-treated patients discontinued prematurely because of adverse medical events (P=.02). No clinically meaningful abnormalities were apparent on vital sign determinations, laboratory findings, or electrocardiographic measurements. CONCLUSION: Sertraline appears to be a safe and effective short-term treatment for children and adolescents with obsessive-compulsive disorder.

Expression of plakoglobin in renal cell carcinoma.

Little is known about the role of molecules involved in cell-cell interactions during the progression of renal cell carcinoma (RCC). We investigated the expression of plakoglobin (a component of the cadherin-catenin adhesion system) in 94 samples of normal kidney tissue from patients with RCC, in 109 primary renal cell carcinomas and in 16 metastases by immunohistochemistry. Expression of plakoglobin was significantly diminished in tumor tissue, particularly in metastatic lesions, as compared to normal kidney tissue (p < 0.001). Follow-up data were available from 87 patients. Patients with a diffuse plakoglobin expression (91-100% positive cells) in primary tumor tissue had a significant better survival rate than patients with a disturbed plakoglobin expression (p < 0.05) as determined by the log rank test. These results indicate that loss of plakoglobin may play an important role in malignant transformation of renal cells. Plakoglobin expression status could give additional information about the individual prognosis.

H-dependent formation of 5-aminolaevulinic acid-induced protoporphyrin IX in fibrosarcoma cells.

pH-Dependent variations in the fluorescence intensity of 5-aminolaevulinic acid-induced 5-aminolaevulinic acid protoporphyrin IX (PP IX) were compared with the cell viability following light irradiation. The fluorescence intensity was determined by flow cytometry and the cell activity was investigated by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 24 h after photodynamic treatment. The results obtained by fluorescence measurements clearly showed that pH values of the incubation medium containing (5-ALA) below and above pH 7.4 led to a significant decrease in the fluorescence intensity. The viability of cells incubated with 0.6 mM 5-ALA in a medium at pH 6.0 was unaffected on exposure to light at lambda = 635 nm up to 15 J cm-2. However, cells incubated at pH 7.4 (with the other treatment parameters the same) were nearly completely destroyed. In addition, depletion of intracellular PPIX was faster in physiological medium than in acid medium.

Urokinase plasminogen activator receptor (uPA-R): one potential characteristic of metastatic phenotypes in minimal residual tumor disease.

Evidence of dynamic development of cytokeratin (CK) 18-positive disseminated tumor cells in bone marrow of curatively resected cancer patients has implicated a subclinical minimal residual disease as a biologically relevant component in solid cancer. However, differentiation between irrelevant shed cells and those cells potentially capable of causing later recurrence has not yet been made. In parallel, accumulating data show functional association of the urokinase plasminogen activator (uPA) system and the membranous uPA receptor (uPA-R) with the capacity of a tumor cell for invasion and metastasis. The present study was designed to find descriptive evidence in vivo concerning whether uPA-R could be one potential characteristic for metastatically relevant phenotypes of disseminated tumor cells. An immunocytochemical double staining for uPA-R and CK18 (immunogold/alkaline phosphatase anti-alkaline phosphatase) was performed on perioperative and follow-up bone marrow aspirations of 78 curatively resected gastric cancer patients, if positive tumor cell status had been shown previously with the single alkaline phosphatase anti-alkaline phosphatase method. Bone marrow cells (10(6)) were examined in each assay. Postoperative qualitative and quantitative development of uPA-R-expressing disseminated tumor cells was followed in relation to uPA-R-negative cells and correlated with later clinical relapse. Double staining could be performed perioperatively or in follow-up, or both, in 58 of 78 patients. Expression of uPA-R on perioperatively disseminated tumor cells significantly correlated with later quantitative increases of tumor cells (P = 0.0009). Overall median tumor cell numbers with uPA-R expression significantly increased during follow-up from a median value of 5.5 to 10.0 in 10(6) cells (P = 0.008), and the mean relative percentage of uPA-R-positive, compared with uPA-R-negative, disseminated tumor cells also increased, from 47.9% at surgery to 68.6% in follow-up (P < 0.001). This was mainly due to patients with later tumor relapse (increase from 63.9 to 80.7%, P = 0.001). Patients without relapse showed slight increases at lower percentage levels (5.7% at surgery, 7.4% in follow-up). Differences for relapsing patients were significant (surgery, P = 0.006; follow-up, P < 0.001). Our results suggest from an in vivo model that uPA-R may be one antigen that enables identification and follow-up observations of metastatically relevant phenotypes of disseminated tumor cells, differentiating their individual potential for causing relapse.

Immunocytochemical phenotyping of disseminated tumor cells in bone marrow by uPA receptor and CK18: investigation of sensitivity and specificity of an immunogold/alkaline phosphatase double staining protocol.

Phenotyping of cytokeratin (CK)18-positive cells in bone marrow is gaining increasing importance for future prognostic screening of carcinoma patients. Urokinase-type plasminogen activator receptor (uPA-R) is one example of a potential aggressive marker for those cells. However, a valid and reliable double staining method is needed. Using monoclonal antibodies against uPA-R and CK18, we modified an immunogold/alkaline phosphatase double staining protocol. UPA-R/CK18-positive tumor cell controls exhibited black uPA-R staining in 15-80% of cases and red CK18 staining in almost 100% of tumor cells. Isotype- and cross-matched controls were completely negative. Bone marrow from healthy donors was always CK18-negative. Reproducibility of CK18-positive cell detection was estimated in a series of specimens from 61 gastric cancer patients comparatively stained with the single alkaline phosphatase-anti-alkaline phosphatase (APAAP) and our double staining method (10(6) bone marrow cells/patient). In four cases, double staining could not reproduce CK18-positive cells. In 34 cases it revealed fewer or equal numbers, and in 23 cases more CK18-positive cells than the APAAP method. Overall quantitative analysis of detected cell numbers (838 in APAAP, range 1-280 in 10(6); double staining 808, range 0-253) demonstrated relative reproducibility of APAAP results by double staining of 97%. Correlation of results between both methods was significant (p < 0.001, linear regression). Sensitivity of double staining tested in logarithmic tumor cell dilutions was one CK18-positive cell in 300,000. Specific uPA-R staining was seen on CK18-positive cells in bone marrow from 29 of 61 patients, and also on single surrounding bone marrow cells. To test the specificity of this staining, bone marrow cytospins from 10 patients without tumor disease were stained for uPA-R with the APAAP method. uPA-R expression was confirmed in all 10 cases, with a mean of 6.5% uPA-R-positive cells in 1000 bone marrow cells (SEM 1.2%). These results suggest that our double staining protocol is a sensitive, reproducible, and specific method for routine uPA-R phenotyping of disseminated CK18-positive cells in bone marrow of carcinoma patients.

Photodynamic effects of 5-aminolevulinic acid-induced porphyrin on human bladder carcinoma cells in vitro.

The efficiency of 5-aminolevulinic acid (ALA) in photodynamic therapy (PDT) was investigated in vitro using urothelial carcinoma cells of various differentiation. HCV29, RT4 and J82 cells were cultured in 96-well plates, incubated with 25-100 micrograms/ml ALA in serum-containing medium for 4 h, and irradiated at 630, 635 and 640 nm wavelength with light doses of 15-100 J/cm2. The degree of reduced tetrazolium bromide corresponding to cell viability was determined with a colorimetric MTT assay 0, 24 and 48 h after PDT. A remarkable reduction of mitochondrial activity occurred in poorly (J82) and well differentiated (RT4) malignant urothelial cells. Twenty-four hours after photodynamic treatment with 100 micrograms/ml ALA and 50 J/cm2, the metabolic activity of malignant cells was nearly extinguished, while HCV29 cells, derived from normal urothelium, behaved similarly to non-irradiated control cells. The photosensitivity of cells depended on presence or absence of fetal bovine serum (FBS) in the ALA-incubation medium. A wavelength of 635 nm was up to 60% more effective compared with 630 nm, which is more frequently applied in PDT. From the results of our in vitro studies, we can define a "therapeutic window" for malignant cells without damaging benign cells. The time delayed effects and the strong wavelength dependence are important factors for a clinical application.

[Computer-controlled laser irradiation unit for studies of light-induced processes in cell cultures]. / Computergesteuerte Laserbestrahlungseinheit für Untersuchungen lichtinduzierter Prozesse an Zellkulturen.

For the photodynamic treatment of tumours, synergistic effects of photosensitizing substances and light (today usually laser light) are used. With the aim of optimizing photosensitizing drugs and therapy, the effects of light and drug dose were studied in cell experiments. To automate and standardize such in vitro experiments, a laser irradiation chamber was developed. Cells cultured from tumour cell lines are placed on micro-titre plates or in petri dishes, together with the photosensitizer, and subsequently irradiated in the irradiation chamber with a well-defined dose of laser light of a wavelength corresponding to the absorbance of the photosensitizing agent. The plates or dishes are irradiated from below. In this way, light dose errors due to refraction from the meniscus of the cell suspension as occurs with irradiation from above, are avoided. During irradiation, speckle effects on the underside of the plates or petri dishes lead to variation in irradiation. A vibrator keeps the light transmission fibre and thus speckle pattern in motion, guaranteeing a homogeneous irradiation of the cells.

Synaptic reorganisation in the rat striatum after dopaminergic deafferentation: an ultrastructural study using glutamate decarboxylase immunocytochemistry.

The ultrastructure of GABAergic and non-GABAergic synapses in the adult rat neostriatum was examined 6-8 months after unilateral removal of the nigrostriatal dopaminergic pathway by 6-hydroxydopamine injection into the medial forebrain bundle. GABAergic profiles were identified by preembedding glutamate decarboxylase (GAD) immunocytochemistry performed on parasagittal vibratome sections. In three representative fields of the striatum, the nature and number of boutons and their postsynaptic partners were determined and the differences between the striata ipsi- and contralateral to the lesion analyzed. The percentage of GAD-immunoreactive boutons was increased from 23% on the intact side to 28% on the lesioned side. In addition, the GABAergic boutons underwent significantly more multiple contacts with several independent postsynaptic profiles, preferentially with dendritic spines. This could reflect a lesion-induced sprouting of local GABAergic axon terminals. On the other hand, although the vast majority of GABAergic boutons underwent synaptic contacts with dendrites (77% vs. 80%), the number of boutons per dendrite or per dendritic circumference remained unchanged. Thus, the higher frequency of GABAergic boutons may simply reflect the loss of the dopaminergic nerve endings, without a heterosynaptic replacement by GABAergic boutons. The deafferentation also induced structural changes of the postsynaptic profiles. Some dendritic spines had a shortened neck; others were completely integrated in the dendrite which now contained a spine apparatus and was contacted by boutons with the features of axospinous synapses. The spine retraction resulted in a quantitative decrease in the number of spines. Analysis of the synaptic curvature revealed that only spines with a flat contact zone were lost. Concurrently, the number of dendrites was increased, of the GAD-containing in particular, suggesting that the denrites of GABAergic interneurons tend to elongate and/or arborize. Taken together, the results of the present study show that the dopaminergic denervation caused a remodeling of the postsynaptic neurons. The relative increase of the number of GABAergic boutons and their synaptic contacts suggests that an altered wiring of the intrinsic GABAergic system contributes to the changes in the striatal output activity.

Immunocytochemical detection and phenotypic characterization of micrometastatic tumour cells in bone marrow of patients with prostate cancer.

Monoclonal antibodies (mAbs) specific for cytokeratins are potent probes for the identification of disseminated individual epithelial tumour cells in mesenchymal organs such as bone marrow. We have used a monoclonal antibody (mAB) against cytokeratin 18 (CK18) for the detection of individual metastatic tumour cells in bone marrow aspirates from 84 patients with carcinoma of the prostate. CK18+ cells were detected in a sensitivity of 1 per 8 x 10(5) marrow cells using the alkaline phosphatase anti-alkaline phosphatase (APAAP) system for staining. We were able to detect CK18+ tumour cells in the marrow of 33% of patients with stage N0M0 prostate cancers. The incidence of CK18+ cells showed a significant correlation with established risk factors, such as local tumour extent, distant metastases and tumour differentiation. For further characterization of such cells in patients with prostate cancer, we developed an immunocytochemical procedure for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate-specific antigen (PSA). In a first step, cells were incubated with a murine mAb against PSA, followed by gold-conjugated goat anti-mouse antibodies. In a second step, a biotinylated mAb to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. CK18+ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 14 patients with carcinomas of the prostate. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hyperplasia (BPH).(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy and safety of b.i.d. doses of venlafaxine in a dose-response study.

In this study, 312 depressed outpatients received either placebo or one of three venlafaxine doses twice daily (b.i.d.) for up to 6 weeks. The total daily doses of venlafaxine were 25, 50-75, and 150-200 mg/day. Hamilton Rating Scale for Depression (HAM-D) and Montgomery-Asberg Depression Rating Scale (MADRS) total scores at Week 6 were significantly lower for the high-dose group than for the placebo group. A positive dose-response trend for the primary efficacy parameters was demonstrated as early as Week 1. Venlafaxine was well tolerated at all dose levels. The most common side effects of clinical interest were nausea and dry mouth. The frequency of nausea in the venlafaxine groups was essentially the same (25-29%), whereas the frequencies of dry mouth, somnolence, and sweating were dose related. The results indicate that b.i.d. doses of venlafaxine are safe and effective in treating depression.

Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells.

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18+/PSA- HT29 colon carcinoma cells. CK18+ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy.(ABSTRACT TRUNCATED AT 250 WORDS)