Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue

Leers, Math P.G.; Schütte, B.; Theunissen, P. H. M. H.; Ramaekers, Frans C. S.; Nap, Marius

doi:10.1002/(sici)1097-0320(19990301)35:3<260::aid-cyto9>3.0.co;2-o

Cited by 53 publications

(37 citation statements)

References 23 publications

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“…27 In a recent study, we have described a new technique for the isolation and subsequent flow cytometric analysis of single cells from formalin fixed, paraffin wax embedded tissue. 11 Using this method, cell recovery was doubled compared with commonly used protocols, the quality of the DNA histograms was significantly improved, and the accessibility of several antigen epitopes, including those from cytokeratins, 11 steroid hormone receptors, 10 and membrane surface antigens, 28 was restored. In our present study we applied this protocol to routinely processed formalin fixed, paraffin wax embedded lymph nodes offered to our department as part of the SLN procedure.…”

Section: Discussionmentioning

confidence: 99%

“…Preparation of cell suspensions The preparation of the single cell suspension has been described previously. 11 Briefly, for every assay 20 sections (50 µm thick) were cut from each paraffin wax block. Subsequently, these tissue sections were dewaxed twice in xylene, for 30 minutes each, and then rehydrated in a descending ethanol series.…”

Section: Specimen Collectionmentioning

confidence: 99%

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Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

Leers¹,

Schoffelen²,

Hoop³

et al. 2002

Journal of Clinical Pathology

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Aim: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. Methods: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 µm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100 000 cells were analysed on the flow cytometer.Results: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple nonSLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. Conclusion: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.

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Section: Discussionmentioning

confidence: 99%

Section: Specimen Collectionmentioning

confidence: 99%

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

Leers¹,

Schoffelen²,

Hoop³

et al. 2002

Journal of Clinical Pathology

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“…Paraffin sections underwent deparaffinization and antigen recovery (27). Frozen sections were washed with phosphate-buffered saline.…”

Section: Mice Wild-type C57bl/6j (H-2kmentioning

confidence: 99%

Neuronal CXCL10 Directs CD8⁺T-Cell Recruitment and Control of West Nile Virus Encephalitis

Klein

Lin

Zhang

et al. 2005

J Virol

388

455

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The activation and entry of antigen-specific CD8؉ T cells into the central nervous system is an essential step towards clearance of West Nile virus (WNV) from infected neurons. The molecular signals responsible for the directed migration of virus-specific T cells and their cellular sources are presently unknown. Here we demonstrate that in response to WNV infection, neurons secrete the chemokine CXCL10, which recruits effector T cells via the chemokine receptor CXCR3. Neutralization or a genetic deficiency of CXCL10 leads to a decrease in CXCR3؉ CD8 ؉ T-cell trafficking, an increase in viral burden in the brain, and enhanced morbidity and mortality. These data support a new paradigm in chemokine neurobiology, as neurons are not generally considered to generate antiviral immune responses, and CXCL10 may represent a novel neuroprotective agent in response to WNV infection in the central nervous system.

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“…Paraffin sections underwent deparaffinization, Ag recovery, and permeabilization as previously described (48,49), and frozen sections were permeabilized, blocked, and stained as previously described (48). For detection of CXCL12 and anti-CD31 (PECAM) or anti-NeuN (neuronal marker), Image-iT FX signal enhancer (Molecular Probes) solution was used according to the manufacturer's instructions.…”

Section: Immunohistochemistrymentioning

confidence: 99%

CXCL12 Limits Inflammation by Localizing Mononuclear Infiltrates to the Perivascular Space during Experimental Autoimmune Encephalomyelitis

McCandless¹,

Wang

Woerner

et al. 2006

The Journal of Immunology

229

262

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The inflammatory response in the CNS begins with the movement of leukocytes across the blood-brain barrier in a multistep process that requires cells to pass through a perivascular space before entering the parenchyma. The molecular mechanisms that orchestrate this movement are not known. The chemokine CXCL12 is highly expressed throughout the CNS by microendothelial cells under normal conditions, suggesting it might play a role maintaining the blood-brain barrier. We tested this hypothesis in the setting of experimental autoimmune encephalomyelitis (EAE) by using AMD3100, a specific antagonist of the CXCL12 receptor CXCR4. We demonstrate that the loss of CXCR4 activation enhances the migration of infiltrating leukocytes into the CNS parenchyma. CXCL12 is expressed at the basolateral surface of CNS endothelial cells in normal spinal cord and at the onset of EAE. This polarity is lost in vessels associated with an extensive parenchymal invasion of mononuclear cells during the peak of disease. Inhibition of CXCR4 activation during the induction of EAE leads to loss of the typical intense perivascular cuffs, which are replaced with widespread white matter infiltration of mononuclear cells, worsening the clinical severity of the disease and increasing inflammation. Taken together, these data suggest a novel anti-inflammatory role for CXCL12 during EAE in that it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thereby limiting the parenchymal infiltration of autoreactive effector cells.

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Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue

Cited by 53 publications

References 23 publications

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

Neuronal CXCL10 Directs CD8⁺T-Cell Recruitment and Control of West Nile Virus Encephalitis

CXCL12 Limits Inflammation by Localizing Mononuclear Infiltrates to the Perivascular Space during Experimental Autoimmune Encephalomyelitis

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Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue

Cited by 53 publications

References 23 publications

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

Neuronal CXCL10 Directs CD8+T-Cell Recruitment and Control of West Nile Virus Encephalitis

CXCL12 Limits Inflammation by Localizing Mononuclear Infiltrates to the Perivascular Space during Experimental Autoimmune Encephalomyelitis

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Neuronal CXCL10 Directs CD8⁺T-Cell Recruitment and Control of West Nile Virus Encephalitis