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The Immunomagnetic Manipulation of Bone Marrow
Published in Adrian P. Gee, BONE MARROW PROCESSING and PURGING, 2020
Immunomagnetic separation of either tumor cells, or T cells in bone marrow should be carried out on noncryopreserved cell suspensions. Once frozen and thawed, the marrow is far too “sticky” to permit use of this approach. This is presumably due to the release of DNA from cells that are lysed as a result of the freezing procedure. However, bone marrow has been stored at 4°C for up to 18 h before immunomagnetic treatment, and this has not proven to be deleterious. It must be remembered that bone marrow is a biological entity, which can vary markedly from individual to individual, particularly if the donors have been exposed to extensive chemotherapy. Manipulation of over 200 pediatric bone marrows has shown that the innate “stickiness” of the marrow varies considerably, but only in one case has it proved difficult to separate “immunobeads” from a marrow, due to excessive nonspecific binding. Some groups routinely add DNase to the marrow samples prior to immunomagnetic manipulation, but a review of the literature indicates that this probably is unnecessary. Our experience suggests that nonspecific binding of the widely used Dynal M 450™ microspheres (Section IV) can be kept to a minimum (1 to 5%) simply by keeping the marrow samples at 4°C during processing.
Circulating Tumor Cells in Individualizing Breast Cancer Therapy
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
James M. Reuben, Massimo Cristofanilli
Over the past few years, immunomagnetic separation technology, with its higher level of sensitivity and specificity, has been used to improve the detection of CTCs compared with the detection of occult CTCs by reverse transcriptasepolymerase chain reaction (RT-PCR) (13-21). In this chapter, we review the methods of detecting CTCs, the prognostic implications of CTCs, and the potential means of exploiting CTCs in developing individualized therapy.
Marrow Purging And Stem Cell Preparation
Published in Siegfried Matzku, Rolf A. Stahel, Antibodies in Diagnosis and Therapy, 2019
Denis C. Roy, Nadine Beauger, Martin Gyger
Immunomagnetic separation relies on the binding of CD34+ cells first to anti-CD34 mAb and then to ferric oxide-treated polystyrene beads coated with sheep anti-mouse IgG (Hardwick et al., 1992; Krause et al., 1996; Auditore-Hargreaves et al., 1994). Magnets applied to the side of the column pull out bead-rosetted CD34+ cells and free CD34— cells are washed out by gravity flow. Chymopapain was first used to cleave a peripheral domain of the CD34 molecule, and separate CD34+ cells from beads and mAbs. As chymopapain is potentially toxic, Pasteurella haemolytica glycoprotease, a non-cytotoxic enzyme, has been used to cleave CD34 epitopes (Marsh et al., 1992). This strategy permitted recovery of up to 78% of CD34+ cells, with a purity of up to 95%. More recently, enzymes were replaced by a releasing agent that competes for antibody binding (PR-34) and eliminates mAb and beads from the surface of CD34+ cells (Hansen et al., 1995). The physiologic role of CD34 epitopes and the impact of such deletions being unknown, this last procedure has the significant advantage of releasing CD34+ cells without altering the CD34 molecule.
The current status of the clinical utility of liquid biopsies in cancer
Published in Expert Review of Molecular Diagnostics, 2019
Anson Snow, Denaly Chen, Julie E. Lang
Due to the rarity of CTCs circulating within the body [96], multiple methods have been created to isolate CTC populations. The two broad categories of isolation techniques are based up physical properties (i.e. size, density, deformability, and electrical charge) [43,97–100] and affinity-based selection [5,96,101–103]. Currently, immunomagnetic separation remains the most widely adopted technique. This process employs antibody-coated magnetic cells to positively enrich for CTCs through epithelial cell adhesion molecules (EpCAM) or negatively enrich for CTCs through CD45. Our group has previously reviewed the various assays in the liquid biopsy field [104]. Despite a multitude of enrichment methods, the anti-EpCAM antibody-based CellSearch assay remains the only Food and Drug Administration (FDA) approved platform. However, one of the issues with EpCAM based isolation methods is it potentially underestimates CTC numbers. Notably, Hyun et al. found that breast cancer CTCs, which undergo epithelial-mesenchymal transition, have low expression patterns of EpCAM [105]. Thus, approaches that rely on EpCAM isolation techniques miss low-EpCAM CTC populations, which is significant given that EMT is thought to play a role in tumor progression in metastasis [106,107].
Bacterial detection using bacteriophages and gold nanorods by following time-dependent changes in Raman spectral signals
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Farzaneh Moghtader, Aysel Tomak, Hadi M. Zareie, Erhan Piskin
Guven et al. [41] have combined immunomagnetic separation (IMS) and SERS to detect E. coli in which gold-coated magnetic spherical nanoparticles carrying anti-E. coli antibodies were prepared and used for IMS. GNRs carrying Raman labels have (5,5-dithiobis-2-nitrobenzoic acid – DTNB) were used for detection of the target bacteria with SERS. The fingerprints of DTNB-labels on the GNRs were followed for detection – not the specific peaks of E. coli. Therefore, they did not attempt to assign SERS peaks of the target bacteria. The correlation between the concentration of bacteria and SERS signal was found to be linear within the range of 101–104 cfu/mL which was quite good alternative approach for specific detection of the target bacteria. Tamer et al. [42] have studied gold coated magnetite nanoparticles further modified with self-assembling molecules against glycoside moieties on the surface of the target bacteria (i.e. E. coli). They were able to demonstrate specific targeting and enhancement in the Raman signals in several specific peaks, but not assigned properly.
Epidemiology of Shiga toxin-producing Escherichia coli O157:H7 in Africa in review
Published in Southern African Journal of Infectious Diseases, 2018
In the present review, sorbitol MacConkey agar was used in isolation of STEC O157:H7 in 21 out of 24 reports from Africa. An immunomagnetic separation technique was employed in seven reports, in which it was used together with sorbitol MacConkey agar. After isolation, the characterisation of E. coli O157:H7 was done by polymerase chain reaction (PCR) to detect the shiga toxin-producing genes (14 reports), O157 antisera for detection of somatic antigen O157 (18 reports) and dot plot DNA hybridisation was used to confirm PCR results (2 reports). Serotyping of O157:H7 antigens was performed in four studies, while Vero-cell cytotoxicity assays were performed to test for cytopathic effects on Vero-cell monolayers in six studies that are included in this review (Table 1). The use of molecular methods (PCR) to detect shiga toxin-producing genes in only 14 out of 24 (58%) studies in this review could have resulted in missed detection and under-reporting of STEC in Africa. All these STEC O157:H7 detection methods required more than 24 h to complete. Moreover, not many laboratories in Africa can afford these diagnostic procedures. There is a need to improve diagnostic facilities in Africa – even by starting with a few reference laboratories in each African country – which will enable quick and accurate detection of STEC O157:H7 infection. This will help to avoid inappropriate management of cases, such as use of antimicrobials which are easily accessed in Africa and often without prescription, for any enteric illness including STEC O157:H7 infection.