Due to the low frequency of circulating tumor cells (CTC), the standard CellSearch method of enumeration and isolation using a single tube of blood is insufficient to measure treatment effects consistently, or to steer personalized therapy. Using diagnostic leukapheresis this sample size can be increased; however, this also calls for a suitable new method to process larger sample inputs. In order to achieve this, we have optimized the immunomagnetic enrichment process using a flow-through magnetophoretic system. An overview of the major forces involved in magnetophoretic separation is provided and the model used for optimizing the magnetic configuration in flow through immunomagnetic enrichment is presented. The optimal Halbach array element size was calculated and both optimal and non-optimal arrays were built and tested using anti-EpCAM ferrofluid in combination with cell lines of varying EpCAM antigen expression. Experimentally measured distributions of the magnetic moment of the cell lines used for comparison were combined with predicted recoveries and fit to the experimental data. Resulting predictions agree with measured data within measurement uncertainty. The presented method can be used not only to optimize magnetophoretic separation using a variety of flow configurations but could also be adapted to optimize other (static) magnetic separation techniques.
The identification of synovial crystals is important for diagnosing rheumatic diseases. Currently, rheumatologists worldwide use compensated polarized light microscopy (CPLM) for crystal identification, but this technique is flawed. Raman spectroscopy might offer an objective, accurate alternative. We have tested Raman hyperspectral imaging on synovial fluid samples of 28 patients with swollen joints, measuring 5–10 crystals in each of these. Reference spectra for identification were measured using patient material, synthetic compounds, and oxalate kidney stones. Additionally, RRUFF and PANGAEA Raman databases were used to identify spectra. We identified the classical pathological crystals monosodium urate for gout and calcium pyrophosphate for calcium pyrophosphate deposition disease (CPPD). Hydroxyapatite, lipid spherules, and calcium oxalate monohydrate have also been observed and had been previously identified in synovial fluids. Other crystals, which were not previously observed in synovial fluid using CPLM, have been identified as well: calcite, aragonite, anatase, rutile, thenardite, dolomite, and a carotenoid. In addition, we found combinations of crystals in 13 out of 28 patients. We propose that the observed large variation of detectable crystals from a small population of patients and a small number of crystals per patient has significant ramifications for the use of compensated polarized light microscopy for the diagnosis of gout and CPPD. Further studies are required to learn the clinical significance of these crystals in human arthritis.
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