The small leukocyte-to-erythrocyte ratio in whole blood requires isolation of leukocytes before their cellular volume can best be determined with a Coulter Counter®.1 A major distinguishing feature of the predominant erythrocytes is their intracellular hemoglobin. The ability of hemoglobin to form oxyhemoglobin by binding oxygen in the lungs and releasing it in tissues where it is needed by forming deoxyhemoglobin enables erythrocytes to support the respiration central to human physiology; the portion of total hemoglobin in the oxy form at any tissue site determines the oxygen saturation there.2 Total hemoglobin normally contains 1-2% of methemoglobin, which can be tolerated at perhaps three times such concentrations before the adverse effects of methemoglobinemia manifest themselves.
Deoxyhemoglobin and methemoglobin are both paramagnetic whereas oxyhemoglobin is diamagnetic, which suggested that if erythrocytes in a test sample contained no oxyhemoglobin, they might be separated magnetically in an appropriate filtration apparatus and so provide a leukocyte population suitable for analysis with a Coulter Counter®. This hypothesis was explored via small filters containing a matrix of martensitic 430 stainless steel wire, typically 50 micra in diameter and of a volume about 12-15% that of the filter chamber, that was randomly wadded to fill the chamber. The filter was clamped between the pole pieces of a Varian V-4005 laboratory electromagnet, and after the filter was flushed with deoxygenating diluent and the electromagnet was provided power, the deoxygenated blood sample was flowed into the filter at a low flow rate; the filtrate was then extracted by depowering the electromagnet and flushing the filter with the diluent flowing at a higher rate to remove the erythrocytes from the filter matrix. At applied field strengths between 1.0 and 2.0 Tesla, useful erythrocyte separations were obtained for typical blood samples,3 but when samples from patients with sickle cell disease were identically deoxygenated, the filter matrices quickly clogged, apparently due to unfavorable magnetic orientation of the elongated sickle erythrocytes.4
The large electromagnet was unsuited to inclusion in instruments, and efforts were made to replace it with permanent magnets. Although a few magnets seemed promising, none yielded the filter field strengths obtained with the electromagnet; furthermore, because the matrix field strength could not be readily minimized, the higher diluent flow rates needed to remove the erythrocytes from the filter matrix often induced unacceptable damage to the out-flowing cells. Two techniques for reducing the required flushing flow rate were explored, diversion of the magnetic flux from through the matrix during filtration to around it during the flushing operation5 and acoustically vibrating the matrix during flushing to help remove erythrocytes from it.6
The high magnetic field strengths required to separate deoxygenated erythrocytes from a blood sample prompted a search for an alternative approach. Trivalent erbium ions have a magnetic moment 1.78 times that for deoxyhemoglobin and 1.64 times that for methemoglobin, while the magnetic moment for trivalent dysprosium ions is 1.96 times that for deoxyhemoglobin and 1.81 times that for methemoglobin. Both lanthanide ions demonstrate high-affinity binding to the membranes of many cell types including not only erythrocytes, but leukocytes and platelets, all three of which should then be magnetically separable from a blood sample according to their occurrence rate in whole blood and how well their physical size matched the matrix design. Due to their predominant occurrence in whole blood, erythrocytes should dominate leukocytes in the filtrate by a factor of least 700, and platelets’ small size would prevent them from being retained at anything approaching their low occurrence rate. Thus, a magnetic filtrate from whole blood appropriately diluted with an erbium or a dysprosium solution should approximate a magnetic erythrocyte separation within usual experimental uncertainty, this with lower field strengths required in the separation matrix, but would likely contain some leukocytes and platelets. So, unless the diluent also included a reagent that inhibited leukocytes from binding the lanthanide, the filtrate would induce artifacts into leukocyte analysis by a Coulter Counter®.
With the separation apparatus described above, filtrates containing erythrocytes in comparable quantities were obtained from lanthanide diluents with magnetic field strengths less than half that required with the aforesaid methods. This suggested that despite their lower separation efficiencies matrices of austenitic stainless steel wires might be substituted for the martensitic matrices used in the separation apparatus described above, and in some experiments unannealed austenitic 316L stainless steel wire, 35 micra in diameter and of a volume about 10-12% of the filter chamber, was randomly wadded to fill the chamber.7 However, it was found that the austenitic matrices gave decreasing filtration efficiencies as the number of operational cycles increased,8 and additional experimentation was mostly done with matrices made of martensitic 430 stainless steel as described above.9 Acceptable filtration efficiencies were demonstrated at field strengths of 0.5 Tesla from small permanent magnets in a separation apparatus occupying slightly more than a cubic inch.10
Although several prior-art chemical methods had been proposed to achieve leukocyte isolation, most induced unacceptable artifacts in the isolated cells. However, lysing diluents had by now become available that selectively removed erythrocytes from whole blood with minimal cellular artifacts, and the decision was made to patent the lanthanide-binding approach11 and avoid the investment required to develop diluents that would inhibit lanthanide binding by leukocytes.
Recently a method was proposed for magnetophoretic separation of deoxygenated whole blood flowing through a microchannel into plasma portions containing paramagnetic erythrocytes, diamagnetic leukocytes, and few cells.12 If implemented in an appropriate apparatus, this approach might offer a versatile microsystem that resolves the difficulty prompting the research summarized herein.
About The Author
Marshall Don. Graham was Technical Advisor to Wallace H. Coulter, June 1978 to November 1997, Coulter Corporation; Principal Staff Development Scientist, emeritus, December 2011, Beckman Coulter, Inc.
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