A microfluidic biosensor to electrically enumerate blood cells at point-of-care for infectious disease diagnosis and management

Cell counting finds many applications in diagnostics of many diseases. One of the most common tests recommended by the physicians is complete blood cell count (CBC). In chemotherapy and radiation therapy, the blood cell production needs to be monitored. Inflammation, leukemia, tissue injury, bone marrow failure and immunodeficiency can be identified by the irregular WBC counts and their differentials. Thus, a microfluidic, disposable, economical CBC would help in monitoring all these diseases with more efficiency and care. Cell counting in general, and ability to count the specific cells, would greatly help in clinical diagnostics. Currently, flow cytometers and hematology analyzers are used for this purpose, but they have not been able to penetrate in the resource-limited settings around the world because of being expensive and because they require trained technicians to operate. Over many years, people have developed microfluidic devices for cell counting, which could provide a portable and economical solution to the problem of cell counting at point-of-care. In this dissertation, we present a technique for counting the white blood cells and differentiating some of its sub-types within a microfluidic device. Starting with the whole blood, the red blood cells are lysed by saponin and formic acid. Quenching solution composed of phosphate buffer saline is infused in the device to halt the lysing process and maintain the pH of the solution. The remaining white blood cells are then passed across microfabricated electrodes within a microfluidic channel. The impedance is measured at 303 kHz and 1.7 MHz signals. Cells are captured in a capture chamber where the specific antibody is immobilized. The remaining cells are counted again and the difference in cell counts give the number of captured cells. We have counted CD4 and CD8 T cells from our biochip using healthy and HIV infected blood samples. The cell counts from the biochip are compared with those from the flow cytometer, showing the high correlation between the two techniques. We have also characterized the coincidence detection and show that cell counting with coincidence detection improves the accuracy of the counting results. We have translated this technology to perform a complete blood cell (CBC) count from a drop of blood. The total leukocyte count and its 2-part differential (lymphocytes, monocytes + granulocytes) were obtained with the single counter. The blood sample is initially lysed, and then quenched, to get the total leukocyte count. At the entrance counter, lymphocytes can be electrically differentiated from monocytes+ neutrophils. We have used blood samples from healthy donors, patients admitted to the intensive care unit (ICU) and patients undergoing chemotherapy to get the complete dynamic range of cells. Erythrocytes and platelets have a very high concentration in the blood sample and require sample dilution for accurate cell counting. Platelet counting was more challenging using the same aperture counting channel because of the 1-2 μm diameter of platelets. We optimized the electronic settings to suit the platelet counting. We have performed on-chip blood dilution with PBS and performed erythrocyte and platelet counting. We have proposed that the same setup can be used in the future for reticulocyte counting that measures the health of bone marrow, especially for individuals undergoing chemotherapy. For the next step in translating our differential immuno-capture technology, we have explored the sepsis diagnosis application. Increased expression of the CD64 antigen on the neutrophils is a biomarker for early diagnosis of sepsis. However, to use our technology to count CD64 neutrophils, as a first step, using blood samples collected from patients admitted to ICU we have shown a high correlation of the CD64 antigen preservation on the neutrophils and monocytes after they get treated with saponin and formic acid lysing buffer. We have also done capture optimization studies and have shown that the percent capture is correlated to the expression level of the CD64 antigen on the cells. In the future, a complete blood cell count will be integrated with the sepsis CD64 capture differential counter in a single chip