High-throughput miniaturized microfluidic microscopy with radially parallelized channel geometry.

In this article, we present a novel approach to throughput enhancement in miniaturized microfluidic microscopy systems. Using the presented approach, we demonstrate an inexpensive yet high-throughput analytical instrument. Using the high-throughput analytical instrument, we have been able to achieve about 125,880 cells per minute (more than one hundred and twenty five thousand cells per minute), even while employing cost-effective low frame rate cameras (120 fps). The throughput achieved here is a notable progression in the field of diagnostics as it enables rapid quantitative testing and analysis. We demonstrate the applicability of the instrument to point-of-care diagnostics, by performing blood cell counting. We report a comparative analysis between the counts (in cells per µl) obtained from our instrument, with that of a commercially available hematology analyzer.

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

Optofluidic device for label-free cell classification from whole blood.

A unique optofluidic lab-on-a-chip device that can detect optically encoded forward scattering signals is demonstrated. With a unique design of a spatial mask that patterns the intensity distribution of the illuminating light, the position and velocity of each travelling cell in the flow can be measured with submicrometer resolution, which enables the generation of a cell distribution plot over the cross section of the channel. The distribution of cells is highly sensitive to its size and stiffness, both being important biomarkers for cell classification without cell labelling. The optical-coding technique offers an easy route to classify cells based on their size and stiffness. Because the stiffness and size of neutrophils are distinct from other types of white blood cells, the number of neutrophils can be detected from other white blood cells and red blood cells. Above all, the enumeration of neutrophil concentration can be obtained from only 5 µL of human blood with a simple blood preparation process saving the usual steps of anticoagulation, centrifugation, antibody labelling, or filtering. The optofluidic system is compact, inexpensive, and simple to fabricate and operate. The system uses a commodity laser diode and a Si PIN photoreceiver and digital signal processing to extract vital information about cells and suppress the noise from the encoded optical scattering signals. The optofluidic device holds promise to be a point-of-care and home care device to measure neutrophil concentration, which is the key indicator of the immune functions for cancer patients undergoing chemotherapy.

External quality assessment of the full blood count, and problems associated with the use of fixed blood preparations.

The experience of using a stored fixed blood preparation for external quality assessment of the full blood count is described. Differences between instruments employing differing technologies are highlighted and the problems of the control of the white cell differential count discussed.

Evaluation of the Sysmex R-1000. An automated reticulocyte analyzer.

The new fully automated reticulocyte analyzer, Sysmex R-1000 (TOA Medical Electronics, Kobe, Japan), was evaluated for its routine use in the Hematological Laboratory at the University Hospital Basel, Switzerland. The operating characteristics, such as within-run precision, linearity, and carryover, fulfilled the manufacturer's specifications and are excellent. Correlation with the standard method, manual reticulocyte counting, is linear for normal and high values. For low reticulocyte counts the regression points show a deviation from their linearity. An absolute zero value is not obtained by the R-1000. The R-1000 measures total RNA content of each cell and expresses the value as low fluorescence ratio (LFR), medium fluorescence ratio (MFR), and high fluorescence ratio (HFR). The analysis of this ratio resolves the problem of zero reticulocytes: A fraction of less than 0.002 (0.2%) with an LFR of 100% represents aplasia; a shift of the intensity of fluorescence to HFR heralds regeneration. Results of samples stored at room temperature remain stable and within the range of the within-run precision for up to 12 hours, when stored at 5 degrees C for more than 48 hours. The authors conclude that the R-1000 is easy to operate, fulfills the criteria for accuracy and precision, and is highly suitable for daily routine use in a large central hematologic laboratory.

An assessment of the Coulter Counter Models S Plus II and III.

The Coulter Counter Models S Plus II and III have been evaluated. No serious safety hazards were identified. Scientific assessment showed some non-linearity in the Hb which caused the MCH and MCHC to vary as samples were diluted. Precision and carry-over were satisfactory. The results obtained compared well with those of the Coulter Counter Model S, except for WBC; reference methods showed better accuracy on the Models S Plus II and III. Platelet counts agreed with those by phase-contrast microscopy and the lymphocyte percentage was similar to that from the blood film except in the lymphoproliferative disorders. The whole blood and pre-dilute modes gave similar results though the platelet count was slightly higher in the whole blood mode. In the National External Quality Assessment Scheme results were in accordance with those from other Model S Plus Users. Time did not allow a detailed evaluation of the cell volume distribution curves but it was noted that the white cell profile was useful for detecting platelet aggregation. Efficiency assessment showed throughputs of 66 and 93 samples per hour on the Models S Plus II and III respectively. The platelet count was clinically useful as was the lymphocyte percentage measurement which rendered some differentials unnecessary. Rejection of the white cell profile was a helpful index of abnormality on the Model S Plus II but occurred non-specifically on the Model III tested.

An assessment of the Ortho ELT-8.

The Ortho ELT-8 is an automated blood counter which appears to be safe, precise and free from carry-over. Red and white cell results generally agree with those on the Coulter Counter, Model S, though discrepancies were noted with the WBC and PCV. Reference methods showed the Model S WBC results tended to be inaccurate on the discrepant samples though neither instrument was predominantly responsible for the PCV discrepancies. The ELT-8 platelet count tended to be higher than with the Thrombocounter/Thrombofuge system. When packed cells were diluted in autologous plasma serious variations in the red cell indices (MCV, MCH & MCHC) were first found due to incorrect voltage-frequency converter setting. Even after this setting had been corrected some variations in the MCH and MCHC were still apparent. In the UK National External Quality Assessment Scheme the ELT-8 results on animal bloods did not agree with those produced by Model S users; this discrepancy probably being due to differences between the light-scattering and aperture-impedance technology.

An evaluation of the Celloscope 401 electronic blood cell counter.

For counting erythrocytes the instrument was precise, with a mean coefficient of variation of 1.21%. Erythrocyte counts showed close agreement with results obtained on a Coulter A electronic counter of proven accuracy. When the Celloscope 401 was modified by the manufacturers to eliminate electrical interference from other laboratory equipment, satisfactory precision and accuracy for white cell counting was obtained. Using cetrimide diluent the coefficient of variation was 1.6% but when using saponin/saline diluent the coefficient of variation was 3.5%. For leucocyte counting there was close agreement between duplicate tests performed on the Celloscope 401 and the Coulter S. The instrument was capable of satisfactory precision and accuracy in platelet counting, provided that the sedimentation method was used to obtain a platelet-rich plasma. The best results were obtained if a two-step dilution was carried out with a first dilution in 10% EDTA and a second in 2.5 mM cocaine in water. Using this method the precision study indicated a coefficient of variation of 3.11%. Close agreement was obtained between platelet counts on the Celloscope 401 when compared with the results obtained either by phase-contrast microscopy or using another electronic counter. Allowing for predilution and duplicate counts on each sample, the rate of throughput was approximately 32 samples per hour. Throughout the test period, the instrument remained electronically and mechanically stable.