The molecular structures of (a) Propidium Iodide (PI), (b) Fluorescein Isothiocyanate (FITC) and (c) Basic Orange 21 (BO21). The measured time trace of the (a) red fluorescence signal and the (b) light extinction signal (intensity versus time) of the microalgae, Phaeocystis.
Introduction to Blood Cell Count
- Blood Cell Count and Its Clinical Significance
- Development of Blood Cell Count Technology
- Needs of Portable Blood Cell Count Technology
- Microfluidics for Blood Cell Counting
- Needs of Future Development
The measurement of WBCs mainly consists of the WBC count (the total number of WBCs in per volume of blood) and the WBC differential count (the absolute numbers or percentages of different types of WBCs). It is worth emphasizing the importance of the WBC count and the WBC differential count in the CBC test [10].
Microfluidic Cytometer for White Blood Cell Count
Introduction
- Flow Cytometry Principle
- Problem of Implementing Flow Cytometry in Portable Analysis
- Microfluidic Cytometer Technology
- Previous Art of Microfluidic Cytometer for Blood Analysis
Second, the volume of blood sample required for flow cytometer analysis is not optimal for point-of-care applications [39–41]. Two major sources of the consumables are the dilution buffer for sample preparation and the enveloping buffer for hydrodynamic focusing.
Sheathless, Fluorescent Microfluidic Cytometer for WBC Count
- Basic Principle
- Dye-Based Assay and Fluorescent Detection of WBCs
The coincidence error of the microfluidic cytometer is tolerable compared to the error tolerance range of the commercial instrument. Dye-based assays are used in the proposed microfluidic cytometer for selective staining of the WBCs in the blood sample.
Experiment and Material
- Device Layout
- Device Fabrication
- Blood Sample
- Fluorescent Dye Assay
- Optical Configuration for Fluorescent Detection
- Portable Detection System
Second, two parts of PDMS prepolymers (Sylgard 184, Dow Corning, MI, USA) were mixed in a ratio of 10:1. An excitation filter (bandpass filter, 440-480 nm) was used before the slit to clean the light illumination wavelength.
Result and Discussion
- System Evaluation with Fluorescent Beads
- Blood Sample Pretreatment with Fluorescent Dye Assay
- Blood Sample Measurement on Microfluidic Cytometer
- WBC Count and WBC 2-Part Differential Count
- Correlation Study with Commercial Blood Cell Counter
Measured fluorescence signals from whole blood sample pretreated with AO dye assay, (a) green fluorescence and (b) red fluorescence. Processed fluorescence signals from whole blood sample pretreated with AO dye analysis (after a 4-point moving average digital signal processing filter): (a). The data of measured fluorescent intensities were first analyzed to count the number of WBC events detected.
For each event, the height of the green fluorescence peak and the height of the red fluorescence peak were recorded. The analysis results included the total number of detected WBC events, NWBC, plus both the green and red fluorescence intensities associated with the WBC events. The error bar shows the variation of three measurements of the same sample on the microfluidic cytometer.
Comparison of measured lymphocyte percentages from a microfluidic cytometer and results from a commercial blood counter (Coulter LH750).
Conclusion
In summary, the leukocyte count and the two-part differential leukocyte count (lymphocyte vs. non-lymphocyte) measured by the microfluidic cytometer showed close agreement with the commercial blood counter results. The microfluidic device was fabricated using the soft lithography process of PDMS and a glass substrate. For the optical measurement, an LED-induced (central wavelength 460 nm, 700 mW), dual-color fluorescence detection scheme was developed.
The measurement wavelength ranges were chosen to cover the green fluorescence (510-560nm) introduced by the AO-DNA binding and the red fluorescence (>590nm) introduced by the AO-RNA binding. After being incubated with the AO assay, blood samples were tested on the portable fluorescence detection system with the sheathless microfluidic device. The WBC events were measured to have an SNR of 14dB for the green fluorescence and an SNR of 20dB for the red fluorescence.
The results of the total WBC count and the two-part differential WBC count (lymphocytes vs. non-lymphocytes) were in good agreement with the results of a commercial automated blood counter (correlation coefficient R2 0.99, maximum error less than 10%).
Dye Assay for WBC Differential Count
Introduction
- Fluorescent Assays for WBC Differential in Flow Cytometer
- Dye Assay for WBC Differential
Comparison of the fluorescent dye assay and fluorescent-conjugated immunostain assay for WBC differential count. In the fluorescent dye assays, the fluorescent label is introduced by the binding of dyes to cellular contents in WBCs (eg, nucleic acid, protein, etc.). The fluorescent dye assays, on the other hand, have limited choice of binging specificity (eg, nucleic acid, protein, etc.).
In the early 1970s, researchers from Bio/Physics Systems demonstrated a 3-part WBC differential with the fluorescent dye Acridine Orange [16]. Above all, the development of a fluorescent staining assay that not only provides differential WBC counts but is also optimal for portable settings is still needed. In addition, the fluorescence wavelength of FITC dye (emission peak 520 nm) differs from the fluorescence wavelength of PI dye (emission peak 620 nm).
Since the fluorescence emissions were introduced by the binding of dyes to cellular contents (eg nucleic acid, proteins, etc.), the measured fluorescence intensities provided a quantitative evaluation of the amount of cellular contents stained in different cells.
Experiment and Materials
- Microfluidic Device and Fluorescent Detection System
- Blood Sample and Fluorescent Beads Sample
The fraction of collected light with a wavelength greater than 565 nm was transmitted through the mirror and measured in the red fluorescence channel. The intensity of green fluorescence and red fluorescence were finally measured by two photon multiplier tubes (PMT, Hamamatsu H5784). Second, individual WBC population types were purified from the WBC fraction by immunomagnetic isolation.
The purity of the WBC populations obtained was shown to be higher than 90% with verification using Wrights staining. The stock solution of Assay I (PI and FITC) was then prepared by mixing the 150M PI stock solution, the 100M FITC stock solution and the PBS solution in a volume ratio of 6:1:5. The staining of the blood sample with the fluorescent assays followed a standard protocol for PI staining of cellular DNA content with adapted modifications [95].
Finally, the sample was incubated with 12L of the test solution (Recipe I or Recipe II) for 7min, and 50L of the sample was then taken for measurement.
Result and Discussion
- System Calibration with Fluorescent Beads
- Blood Sample Staining with Fluorescent Assays
- WBC 4-Part Differential
- Correlation Study of 4-Part WBC Differential
- Cluster Overlap of Monocyte and Lymphocyte
- Improved WBC 4-Part Differential
- Validation of 4-Part Differential with Purified WBC population
- Correlation Study of Improved 4-Part Differential
- WBC 5-Part Differential Feasibility
- Correlation Study of Total WBC Count
Recorded time trace of the measured fluorescent signals (intensity versus time) from the 5m fluorescent beads. Fluorescent images of the WBCs after staining with the dye test (Recipe I: .PI and FITC). The scatter plot of the fluorescence measurement data (green fluorescence intensity versus red fluorescence intensity).
For comparison, two tests were performed on the blood sample from the same donor. Comparison of the differential WBC pattern of staining of blood samples with (a) PI versus (b) PI and BO21. Comparison of the differential WBC pattern of staining of blood samples with Recipe I (PI and FITC) versus Recipe II (PI, FITC and BO21).
Overall, the maximum inaccuracy of the differential counting results was less than 10% compared to the commercial counter results.
Conclusion
Nevertheless, the maximum error of the total WBC count, compared to the commercial count, was less than 10% and is considered to be within the acceptable range for clinically significant (10%). The associations of the differential clusters with the four types of WBCs (lymphocyte, monocyte, neutrophil and eosinophil) were verified by first the spike experiments with purified WBC types, and then in a correlation study with the count results of a commercial blood counter (Beckman) Coulter LH750). The stab experiments confirmed that the purified WBC types indeed led to and corresponded to the increased count of the corresponding target cluster.
The correlation study showed good agreement with the commercial blood count results of WBC 4-part differential (correlation coefficients R2. >0.9, maximum error <10%). In addition, counts of the fifth WBC type, basophil, were also investigated by testing blood samples spiked with purified basophil cells. In normal human whole blood, the basophilic cells are rare (<1% of total WBCs) and rarely form a significant cluster.
In the enriched sample, the basophil cluster became prominent (~30% of total white blood cells), and the measurement results showed a distinct basophil cluster in addition to the four previously measured clusters.
On-Chip Blood Cell Count
Introduction
- Design Principle of Cartridge Chip
- Key Components of Cartridge Chip
- Principle
- Material and Experiment
- Results and Discussions
- Conclusion
Microscope pictures of blood in a PDMS channel (channel height 100m) (a) before and (b) after thermal coagulation. After illumination, the part of the blood sample that was under the laser spot solidified and turned a darker color. The blood sample without laser illumination was easily pushed out of the channel by an applied back pressure of less than 0.1psig.
These results showed that the laser illumination indeed introduced the thermal blockage of the blood sample, and the blockage was sufficient to be used as a closed valve in the microfluidic channel. Two different geometry designs of the valve zone (as shown in Fig. 4.15) were tested for the laser-induced clogging. 4.16(IV), a fixed amount of blood sample was delivered, the volume of which was equal to the volume of the sample chamber.
Photomicrographs of the PDMS device using the laser-induced blockage valve in the administration of a fixed volume of blood sample.
On-Chip Blood Staining with Fluorescent Dye Assay
- Mixing in Microfluidics
- Theory of Passive Hydrodynamic Focusing Mixer
- Velocity Distribution in Hydrodynamic Focusing Mixer
- Diffusion Analysis in Hydrodynamic Focusing Channel
- Design of Hydrodynamic Focusing Channel
- Material and Experiment
- Result and Discussion
- Conclusion
The flow direction of the mixing channel is in the x-axis and the cross-section of the channel is in the y-z plane. At the end of the mixing channel, a sheathless cytometer channel (30 µm wide) was used for the fluorescence measurement of the sample. 4.23(c), the width of the blood sample stream was approximately 20 µm at the inlet of the mixing channel (limit position r≈0.2).
A narrower flow width of the blood sample reduces the diffusion path, thereby increasing mixing efficiency. This lateral diffusion caused a continuous decrease in cell concentration in the sample stream, thus a decrease in viscosity [109]. Furthermore, the total length of the mixing channel of the micro-mixer was 60 mm.
On-chip staining of the blood sample with the dye test (Acridine Orange) was demonstrated here using a hydrodynamic focusing mixer.
Microfluidic Cytometer for Biosensing of Microalgae
Introduction
- Microalgae and Harmful Algal Bloom
- Microalgae Sensing by Microfluidic Cytometer
- Microalgae Population Monitoring and Algal Bioassay
The microfluidic cytometer we developed was found to be suitable for fluorescence measurement of the microalgae, providing a portable alternative to traditional flow cytometer for on-site assessment. The size of the entire system, as shown in fig. 5.2, was suitable for use in the field. First, the microfluidic cytometer was able to provide sufficient signal integrity (eg, SNR) for measuring chlorophyll fluorescence from microalgal cells.
But it can demonstrate the feasibility of using the microfluidic cytometer for microalgal population monitoring by distinguishing certain types of microalgal cells. For the application of the algal bioassay, it is important to demonstrate that the microfluidic cytometer could simultaneously measure the Chlorophyll fluorescence from the microalgae cells, and additional fluorescence from cells' response to the test condition. The fluorescence of the Fluorescein (emission peak 521nm) can be measured in the green channel of the microfluidic cytometer.
The molecular structures of (a) Fluorescein diacetate (FDA) and (b) Fluorescein, and the basic principle of the hydrolysis process.
Material and Experiment
The measured green fluorescence of FDA staining showed an SNR close to 30 dB. FDA fluorescence was due to esterase activity in microalgal cells. Grimaldi, E., et al., Evaluation of monocyte counts by two automated hematology analyzers compared with flow cytometry. Harris, N., et al., Performance evaluation of the ADVIA 2120 hematology analyzer: an international multicenter clinical trial.
Suh, I.B., et al., Evaluation of the Abbott Cell-Dyn 4000 hematology analyzer for detection and therapeutic monitoring of Plasmodium vivax in the Republic of Korea. Rotstein, R., et al., Utility of telemedicine for the detection of infection/inflammation at the point of care. Osei-Bimpong, A., et al., Point-of-care method for total white blood cell count: evaluation of the HemoCue WBC device.
Geacintov, N.E., et al., Dynamics of Binding of Acridine Dyes to DNA Investigated by Triplet Excited-State Probe Techniques.
