This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

Experiments with blood samples were performed in compliance with the relevant laws and institutional guidelines. The samples were the residual blood samples that had been acquired and processed in clinical tests at the institution.
1. PT-AS Optical Setup
NOTE: One may use an empty micro-capillary tube for an initial PT-AS setup.
<ol>
	<li>Mount an empty micro-capillary tube with inner and outer diameters of 200 and 330 &#956;m, respectively, and a length of greater than ~5 cm on a capillary tube fixture. Commercially-available fiber fixtures can be used as the tube fixture.</li>
	<li>Securely anchor a 650 nm laser pointer, i.e., probe light source, to illuminate the capillary tube. The probe beam should be larger than the capillary tube. Place a screen (e.g., white paper) behind the capillary tube to observe an angular periodic pattern.</li>
	<li>For the detection part, remove any lenses in a webcam to directly capture the scattering pattern. Position the webcam behind the capillary tube at an angle of 25-35&#176; relative to the probe beam direction. Ensure that the angular periodic pattern produced by the capillary tube can be measured with the detector (Figure 1). Observe the angular periodic pattern in the middle of the image sensor when the image sensor is properly positioned.</li>
	<li>Position a 532-nm PT excitation light source to illuminate the capillary tube. Position the PT light source at any angle, as long as the PT excitation light overlaps with probe beam on the capillary tube and does not reach the detector directly. PT excitation of blood samples using high optical power typically improves the PT-AS sensitivity, as it leads to a larger change in the RI.
	<ol>
		<li>Use the highest optical power of the employed PT excitation light source. In addition, ensure that the PT excitation light overlaps the probe light on the capillary tube. Use a beam size of the PT excitation light at least twice that of the probe light to heat the entire probe volume.</li>
	</ol>
	</li>
	<li>Place a long-pass filter in front of the detector to block the 532-nm light and measure only the 650-nm probe light.</li>
	<li>Install an optical chopper in the path of the PT excitation light before illuminating the capillary tube. The optical chopper is employed to modulate the PT excitation light intensity.</li>
</ol>
2. Blood Sample Preparation
<ol>
	<li>Draw 6 ml of fresh whole blood in anemic condition into ethylenediaminetetraacetic acid blood sampling tubes, and mix the samples well. No other processing is required.</li>
	<li>Measure the blood samples using the PT-AS sensor within 24 hr of extraction to prevent coagulation.</li>
</ol>
3. PT-AS Measurement Protocols
<ol>
	<li>Load a micro-capillary tube with a blood sample to measure. Fill the capillary tube with the blood through capillary action by placing the tube into the blood sample. The minimum sample volume required for measurement is determined by the inner diameter of the capillary tube and the probe beam size.
	<ol>
		<li>Employ a tube with an inner diameter of 200 &#956;m. The probe beam size was 2 mm in the representative results, suggesting that the measurement can be performed with a sample volume of &#62;63 nl.</li>
	</ol>
	</li>
	<li>Mount the capillary tube at the designated position in the fixture.</li>
	<li>Turn on the 650 nm probe laser to illuminate the blood-loaded micro-capillary tube. The angular periodic pattern should be observed with the webcam.</li>
	<li>Turn on the 532-nm PT excitation laser to illuminate the tube.</li>
	<li>Run the optical chopper to modulate the intensity of the PT excitation light at 2 Hz. 
	&#8203;NOTE: The rationale for the selection of this operating condition is described in Discussion and Kim et al.12.
	<ol>
		<li>Mount a chopper wheel in the motor head assembly of the optical chopper system.</li>
		<li>Turn on the chopper control box, and use the control knob in the console to set the modulation frequency.</li>
		<li>Run the chopper using the control knob.</li>
	</ol>
	</li>
	<li>Record the fluctuating scattering pattern via the webcam for 5 sec in MPEG-4 (mp4) format.</li>
</ol>
4. Signal Processing
NOTE: PT-AS signal processing was performed using a lab-developed MATLAB code.
<ol>
	<li>Load the video file to extract the images. For each image [see Figure 2(a) for a representative image], obtain the averaged scattering pattern by computing the mean of the pixel values along the vertical direction [Figure 2(b, c)].</li>
	<li>Evaluate the Fourier transform of the averaged scattering pattern, and compute the phase at the peak spatial frequency. Perform these operations for all the frames of all the recorded images.</li>
	<li>Using the phase values obtained from all the images, plot the temporal phase fluctuation [Figure 2(d)]. Note that the phase fluctuates at the PT modulation frequency. Take the Fourier transform of the phase fluctuation in the time domain, and obtain the magnitude at the modulation frequency. This signal is referred to as the PT-AS signal [Figure 2(e)].</li>
	<li>Measure the [Hb] of a blood sample by converting its PT-AS signal into the corresponding [Hb] using the calibration curve that is obtained in Protocol 5.</li>
</ol>
5. PT-AS Calibration
<ol>
	<li>Prepare blood samples, having [Hb] values that are uniformly distributed in the detection range of the PT-AS sensor (e.g., 0 - 18 g/dl).</li>
	<li>Before calibration, quantify the [Hb] values of the samples using a reference hematology analyzer. Measure the PT-AS signals of the samples.</li>
	<li>Derive a calibration curve relating [Hb] to the PT-AS signal by performing a linear least squares fit, [Hb] = A[PT-AS Signal] + B, of the experimental results. For the operating conditions specified in Table 1, the relationship between [Hb] and the PT-AS signal was found to be [Hb] = 5.13 [PT-AS signal] - 0.09. Use MATLAB code to perform the linear fit.</li>
</ol>

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No conflict of interest is declared.

The PT-AS sensor represents an all-optical method capable of direct [Hb] measurement of unprocessed blood samples. The method quantifies [Hb] in blood using the intrinsic PT response of hemoglobin molecules in erythrocytes. Under illumination by 532-nm light, Hb molecules absorb the light energy and produce heat. The resultant temperature rise changes the RI of the blood sample. The high RI sensitivity of BSI was exploited to measure this RI change in blood. Previously, we demonstrated that the PT-AS sensor enables [Hb] measurement with a detection limit of 0.12 g/dl over the range of 0.35-17.9 g/dl, which is comparable to that of commercial [Hb] sensors on the market.
A notable feature of the PT-AS sensor is that it does not require any preconditioning of blood samples or chemicals. Hence, the sensor enables direct, rapid (&#60;5 sec), and environment-friendly measurement. The use of glass-based micro-capillary tubes as a sample container enabled [Hb] assay at a low operating cost. The minimum sample volume in the PT-AS sensor is determined by the inner diameter of the capillary tube and the measurement beam size on the capillary tube. It is estimated to be ~63 nl in the representative results. In comparison to the sample volumes required in the commercial instruments (e.g., 50-200 &#956;l for the reference hematology analyzer), the PT-AS sensor enables [Hb] measurement with a significantly reduced sample volume. Several rapid and low-cost [Hb] detection techniques have been reported11,17,18 but still require the sample volumes of 2-10 &#181;l for operation.
Several features of the PT-AS sensor implementation should be noted. One should ensure that the size of the PT excitation light beam is at least twice that of the probe light beam on the capillary tube. The two light beams should overlap on the capillary tube, as no or partial overlap of the two light beams on the tube will result in either no or a smaller PT-AS response. One should also make sure that the angular scattering pattern is not saturated on the detector. Adjustment of the scattering pattern orientation along the horizontal or vertical direction may be necessary; otherwise, the acquired image should be rotated in the signal processing stage. Note that scattering of 532-nm PT excitation light by the tube also generates an angular scattering pattern on the detector. Thus, a long-pass filter is required to block the 532-nm light. Larger image sensor captures more angular periodic patterns. Fourier transform of the angular pattern would thus produce higher signal at the corresponding spatial frequency, which allows phase measurement with higher precision. Moreover, a higher frame rate would typically result in a PT-AS measurement with an improved SNR, as it enables more sampling of the temporal phase fluctuation. Therefore, the use of a large, high-speed image sensor with a high pixel density is advantageous.
Some comments should also be made on the measurement time and PT modulation frequency. As described in Kim et al.12, the PT-AS signal refers to the magnitude of the Fourier transform of the phase fluctuations of the angular scattering pattern measured at the PT modulation frequency. The noise is defined as the peak amplitude of the Fourier transform of the phase measurement before PT excitation12. The SNR of the PT-AS signal is evaluated by dividing the magnitude of the PT-AS signal by the noise. A longer measurement time typically yields measurements with a higher SNR, but increases the total [Hb] assay time. The measurement time was set to be 5 sec to achieve an SNR greater than 3 even for blood samples of [Hb] &#60; 1 g/dl. The optimal PT modulation frequency can be found by examining the SNR of the PT-AS sensor as a function of the PT modulation frequency. The optimal modulation frequency for the representative results was found to be 2 Hz. Operation with a PT modulation frequency less than 2 Hz did not produce a high SNR owing to low-frequency noise such as excessive motion of the optical chopper and vibration.
In this demonstration, the PT-AS sensor was demonstrated in a benchtop configuration using a commercial laser pointer and webcam. The optical setup is straightforward, and, because no chemicals are involved, the measurement procedures are simple. On the other hand, it should be emphasized that the sensor can potentially be packaged in a compact handheld device. The light sources for probe and PT excitation can be replaced by low-cost laser diodes or light-emitting diodes. A miniaturized complementary metal-oxide-semiconductor image sensor with built-in computational power can also be utilized as a detector. Integrating these components in a small form factor would generate a new portable, chemical-free, and inexpensive platform for [Hb] assay. In addition to [Hb] assay, the detection principle of the PT-AS sensor may be extended to sensing various biomarkers and chemicals that exhibit PT responses. For example, PT assay of organophosphates and pesticides has also been demonstrated19, and can be readily realized with the PT-AS scheme.

A blood test is commonly performed to evaluate overall human health and to detect biomarkers related to certain diseases. For example, the cholesterol concentration in blood serves as a criterion for hyperlipidemia, which is closely related to cardiovascular diseases and pancreatitis. The blood glucose contents should be measured frequently, as the glucose level is associated with complications such as diabetic ketoacidosis and hyperglycemic hyperosmolar syndrome. Serious illnesses such as malaria, human immunodeficiency virus and acquired immune deficiency syndrome are diagnosed by blood examinations, and quantification of blood components including erythrocytes, thrombocytes, and leukocytes enables screening of pancreatic and renal diseases.
Hemoglobin (Hb), a critical component of blood, makes up about 96% of erythrocytes, and transports oxygen to human organs. Significant alteration of its mass concentration ([Hb]) may indicate metabolic changes, hepatobiliary disease, and neurological, cardiovascular and endocrinological disorders1. [Hb] is therefore routinely measured in blood tests. In particular, anemic patients, dialysis patients, and pregnant women are strongly recommended to monitor [Hb] as a vital task2.
Various [Hb] detection methods have thus been developed. The hemoglobin cyanide method, one of the most common techniques for [Hb] quantification, employs potassium cyanide (KCN) to destroy the lipid bilayer of erythrocytes3. The cyanide hemoglobin produced by the chemical exhibits high absorption around 540 nm; hence, [Hb] measurements can be made via colorimetric analysis. This method is widely employed owing to its simplicity, but the employed chemicals (e.g., KCN and dimethyllaurylamine oxide) are toxic to humans and the environment. The hematocrit scheme measures the volume ratio of red blood cells compared to the total blood volume through centrifugal separation; however it requires a relatively large blood volume (50-100 &#956;l)4. Spectrophotometry methods measure [Hb] precisely without any chemicals, but measurements at multiple wavelengths and a large blood volume are required5,6. Similarly, several optical methods for measuring [Hb] have been proposed including detection methods based on light-scattering, but their measurement accuracies depend strongly on the accuracy of the theoretical blood model.
To overcome these limitations, [Hb] detection methods based on the photothermal (PT) effect of Hb have recently been proposed7. Hb, which is composed mainly of iron oxides, absorbs light at 532 nm and converts the light energy into heat8-10. This PT temperature increase can be detected optically by measuring a change in the refractive index (RI) of blood samples. Yim et al. employed spectral-domain optical coherence reflectometry to measure the PT optical path-length change in a blood-containing chamber11. Although the method enables chemical-free and direct [Hb] measurement, the use of a spectrometer and an interferometric arrangement may hinder its miniaturization. We recently presented an alternative [Hb] detection method, termed photo-thermal angular light scattering (PT-AS) sensor, which is more suitable for device miniaturization12. The PT-AS sensor exploits the high RI sensitivity of the back-scattering interferometry (BSI) to measure PT changes in the RI of a blood sample inside a capillary tube. BSI have been utilized to measure RI of various solutions13-15 and to monitor biochemical interactions in free solution16. The PT-AS sensor employs similar optical arrangement as in BSI, but combines photothermal excitation setup to measure PT increase of RI in blood samples. Operating principles of the BSI and the PT-AS sensors are described in detail elsewhere12,15. PT-AS sensor demonstrated high-sensitivity [Hb] measurement over a wide detection range (0.35-17.9 g/dl) and is capable of operating with sample volumes of &#60;100 nl. No preconditioning of blood sample is required, and the measurement time is only ~5 sec. Here, the experimental setup and a detailed measurement protocol are described. Representative PT-AS results are provided using blood samples from anemic patients, and the results are compared against those from a hematology analyzer to assess the accuracy of the PT-AS sensor.

<table><tbody><tr><td>650 nm laser pointer</td><td>LASMAC</td><td>LED-1</td><td>Probe light</td></tr><tr><td>Hollow round glass capillaries</td><td>VitroCom</td><td>CV2033</td><td>Blood sample container</td></tr><tr><td>Webcam</td><td>Logitech</td><td>C525</td><td>CMOS optical sensor</td></tr><tr><td>Optical chopper system</td><td>Thorlabs</td><td>MC2000-EC</td><td>Optical chopper</td></tr><tr><td>Plastic long-pass filter</td><td>Edmund Optics</td><td>#43-942</td><td>To reject 532-nm PT excitation light</td></tr><tr><td>Fiber clamp</td><td>Thorlabs</td><td>SM1F1-250</td><td>Capillary tube fixture</td></tr><tr><td>EDTA coated blood sampling tube</td><td>Greiner Bio-One</td><td>VACUETTE 454217</td><td>Blood sampling &amp;amp; anticoagulating</td></tr><tr><td>Hematology analyzer</td><td>Siemens AG</td><td>ADVIA 2120i</td><td>Reference hematology analyzer</td></tr></tbody></table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A hemoglobin assay was performed using the PT-AS sensor, and its measurements were compared with those from a hematology analyzer. The experiment was conducted with a PT excitation light intensity of 1.4 W/cm2, PT modulation frequency of 2 Hz, and measurement time of 5 sec. Table 1 summarizes the experimental conditions. The beam sizes of the probe and PT excitation light were 5.5 and 2 mm, respectively. The webcam recorded the images at a frame rate of 30 fps. For measurement, anemic blood samples with three different Hb concentrations were employed. Before the PT-AS measurements, the [Hb] values of the samples were first measured as 5.3, 7.5, and 9.9 g/dl by the hematology analyzer.
Figure 3(a) shows representative time-lapse phase fluctuations of the angular scattering patterns under the modulated PT light illumination. This information was obtained by taking the Fourier transform of the angular scattering pattern and measuring the temporal phase fluctuations at the peak spatial frequency. Note that blood samples with a higher [Hb] exhibit larger phase shifts. The corresponding PT-AS signals were evaluated and converted into [Hb] values. Eleven measurements were performed for each sample, and the mean [Hb] values were found to be 5.46, 7.23, and 9.85 g/dl, respectively. The results agreed well to those obtained using the hematology analyzer [Figure 3(b)]. The [Hb] measurement precision of the PT-AS sensor was found to be &#60;0.89 g/dl. This fluctuation may be in part accounted for by the number fluctuation of erythrocytes in the probe volume and the intensity fluctuations of the employed light sources. Table 2 presents a detailed comparison of the PT-AS measurements against those from the hematology analyzer.
<img alt="Figure 1" src="/files/ftp_upload/55006/55006fig1.jpg" /> 
Figure 1: Schematic of PT-AS sensor. 650-nm probe light from a laser pointer is directed to a blood-loaded capillary tube. The light is then scattered by the blood-containing tube, generating a periodic pattern on a webcam. Upon illumination with 532-nm light, at which Hb molecules exhibit high absorption, Hb molecules absorb the light energy and convert it into heat. The resultant temperature rise changes the RI of the blood. Because the angular periodic pattern varies with the RI and the physical size of the tube, [Hb] in the blood is quantified by measuring this PT shift in the angular periodic pattern. An optical chopper is employed to achieve [Hb] measurement with a high signal-to-noise ratio. A low-cost plastic long-pass filter is located directly in front of the webcam to detect only the probe light. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55006/55006fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55006/55006fig2.jpg" /> 
Figure 2: PT-AS signal processing procedures. (a) Representative webcam images with PT excitation light on and off. The angular scattering pattern shifts because of the PT response of Hb molecules. (b) Each image is averaged along the vertical (y) direction to obtain the averaged pattern. (c) Representative averaged periodic patterns with PT excitation on and off. (d) The averaged periodic pattern is then Fourier transformed, and the phase at the peak spatial frequency is examined as a function of time. Under the modulated PT light illumination, the phase of the periodic pattern fluctuates at the modulation frequency. (e) The measured phase fluctuation is Fourier-transformed, and its magnitude evaluated at the modulation frequency, referred to as the PT-AS signal, is converted into [Hb]. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55006/55006fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55006/55006fig3.jpg" /> 
Figure 3: PT-AS measurement of anemic blood samples. (a) Representative phase fluctuations of the angular scattering patterns measured for three blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl). The blood samples with higher [Hb] values produce larger phase variations. (b) Comparison of [Hb] values measured using the PT-AS sensor with those from the reference hematology analyzer. Eleven PT-AS measurements were performed for each sample. The error bar denotes the standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55006/55006fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Experimental conditions</td>
		</tr>
		<tr>
			<td>PT modulation frequency</td>
			<td>2 Hz</td>
		</tr>
		<tr>
			<td>PT light intensity</td>
			<td>1.4 W/cm&#178;</td>
		</tr>
		<tr>
			<td>PT beam size</td>
			<td>5 mm</td>
		</tr>
		<tr>
			<td>Probe beam size</td>
			<td>2 mm</td>
		</tr>
		<tr>
			<td>Measurement time</td>
			<td>5 sec</td>
		</tr>
		<tr>
			<td>Frame acquisition rate</td>
			<td>30 fps</td>
		</tr>
	</tbody>
</table>
Table 1: Experimental conditions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Hematology analyzer (g/dl)</td>
			<td colspan="2">PT-AS Sensor</td>
		</tr>
		<tr>
			<td>Mean (g/dl)</td>
			<td>SD (g/dl)</td>
		</tr>
		<tr>
			<td>5.3</td>
			<td>5.46</td>
			<td>0.72</td>
		</tr>
		<tr>
			<td>7.5</td>
			<td>7.23</td>
			<td>0.89</td>
		</tr>
		<tr>
			<td>9.9</td>
			<td>9.85</td>
			<td>0.84</td>
		</tr>
	</tbody>
</table>
Table 2: Comparison of [Hb] measurements by the PT-AS sensor with those by the hematology analyzer.

Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.7K Views.  Yonsei University. A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Video: A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Immunology and Infection

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Neuroscience

A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the most commonly used therapy to treat or prevent these devastating complications. Other therapies include hydroxyurea therapy as well as bone marrow transplantation. Chronic intermittent transfusion is especially indicated to prevent recurrent strokes. However, transfusion therapy is associated with significant adverse effects (e.g., alloimmunization and iron overload). The point-of-care (POC) lateral flow immunoassay used here quantifies the %HbS in 15 min using a small patient blood sample. Utilizing this information, the healthcare provider is able to adjust blood transfusion volume for SCD patients to achieve the desired target HbS (most often &#60;30%), while reducing the risk of transfusion-related complications. When compared to laboratory hemoglobin electrophoresis data for 38 whole blood samples, the POC test performed with very high correlation and linear fit (slope = 0.9949, R2 = 0.9751). The strong agreement between the two hemoglobin S percentage (%HbS) quantifying methods shows that 89.5% of samples fall within &#177;5.2% HbS bias. The calibration for quantifying %HbS is built into the device to allow for an automated quantification of %HbS. This time- and cost-effective POC test thus allows the healthcare provider to make timely informed decisions when treating SCD patients, using accurate and updated data.

We have developed a point-of-care immunoassay to rapidly quantify hemoglobin S (HbS) levels during transfusion therapy for sickle cell disease (SCD) patients. By applying a small amount of treated blood to the device, the healthcare provider can determine the %HbS in a SCD patient to immediately inform clinical decisions.

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the ...

Medicine

Optimizing VIS-SWIR Spectroscopy for Biological Molecules

Characterization of Biological Absorption Spectra Spanning the Visible to the Short-Wave Infrared

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. However, because of the lack of sensitivity of hardware at longer wavelengths, absorption spectroscopy has typically been applied for wavelengths in the visible (VIS) and near-infrared (NIR) range from 400 to 1,000 nm. Hardware advancements in the short-wave infrared (SWIR) range have enabled investigators to explore wavelengths in the ~1,000 nm to 3,000 nm range in which fall characteristic absorption peaks for lipid, protein, and water. These molecules are difficult to visualize in the VIS-NIR and can provide label-free sources of biological contrast. Furthermore, lower SWIR absorption has been observed for melanin, the primary chromophore responsible for skin pigmentation. In vivo&#160;optical devices like clinically standard pulse oximeters have been found to have reduced accuracy in people with darkly pigmented skin, possibly because of the stronger melanin absorption in the VIS range. Thus, error associated with skin pigmentation could be reduced by using devices operating in the SWIR. Optical instrument design is facilitated by the understanding of the absorption properties of core tissue components from the VIS to the SWIR range. This article describes protocols and instrumentation for obtaining VIS-SWIR absorption spectra of common tissue absorbers: oxygenated hemoglobin, deoxygenated hemoglobin, melanin, water, and lipid.

Biological samples have distinct optical characteristics in the short-wave infrared (SWIR) compared to the visible (VIS) and near-infrared (NIR) wavelength ranges. However, recording pure SWIR absorption spectra is challenging and rarely conducted for biological molecules. This article presents a method to characterize the VIS-SWIR absorption spectra of biological absorbers.

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...

POC Method and Integrated Decision Support Tool for Estimating Anemia

A Point-of-Care Method with Integrated Decision Support Tool to Estimate Anemia at Population Level

Robust point-of-care methods are required to estimate anemia at the population level. The accurate methods are lab-based and cannot be used at the point of care. To address this caveat, a novel method based on pooled capillary blood and a portable autoanalyzer was developed for the estimation of Hb. Additionally, custom software was developed for near-real-time integration of the Hb values from the auto analyzer to the server. Moreover, a decision support tool that can immediately categorize the participants into different stages of anemia was developed. The decision support tool was designed based on the World Health Organization (WHO) cut-off for anemia at the population level and was available for all age and gender groups. This simple and user-friendly tool could&#160;easily be used by front-line health workers who have limited technical skills. Overall, the method developed could&#160;be used at the point of care and is accurate. This high-throughput method could&#160;be used for screening anemia at the population level for all age and gender groups.

Author Spotlight: Developing a Point-of-Care Hemoglobin Estimation Method for Anemia Management

An accurate hemoglobin estimation method is lacking at the point of care and may hinder population-based programs for treating anemia. Therefore, we developed a point-of-care method based on pooled capillary blood and an auto-analyzer integrated into a custom software application to categorize the hemoglobin values into different grades of anemia.

Robust point-of-care methods are required to estimate anemia at the population level. The accurate methods are lab-based and cannot be used at the point ...

Biology

Bathophenanthroline Sulfonate-Based Colorimetric Assay: A Simple and Rapid Method for Quantitation of Non-Heme Iron in Mouse Liver Tissue

Source: Duarte, T. L., Neves, J. V. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/63469">Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay.</a> J. Vis. Exp. (2022)
In this video, we describe a colorimetric-based method to determine non-heme iron content in mouse liver tissue. Bathophenanthroline sulfonate is used as the chromogenic substrate in the presence of a reducing agent, which converts ferric iron to chelatable ferrous iron.

Source: Duarte, T. L., Neves, J. V. Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay. J. Vis. Exp. (2022)
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A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

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