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

Summary

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.

Abstract

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.

Introduction

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 μl)⁴. Spectrophotometry methods measure [Hb] precisely without any chemicals, but measurements at multiple wavelengths and a large blood volume are required^5,6. 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 heat^8-10. 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 solutions^13-15 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 elsewhere^12,15. 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 <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.

Protocol

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.

1. Mount an empty micro-capillary tube with inner and outer diameters of 200 and 330 μm, 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.
2. 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.
3. 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° 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.
4. 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.
   1. 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.
5. Place a long-pass filter in front of the detector to block the 532-nm light and measure only the 650-nm probe light.
6. 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.

2. Blood Sample Preparation

1. 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.
2. Measure the blood samples using the PT-AS sensor within 24 hr of extraction to prevent coagulation.

3. PT-AS Measurement Protocols

1. 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.
   1. Employ a tube with an inner diameter of 200 μm. The probe beam size was 2 mm in the representative results, suggesting that the measurement can be performed with a sample volume of >63 nl.
2. Mount the capillary tube at the designated position in the fixture.
3. 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.
4. Turn on the 532-nm PT excitation laser to illuminate the tube.
5. Run the optical chopper to modulate the intensity of the PT excitation light at 2 Hz.
   ​NOTE: The rationale for the selection of this operating condition is described in Discussion and Kim et al.¹².
   1. Mount a chopper wheel in the motor head assembly of the optical chopper system.
   2. Turn on the chopper control box, and use the control knob in the console to set the modulation frequency.
   3. Run the chopper using the control knob.
6. Record the fluctuating scattering pattern via the webcam for 5 sec in MPEG-4 (mp4) format.

4. Signal Processing

NOTE: PT-AS signal processing was performed using a lab-developed MATLAB code.

1. 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)].
2. 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.
3. 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)].
4. 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.

5. PT-AS Calibration

1. 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).
2. Before calibration, quantify the [Hb] values of the samples using a reference hematology analyzer. Measure the PT-AS signals of the samples.
3. 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.

Results

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....

Discussion

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] ...

Materials

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