A novel, low-cost paper-based liver function point-of-care system

ABSTRACT

As one of the important means of quantitative evaluation of liver injury, biochemical analyzer is a conventional testing method. However, it has not been widely used in low-middle-income countries (LMICs) due to the limited personnel, lack of funding and complicated environmental problems. How to acquire the information of liver injury quickly with simple and efficient method providing powerful support for clinical decision-making is still a challenge for LMICs. To solve this problem, we designed a novel and easy-to-use point of care system for liver injury. This system consists of a detection device which is based on a two-photon macro photochemical sensor and a paper-based test card with a built-in system for blood cell filtration. The two-photon structure is used to reduce the overall volume and cost of the system, it makes up for the inter-station errors introduced during the instrument assembly process. The blood cell filtration system reduces the operational complexity, and the blood can be filtered, react and change color directly on the test card. The detection device obtains the continuous light reflection signal of the paper-based test card and quantifies the signal to complete the quantitative test of liver damage indexes. We simulated the national environment of LMICs to evaluate the performance of the system: under the environment of 35℃ and 90% relative humidity, 40 Heparin whole blood, the correlation R² between our system and MindrayBS350s was greater than 0.95. Both of the Randox Quality Control level 2 and level 3 repeatability CV were less than 7.5%. The results show that the system is not only small in size, low in cost and simple in operation, its measurement performance and stability meet the clinical requirements of high temperature and high humidity environment, and so it can be used in LMICs for primary liver function screening and liver disease progression assessment.

Keywords: Reflection spectrum; Photochemical sensor; Multi -signal processing; Rapid detection of liver injury; POCT analyzer; LMICs

1. INTRODUCTION

Liver diseases have become a huge burden on global public health by killing more than 2 million people globally each year^1,2. A large proportion of those deaths can be treated (such as chronic hepatitis B), cured (such as chronic hepatitis C) or prevented (such as drug-induced liver failure) with timely diagnosis and treatment³. Diagnosis of liver diseases is currently performed by non-invasive methods including ultrasound and instantaneous elastic imaging, as well as measurement of biochemical markers in blood. However, these diagnostic techniques require sophisticated equipment with well-trained personnel, which are often not available in low middle income countries (LMICs) with limited resources making the detection, diagnosis and monitoring of liver injury very difficult^4-7. The consequent result may be that many people remain undiagnosed until the advanced stages of the diseases leading to significant delays in clinical decision making and significant costs for the subsequent treatment. Many scholars recognized the problem of liver disease detection in LMICs³, and pointed out the urgent need to develop a rapid, effective and portable method for fast measurement of liver function injury for these countries.

*liyingchun@konsung.com;

The three biomarkers of Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin (ALB) are commonly used in clinical detection of liver injury, elevation of one of them or all is the most common abnormal phenomenon in liver function examination. Detection ALT, AST and ALB can be used for preliminary judgment of liver injury and evaluation of the progression of liver diseases^8-13. There are currently two kinds of FDA-approved clinical chemistry devices for liver function that usually used in LMICs: LDXH (Alere, San Diego, CA) and Roche ReflotronH Plus (Roche Diagnostics, Indianapolis, IN). These two kinds of devices can perform measurements of ALT, AST on fingertip blood without the need of the separation of serum. However, for LMICs with limited resources, it is too expensive (equipment 3000-6000 US dollars, test pieces >4 US dollars)¹⁴ and complex to apply, which require well-trained technicians, stable power supply, and only one marker tested at a time.

We have developed a rapid detection platform and test card for the point-to-point measurement of ALT, AST and ALB based on reflectance photometry, which is suitable for the environment of LMICs with the great merits of low-cost, great simplicity to operate, capability of conducting whole blood/fingertip blood tests and independence of laboratory power supply. It is a portable Point of Care Testing (POCT) testing system including a POCT analyzer and the corresponding paper-based test card. By using this system, the blood to be measured can be drained to a specific region, and gradually the filtration of the blood cell shall be completed, then there would be enzyme-linked reaction and the color rendering. Finally, the reflection spectral curve of the paper-based test card obtained by POCT device is quantified and then the concentration of ALT, AST and ALB would be quantified. The great advantages of low cost, easy portability of the device with unrestricted testing site and fast speed will help expand and decentralize the clinical screening, assessment and treatment services of the liver diseases in low-income countries, and this system will provide powerful support for vast tests in low-income countries with limited economic resources.

2. METHODS

2.1 Principles
This system is based on the Kubelka-Munk theory (K-M theory). The K-M theory is generally used for the analysis of diffuse reflectance spectra obtained from weakly absorbing samples providing a correlation between reflectance and concentration. The concentration of an absorbing species can be determined using the Kubelka Munk formula:
 

Where R = reflectance, k = absorption coefficient, s = scattering coefficient, c = concentration of the absorbing species and A = absorbance¹⁵.

The blood will react in the testing region of the paper-based test card of the system resulting the change of color, and the degree of change is proportional to the related concentrations. According to the K-M theory, for a series of test pieces systems of the same material and thickness, irradiated by the same light source, k will be proportional to the substance concentration c, F(R) proportional to c, and then the concentration of the absorption species can be quantified through the value of reflectivity.


2.2 Paper-based liver function tests
Paper-based test cards are made of multiple layers of patterned paper (Figure 1), each layer with different functions and materials. Multilayer layers of the paper are vertically combined/mounted by polymeric materials to form a reagent test card. The top layer utilizes an isotropic hydrophilic gauze to form a microfluidic hydrophilic pathway directing blood to the designated detection region for plasma separation and testing. There are four independent regions in the test card, three of which are testing areas, which can measure ALT, AST and ALB respectively; the remaining one is an exception identification region to ensure the normal performance of the device. Each of the four zones has a unique environment (such as the reagents, buffers, indicators) to ensure that the measurement process is fast and independent of each other. Using this system, the total time from sample addition to the completion of reaction is less than 3min. The measure of ALT and AST is based on peroxidase method¹⁶, and ALB based on the bromocresol green method¹⁷. The analyzer quantifies the concentration of the substance to be measured by capturing the color's change of the test card^18,19.

Figure 1. The structure of paper-based test card (the left one), and the color change of the test cards for ALB, AST, ALT respectively 3 mins after the addition of the sample

2.3 Low-cost point-of-care Liver function tests platform
POCT device is used to capture color changes of the paper-based test cards and transforms them into concentration outputs. The device includes sampling module, photoelectric detection module, signal analysis module, power module and display module, and each module is independently designed for easy maintenance. The sample module is used to fix the test card, and the photoelectric module is to obtain various information and convert the optical information into electrical signals. After inserting the paper-based test card into the sample module, the blood drops will be added. The blood enters the paper- based test card, goes through blood-pulp separation and chemical reaction, then the color changes generated are captured by the photoelectric detection module of the POCT device. By the signal to concentration conversion model of the signal analysis module, the concentrations of ALT, AST and ALB are acquired and finally displayed by the display module.

For the photoelectric detection module, the core of a POCT device, we specially designed a 4-channel micro-two-photon dual-light source diffuse photochemical sensor which is only of the size of a coin. Each channel consists of 2 light sources of different wavelengths, 2 light sources of the same wavelength and 1 light receiving sensor, as shown in Figure 2. Unlike the conventional biochemical equipment, the design of the micro photochemical sensor here is for low power consumption and with lithium battery power supply it will be free from the conventional dependence on power supply. And thus, the portability and applicability of the device are improved. After the light emitted by the dual light source is processed by the lens, it illuminates the surface of the subject to be measured. The reflected light of the subject is collected by the sensor after processed by lens, then it is converted to electrical signal, and recorded as a function of wavelength. At this time, what the sensor receives is a superposition of the signals from the light sources on the left and right ends, and the reflected signal is strengthened. Because the distance between the two light sources is predesigned so that the corresponding reflected signals of them are complementary. By this design, the signal difference between different stations caused by the distance error during the assembly process of the instrument can be reduced to a minimum, so there is no need to calibrate between different equipment decreasing the production cost, and at the same time the maintenance of the equipment after leaving the factory will become simpler and more convenient.

Figure 2. The photoelectric detection module and the schematic diagram of the single-channel two-photon macro photochemical sensor.

2.4 Study design
In order to verify the feasibility of this equipment in LMICs, we tested the accuracy of whole blood measurement and the repeatability of the Randox Quality Control under the environment of 90% relative humidity at 35°C. The whole blood samples, heparin sodium whole blood, were collected in May 2023 in the third people's hospital of Zhenjiang. The samples were divided into two copies, one was centrifuged and then tested on the machine MindrayBS350s (Mindray, Shenzhen, China) at normal temperature, the other tested with the POCT system proposed in this paper at 90% relative humidity at 35°C and the results were recorded. Here, Randox Quality Control (Randox, Northern Ireland, UK) level 2 (Lot 1503UN) and level 3 (Lot 1223UN) were tested at 35°C and 90% relative humidity with POCT analyzer and the test cards for 10 times respectively.

2.5 Statistical analysis
Data analysis and the generation of the regression plots were performed using Excel2013. Regression plots were plotted and correlation coefficient R² were calculated for the differences observed in results between the system of this study and MindrayBS350s, Coefficient of variation (CV) were calculated for the measurement performance evaluate.

3. RESULTS

3.1 Operating conditions and assay performance
In the 40 cases of heparin blood, the correlation, R², of ALT, AST and ALB with MindrayBS350s was 0.9909, 0.9918, and 0.9913 respectively at 35°C and 90% relative humidity, as shown in Figure 3. The R2 of the three items were all greater than 0.95, and indicated that the results of the device and its supporting reagents had a good correlation with that of the clinical biochemical device of MindrayBS350s in this environment.

Figure 3. Correlation of with the MindrayBS350s for the complete ALT, AST and ALB in 35°C and 90% relative humidity.

3.2 Precision
In-batch precision indicates in-batch measurement error, also known as repeatability, it is determined repeatedly by the same operator on the same sample. At 35°C and 90% relative humidity, the precision of the equipment and test cards is evaluated using Randox quality control to verify that the environment introduces negligible random errors. The results are shown in table1, the Randox quality control ALT Level 2 CV is 5.8%, Level 3 4.5%; AST Level 2 CV 6.5% and Level 3 CV 2.5%. ALB Level 2 is 5.7%, Level 3 7.3%. ALT and AST are in line with China's dry chemical analyzer industry standard²⁰: the variability coefficient of ALT and AST in the concentration range of 40U/L~60U/L less than or equal to 12%, and the CV with the concentration range of 100U/L~300U/L less than or equal to 5%; All of the above analytes meeting the requirement that CV less than 7.5% listed in the manual of most dry biochemical POCT analyzers which have been on the market. The results show that under the conditions of 35°C and 90% relative humidity, the CV of the equipment and the test card are within the acceptable range, and the random error of the test card under such environment is acceptable.
 
Tabel1 ALT, AST and ALB Randox quality control experiment and the results.

4. DISCUSSION AND CONCLUSIONS

Biochemical analyzers have been used as the preferred method for the detection of the hematological markers of liver such as ALT, AST, and ALB for decades. After human peripheral venous blood is collected, it is transported to a central laboratory, where the serum is separated by trained technicians and tested by a biochemical analyzer. These seemingly routine detection methods are very difficult for LMICs because: expensive equipment, stable test environment required, and well-trained technicians to perform the tests and necessary maintenance. Moreover, even though the detection can be carried out, the scattered environment of LMICs will delay the detection results.

In this paper, a system with low-cost, rapid detection speed for liver injury indexes is designed. Compared with the existing POC devices and previously published studies ^{21, 22, 23}, our POCT devices can perform quantitative tests of ALT, AST and ALB simultaneously. The design of low power consumption and lithium battery makes that the related tests can be performed anytime and anywhere independent of the power supply of the testing site. Because peripheral blood can be tested directly, there is no need for a complicated centrifugation process before testing. And the primary hygienists after simple training can perform the test well. The cost is <$700/instrument, and the paper-based test card <$3/piece. Furthermore, we have done the experiment work at the high temperature and high humidity environment of LMICs in the laboratory to verify its effectiveness in LMICs. At 35℃ and 90% humidity, the correlation between the ALT, AST and ALB and the clinical large biochemical equipment MindrayBS350s was all >0.95 respectively, and the repeatability of quality control of Randox all <7.5% in line with China's dry chemical analyzer industry standard, the CV of ALT and AST in the concentration range of 40U/L~60U/L less than or equal to 12%, and the CV with the concentration range of 100U/L~300U/L less than or equal to 5%, meeting the requirement that CV less than 7.5% listed in the manual of most dry biochemical POCT analyzers which have been on the market. This device and its accompanying paper-based test card can be used in LMICs to meet the needs of rapid detection of liver injury indicators.

To sum up, the POCT analyzer and its supporting reagents or test card proposed in this paper have the following merits:
1. Primary health care workers can be used with simple training; 2. The paper-based system is easy to be operated and maintained; 3. The three quantitative assays, ALT, AST and ALB, can be tested at the same time, the test time is within 3 mins, and the blood volume < 45uL; 4. With a broader detection range: ALT 10-800U/L, AST 10-800U/L and ALB 10- 60g/L; 5. The sample can be tested at 35℃ and 90%RH without affecting the test results; 6. Lithium battery power supply, no dependence on laboratory environment; 7. The last but not the least, the testing costs of the equipment are very low. At present, the above advantages are urgently needed by LMICs. This device not only provides a novel and effective way for the rapid detection of liver injury in LMICs, but also promotes the development of POCT devices for monitoring other clinically important analytes.

ACKNOWLEDGEMENTS

The study of operating conditions and assay performance in this paper was funded by the Foundation for Innovative New Diagnostics (FIND).

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