Evaluation of Paper-Based Point of Care Screening Test for Sickle Cell Disease

Abstract

The aim of the study is to evaluate the stability and longevity of the paper-based screening test for the sickle cell disease in relation to different temperatures and storage time. Blood stain patterns were interpreted after spotting the blood-buffer mixture (phosphate buffer, saponin and sodium metabisulfite) on chromatographic paper (Whatman no. 3). The stability of the buffer was tested after keeping the buffer at different temperature for 24 h. Longevity of the buffer was tested post storage for various time intervals. Test indicated reproducibility with the buffer stored at 4°C. The 15% metabisulfite buffer was found to be stable up to 180 days at 4°C and showed accurate identification of all genotypes. The tests revealed 100% sensitivity and 100% specificity in identification of HbS. However, the sensitivity of differentiation between sickle cell trait (AS) with disease (SS) was found to be 97.7% with 100% specificity. The paper-based screening test may be used as a method of choice for the screening of sickle cell anemia in community-based screening programs. The low-cost, rapid, and accurate point of care testing tools offer an avenue for effective screening in developing nations.

Introduction

Sickle cell disease is an inherited blood disorder characterised by the presence of unusual sickle shaped RBCs under hypoxic conditions. Sickle cell disease patients are at increased risk of severe haemolytic anemia, acute painful crises and organ infarction and bacterial infections [1]. Due to its high prevalence in certain ethnic groups, living in a habitat far away from health amenities and being educationally and socio-economically backward, majority of these patients remain undiagnosed. Thus, leading to high morbidity and mortality [2, 3]. According to an estimate around 37,500 sickle cell disease patients are born annually and which inflict around 360 million Rupees ($ 5.4 million) economic burden on India annually [4]. It also causes great psycho-social burden to patients and their families.

With the advances in medical sciences and development of health resources and government health insurance policies (Ayushman Bharat), it is now possible to provide adequate treatment, prevent and control these disorders in India. In this connection, Government of India has also issued guidelines in 2016 aimed at informing and providing broad guidance on prevention and management of these disorders [5].The crucial step dwells in identification of the affected individuals followed by treatment, counselling and antenatal diagnosis for prevention.

Typical current standard practices for diagnosis of sickle cell disease are Cation Exchange-High Performance Liquid Chromatography (CE-HPLC), capillary electrophoresis and cellulose acetate gel hemoglobin electrophoresis. These tests require trained personnel and state-of-the-art facilities, both of which are lacking in rural areas of India, where the disease is most prevalent. In addition, these tests (HPLC and capillary electrophoresis) are cost intensive (around 4 million Rupees) at initial set up. Furthermore, all these methods are not point of care testing methods. Therefore, the need of hour is to develop an inexpensive, rapid, and portable method for screening of sickle cell disease in high endemic areas.

Recently, Torabian et al. [6] reported an easy-to-use, inexpensive, rapid method of identification of sickle cell that could accurately distinguish between normal (AA),  Sickle cell trait (AS) and sickle cell disease (SS) individuals in a population of adults and children over one year of age. This test is based on the visual interpretation of the blood stain patterns produced on chromatography paper by a drop of mixture of blood and solubility reagent and able to distinguish insoluble sickle hemoglobin (HbS) from soluble forms (HbA, HbF, HbC and others). However, they did not test validity at different temperature settings.

India is a tropical country with a large variation in temperature throughout the year and at different geographical locations. Any variation in temperature may result in erroneous findings rendering the assay ineffective for community screening. Therefore, this study was planned to evaluate the sensitivity and specificity of the paper-based screening test at different temperature and storage conditions.

Material and Methods

Subjects

After the approval of institutional ethical committee, sickle cell disease (SS) patients, sickle cell trait (AS) and normal healthy controls (AA) were recruited for the study. The subjects were either referred to our department from the government medical college or recruited from community-based screening program. Patients with history of blood transfusion in the previous three months were excluded from the study. Individuals with less than one year of age were also excluded as they have high level of fetal hemoglobin. Written informed consent was taken from all the individuals. Venous blood samples were collected in vacutainer containing Ethylene diamine tetra acetic acid Potassium salt (K₃EDTA) as an anti-coagulant and were used within 4–5 h or within 21 days when kept at 4°C. The diagnosis was confirmed by cellulose acetate gel hemoglobin electrophoresis (Cellas Gel, Cleaver Scientific, Rugby, Warwickshire, UK) at alkaline pH (8.4) in all the samples.

Standardisation of the Paper-Based Test

Screening test was performed as described previously by Torabian et al. [6]. Briefly, the solubility buffer was prepared by mixing 1.24 M potassium phosphate monobasic (KH₂PO₄) (Sigma Aldrich), 1.25 M potassium phosphate dibasic (K₂HPO₄)(Sigma Aldrich), 4% saponin (Weight/Volume) (Sigma Aldrich), and different concentration of sodium metabisulfite (10%, 12% and 15% weight/volume) (Sigma Aldrich) in deionized water. Saponin is a plant-based detergent and lyse red blood cells, results in release of hemoglobin into buffer. Sodium metabisulfite is a reducing agent which converts the hemoglobin into deoxy hemoglobin. Phosphate buffer (KH₂PO₄ and K₂HPO₄) having ionic strength polymerizes the deoxy hemoglobin.

Twenty µL of whole blood was mixed with 180µL of the above solubility buffer in 1.5 mL centrifuge tube and vortexed for 30 sec and incubated for 5 min at room temperature. Twenty µL of this solution was spotted onto dry chromatographic paper (Whatman filter paper no 3) using a pipette and allowed to dry for two minutes at room temperature. Different blood stain patterns were formed due to difference in mobility of insoluble sickle hemoglobin (HbS) polymers and soluble forms of hemoglobin (HbA, HbF, HbD etc.) through paper substrate and concentration of respective hemoglobin variants. Soluble hemoglobin moves on the chromatographic paper whereas insoluble hemoglobin (HbS) concentrates at the point of spotting. Blood stain patterns were visually interpreted after two minutes of spotting. All the interpretation was done independently by the three investigators. When there was no agreement in all three, results deduced by two investigators was recorded as final.

Solubility buffer were prepared using different concentration (10%, 12% and 15% weight/volume) of the sodium metabisulfite in phosphate buffer saline and saponin. Test was performed as described above using the known samples. For evaluating the stability of the buffer at different temperature, the test was done after storage of buffer at different temperature (0°C, 4°C, room temperature [25–30°C] and 40°C) for 24 h. Similarly, longevity of buffer was tested by storing the buffer and repeating the test at different time point (Day 15, 30, 60, 90, 120 and 180 days) using known samples. A total of 10 sickle cell disease (SS), 10 sickle cell trait (AS) and 10 normal (AA) known samples were tested in each batch.

After preliminary testing of stability and shelf life, sensitivity and specificity of the test was done on 401 unknown samples (232 AA, 82 AS, 87 SS). Results were confirmed and compared with the cellulose acetate gel hemoglobin electrophoresis (Cellas Gel, Cleaver Scientific, Rugby, Warwickshire, UK) at alkaline pH(8.4).

Statistical Analysis

Sensitivity (True Positive/True Positive + False Negative), specificity (True Negative/True Negative + False Positive), Positive Predictive Value (True Positive/True Positive + False Positive), Negative Predictive Value (True Negative/True Negative + False Negative) and Accuracy (True positive + True Negative/True positive + False Positive + True Negative + False Negative) of test was calculated with reference to cellulose acetate gel hemoglobin electrophoresis.

Results

For standardisation of concentration of the solubility buffer, assay was performed using 3 different concentrations (10%, 12% and 15% weight/volume) of sodium metabisulfite. Figure 1 shows the outcome of tests performed with both 12% and 15% solubility buffer. It clearly shows equally visually identifiable spots on filter paper for all samples tested (sickle cell disease, trait and normal). As both 12% and 15% sodium metabisulfite produced good visual identity, stability of the buffer at different temperatures (0°C, 4°C, room temperature [25–30°C] and 40°C) was tested using the known samples. The assay was repeated with buffers stored at different temperatures for 24 h. Buffer stored at 4°C produced optimally visible spots (Fig. 2).

Fig. 1.

Paper-based screening test using different concentration of Sodium Metabisulfite (MS). A total of 10 sickle cell anemia (SS), 10 sickle cell trait (AS) and 10 normal (AA) samples were tested

Fig. 2.

Effect of temperature on test results. A total of 10 sickle cell anemia (SS), 10 sickle cell trait (AS) and 10 normal (AA) samples were tested each time

Length of storage of above buffer at 4°C (Day 15, 30, 60, 90,120 and 180 days) was also tested. Dry blood spotting efficiency of 12% MS buffer with storage longer than 90 days was lower than 15% MS buffer (Fig. 3). The 15% MS buffer was found to be stable up to 180 days i.e. 6 months and showed accurate identification of all genotypes.

Fig. 3.

Effect of length of storage of buffer on test results. A total of 10 sickle cell anemia (SS), 10 sickle cell trait (AS) and 10 normal (AA) samples were tested in each batch

Post identification of stability and shelf life of the buffer, all further experiments were carried out with 15% sodium metabisulfite buffer all further experiments were done with 15% MS buffer. A total of 401 (232 AA, 82 AS, 87 SS) samples were tested (Table 1). The tests revealed 100% sensitivity and 100% specificity in identification of HbS. However, the sensitivity for differentiating the sickle cell trait (AS) with disease (SS) was 97.7% with 100% specificity. The accuracy for differentiating AS with SS genotype was 98.82% (Table2).

Table 1.

Showing paper-based screening test with different hemoglobin pattern using 15% metabisulphite (MS) buffer

Hb pattern (N = 401)	Cellulose acetate gel electrophoresis (n)	Paper based screening (n)
SS	87	85
AS	82	84
AA	232	232
Total	401	401

Table 2.

Sensitivity and specificity of the assay

	AS + SS versus AA (%)	AS versus SS
Sensitivity	100.0	97.7
Specificity	100.0	100.0
PPV	100.0	100.0
NPV	100.0	97.62
Accuracy	100.0	98.82

Discussion

Yang et al. [7] in 2013 described a simple, quick and inexpensive paper-based sickle cell screening method that could differentiate between the sickle cell disease (SS), sickle cell trait (AS) and normal individuals (AA). They mixed the whole blood with the Hb solubility assay (SickleDex) solution and applied the mixture onto a paper. Blood stain patterns were visually interpretable.

Further, Piety et al. [8] improved this assay using Hb solubility buffer (phosphate buffer saline, saponin and sodium hydrosulfite) and digitised the images of the blood stain pattern that could be quantified for HbS using the mean red colour intensity in the centre of the spot. They reported 93% sensitivity and 94% specificity for visual evaluation and 100% sensitivity and 97% specificity for automated analysis.

However, main bottle neck in their assay was the stability of the buffer used. As sodium hydrosulfite quickly oxidizes in aqueous solution, the buffer was not stable for more than one day, as a result fresh buffer was required for performing the test each time. Torbian et al. [6] in 2017, overcame this difficulty by replacing the sodium hydrosulfite with sodium metabisulfite. Sodium metabisulfite is stable up to a year as a solid at room temperature and at least 6 months in aqueous solution in presence of oxygen. In the present studies, we found that solubility buffer with 15% sodium metabisulfite was stable for 180 days when stored at 4°C. Buffer was not stable at room temperature in present study unlike in the previous study.

Paper-based screening test is a cost-effective method for screening and it costs less than Rs. 20 per test (Table 3) and is able to identify HbS patterns within 10 min. Buffers can be prepared in any laboratory set up and does not require any high-end equipment. Once the buffer is prepared in the laboratory, it can be used in field for 6 months when stored at 4 °C. Moreover, untrained or inexperienced workers were also able to precisely identify the blood stain patterns visually and correlate with genotype.

Table 3.

Cost of assay for 100 tests

Reagents and consumables	Cost
Sodium metabisulfite	Rs 16/ =
Saponin	Rs 100/ =
Potassium phosphate dibasic (K2HPO4)	Rs 172/ =
Potassium phosphate monobasic (KH2PO4)	Rs 54 / =
Chromatographic paper	Rs 1000/ =
Pasteur pipette	Rs 50/ =
Microcentrifuge tubes	Rs 180/ =
Lancet	Rs. 100/ =
Total	Rs 1672/ =

However, the present test also has certain limitations. This test does not differentiate between HbA and HbC, this technique will not be able to differentiate sickle cell trait (AS) from sickle cell anemia-SC (SCA-SC). In West Africa, for example, nearly 40% of the SCD affected individuals have SCA-SC genotype. This test will, therefore, not be useful for persons co-inheriting HbC and HbS. Therefore, this screening test is useful in areas with very low frequency of HbC as in India, East and Central Africa. This test also cannot differentiate between the sickle cell trait and sickle beta thalassemia or Sickle HbD disease. Furthermore, this assay may not be suitable for new-born screening as sickle cell disease new-borns have very low HbS and high HbF. The buffer is stable only at 4°C, therefore the assay may not be useful in resource poor settings.

Currently sickling and solubility test are being used for community screening programs in India. Sickling slide test is based on the principle that in the presence of reducing agent RBC’s become sickle shaped and can be viewed under microscope. However; longer incubation periods (up to 12 h) are required for detection of sickle cell trait. Moreover, it is time consuming and labour intensive and requires a trained person. Furthermore, this test cannot differentiate between AS and SS. Similarly, solubility test based on the principle of decreased solubility of HbS in hypotonic buffers results in turbidity. Both these tests often produce false negative results in patients with severe anemia or low RBC count. Lastly, working buffers are to be freshly prepared due to short half-life. Turbidity of the solution can also be due to several other clinical symptoms (leucocytosis, paraproteinaemia, hyperlipidaemia and polycythaemia) that compromise the test findings.

Other point of care testing methods for the screening of SCD are either under development and/or at testing stages. The current screening test focuses on adapting available diagnostic tools for feasible operation at the field, especially in resource poor settings. The evolving technologies have veered towards overcoming concerns of cost, fabrication complexity, portability, as well as the need for highly-trained operators associated with conventional techniques.

Sickle SCAN™ is based on direct lateral flow chromatographic qualitative immunoassay technique to rapidly detect the presence or absence of HbA, HbS and HbC [9].The advantage of the Sickle SCAN™ is its ability to identify the HbS even in presence of high HbF (96%) and is suitable for new born screening [10]. HemoTypeSC is another emerging rapid diagnostic test based on competitive lateral flow immunoassay technique which means the presence of a line indicates the absence of Hb type and the absence of it indicates the presence of Hb type which is reverse in SickleSCAN™ [11]. Another difference between Sickle SCAN™ and HemoTypeSC is the type of antibodies used. Hemoglobin variants specific polyclonal antibodies are used in former whereas the monoclonal antibodies are used in the later [9, 11]. The major drawback of the both SickleSCAN™ and HemoTypeSC point of care testing device is their high cost. The average cost of both the kits are around Rs. 250–300 ($3 to $4) per test which is on the higher side than the paper-based screening test.

HemeChip diagnostic system (Hemex Health Inc., Portland, OR) is another point of care device based on microchip electrophoresis assay. HemeChip separates hemoglobin variants on a piece of paper that is housed in a micro-engineered chip with a controlled environment and electric field [12, 13]. The advantage of HemeChip diagnostic system is that it can detect and quantitate different hemoglobin variants HbA, HbS, HbF, HbA₂and HbC as well. The ability of quantification of HbA₂ makes the device useful for the thalassemia screening also. The average cost of the test is around Rs. 150–225 ($2 to $3). The main limitation of this test is high cost of the equipment and the requirement of low power electricity making it less suitable for screening at field site in rural areas where electricity is a major concern. Recently manufacturers have made use of rechargeable portable batteries as a power source for the instrument setup making it powerful point of care device.

Kumar et al. [14, 15] developed a density-based separation in aqueous multiphase system for the identification of sickle cell disease. The method relies on the fact that sickle cell RBCs have higher density as compared to normal RBCs and can be separated in aqueous multiphase polymer and enables the visual interpretation of SCD. However, the main drawback of this test is that it cannot distinguish between AA and AS genotype. Furthermore, in presence of high HbF, which protects sickling in SCD, the number of dense cells is reduced, therefore, it is not useful for testing in new-borns.

In conclusion, paper-based assay is easy to use, electricity free, low cost screening test for sickle cell with high accuracy. This point of care testing device may be preferable over solubility and sickle slide test as it may differentiate the sickle cell trait and sickle cell disease. This may be a method of choice for mass screening for SCD where HbSC and HbSD disease is not prevalent.

Authors' Contribution

RK has designed the study and wrote the draft of the manuscript. SM and AG have done the laboratory work. SR modified the manuscript and approved the final version of the manuscript.

Funding

ICMR-NIRTH, Jabalpur (intramural).

Compliance with Ethical Standards

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study has been approved by the institutional ethical committee.

Informed Consent

Written Informed consent have been taken from all the participants.