A method to determine the concentration of sickle- cell haemoglobin in blood

Abstract

The present invention relates to a method to determine the concentration of sickle-cell haemoglobin in blood. Said method is based on a smear technique involving drawing a smear at a particular speed, wherein the length of the smear is a function of the concentration of the HbS. It also provides a smear card having graduations to measure the smear length and the corresponding viscosity (in cP) and a device comprising such smear card; reducing agent and substrate.

Description

[0001] A METHOD TO DETERMINE THE CONCENTRATION OF SICKLE-CELL HAEMOGLOBIN IN BLOOD

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to a blood smearing technique using a blood smearing device for determining the concentration of the Hemoglobin protein (HbS) in a blood sample of a sickle-cell diseased person.

[0004] BACKGROUND OF THE INVENTION

[0005] Sickle cell disease (SCD) is caused by a point mutation in the HBB gene on chromosome 11 that encodes the P-globin chain of haemoglobin, causing the hydrophilic amino acid glutamic acid to be replaced with the hydrophobic amino acid valine at the sixth position of the P-globin chain. Two molecules of wild-type a-globin combine with two mutant P-globin subunits forms haemoglobin S (the normal haemoglobin A is formed with two wild-type P-globin subunits). Under low oxygen condition, the absence of a polar amino acid at position six of the P- globin chain results in non-covalent polymerization of haemoglobin leading to the formation of long aggregated chains of haemoglobin molecules. The aggregation distorts red blood cells into a sickle shape and increases their rigidity. Individuals with one copy of the mutated gene contain both normal and abnormal haemoglobin in each of their red blood cells (RBCs).

[0006] The one healthy allele produces enough wild-type P-globin subunits that their RBCs can work normally although other RBCs with the mutant P-globin chain will remain defective. Such individuals with one healthy allele are said to have Sickle Cell Trait (SCT). When both alleles contain the point mutation, all RBCs have the mutant P-globin subunits and hence defective. This leads to Sickle Cell Anaemia (SCA). An individual with SCT has a lower concentration of HbS protein (35-45% of the total haemoglobin with the rest mostly normal haemoglobin, HbA) and the double mutation results in a high concentration of the HbS protein (>80%) - Table 2.28 in Pagana et al [Pagana, Kathleen D., Pagana, Timothy J., and Pagana, Theresa N.,. "Mosby's Manual of Diagnostic and Laboratory Tests (7th edn)." (2023)].

[0007] The loss of elasticity in red blood cells in the deoxygenated condition is central to the pathophysiology of the disease. Normal red blood cells due their high elasticity can deform and pass through capillaries. In case of sickle cell disease, the cells become rigid in the low-oxygen condition and get stuck as they pass through narrow vessels, leading to acute and chronic pain, severe anaemia, kidney dysfunction, acute chest syndrome, stroke and other cardiovascular diseases, increased susceptibility to infectious diseases, pregnancy complications, and maternal mortality. An estimated 7.74 million cases of sickle-cell anaemia were prevalent in 2021. The highest sickle cell disease disability burden is concentrated in western and central sub-Saharan Africa and India [Thomson, Azalea M., Theresa A. McHugh, Assaf P. Oron, Corey Teply, Nikhil Lonberg, Victor Vilchis Telia, Lauren B. Wilner et al. "Global, regional, and national prevalence and mortality burden of sickle cell disease, 2000-2021: a systematic analysis from the Global Burden of Disease Study 2021." The Lancet Haematology (2023).].

[0008] A number of techniques have been proposed to screen and / or diagnose the sickle cell disease. US20080255766A1 describes a method of diagnosing sickle cell in a human subject, preferably a newborn, suspected of suffering from sickle cell, which comprises: (a) obtaining a fluid sample (b) determining the concentration of Eotaxin in said fluid sample; (c) deciding if the determined concentration of Eotaxin in said fluid sample is statistically different from that found in a control group of human subjects, whereby a statistically different depressed concentration of Eotaxin supports a positive diagnosis of sickle cell. An article of manufacture is provided which comprises binding reagents specific for at least one of Eotaxin and Monocyte Chemotactic Protein- 1, preferably both biomarkers.

[0009] US2018 / 0267021 describes high throughput methods and devices for evaluating mechanical, morphological, kinetic, rheological or hematological properties of cells, such as blood cells under regulated gas conditions. In some aspects, the invention relates to methods and devices for diagnosing and / or characterizing a condition or disease in a subject by measuring a property of a cell from the subject, under controlled gas conditions. This involves passing a particle suspension is through a small chamber with gas bubbling and then the cells are observed for sickling.

[0010] US2016 / 0116489 describes screening methods and devices for detecting and diagnosing hemoglobinopathies for sickle cell disease and related phenotypes. The patent describes lateral flow immunoassay devices for the detection of hemoglobinopathies, methods for screening for hemoglobinopathies, kits for the detection of a hemoglobinopathy in a sample and immunogenic peptides for producing antibodies against hemoglobin variants. The method is based on lateral flow immunoassay and uses specific polyclonal antibodies (pAbs) and / or monoclonal antibodies (mAbs) having an afinity to HbS, HbC, and HbA. This detection antibody can be conjugated to colored (or fluorescent) nanoparticles. Other pAbs and / or mAbs against the initial N-terminal amino acid (AA) sequence of human sickle cell hemoglobin (HbS), human hemoglobin C (HbC), and adult normal hemoglobin (HbA) can be used as capture anti bodies.

[0011] While the above discussed methods may provide an effective targeted screening, they require laboratory set up, specialized instruments and are prohibitively expensive for use in resource constrained settings.

[0012] WO2017 / 106334 Al provides a method of screening for sickle-cell disease comprising contacting an end of a capillary tube with deoxygenated blood; observing flow rate of the deoxygenated blood within the capillary tube; and performing one or more steps selected from the group consisting of: comparing the flow rate to a predetermined flow-rate threshold for the capillary tube indicative of sickle-cell disease; calculating viscosity based on the flow rate and comparing the viscosity to a predetermined viscosity threshold for the capillary tube indicative of sickle-cell disease; and comparing the flow rate to a second flow rate for oxygenated blood from a same subject within a substantially identical capillary tube.

[0013] The time of rise of oxygenated and de-oxygenated blood in capillary is used to distinguish healthy, trait and sickle cell anaemia blood samples. The time of rise is equivalent to the flow rate of the sample and is related to the viscosity of the sample. Higher viscosity sample is expected to rise slower while low viscosity sample is expected to rise faster in the capillary. The patent relates the time of rise of sample to the HbS concentration. While there is an observable difference in the rise time between the healthy and sickle diseased samples (both trait and sickle cell anaemia), the method is unable to distinguish the SCT from SCA samples. Instead, the rise time is shown to be dependent on the kinetics of the deoxygenation. Hence the rise time has to be obtained at different times after sickling treatment to distinguish the trait and sickle cell anaemia samples, which is time consuming and hence disadvantageous.

[0014] A more recent publication from the same research group, (Brown et al, Biophysical Journal 120, 2138-2147, June 1, 2021 ) has shown that SCT blood upon deoxygenation takes longer to rise compared to the anaemia (SCA) sample upon deoxygenation, although the viscosity is higher in the latter case due to the higher concentration of HbS protein. This discrepancy is attributed to the segregation of the red blood cells in the capillary. Thus, it fails to provide a one- to-one correspondence between the rise time of deoxygenated blood sample and the HbS concentration using the capillary rise method.

[0015] US2016 / 01873 describes a device useful for evaluating the size, shape, surface texture, mobility, rigidity, flexibility, tensile resistance or turnover rate of red blood cells in a blood sample from an individual. The device of the present invention may be used to determine if the individual suffers from a red blood cell abnormality, such as SCD. The method uses a packed bed in a capillary and lets the blood flow through it. Rigidity of the cells in case of sickle cell disease causes the flow to be lower compared to the blood of a healthy individual. The said method requires a laboratory set up, specialized instruments and suffers from similar drawbacks as stated above.

[0016] Currently, the most prevalent screening tests include the sickle solubility test [Tubman, Venee N., and Joshua J. Field. “Sickle solubility test to screen for sickle cell trait: what’s the harm?.” Hematology 2014, the American Society of Hematology Education Program Book 2015, no. 1 (2015): 433-435]. In this method, the HbS lysed from red blood cells is mixed in a solution containing sodium hydrosulphite. The HbS precipitates causing the solution to become turbid. Visual inspection confirms the presence of HbS although automatic readers have also been developed. Another test is based on mixing a solution of sodium metabisulphite (SMBS) with blood. The former causes deoxygenated condition resulting in sickling of the red blood cells. Examination of a blood smear under a microscope confirms the presence of HbS. The aforementioned test has high sensitivity and specificity although false-negatives are seen in individuals with severe aneamia, in those with a haemoglobin HbS fraction less than 10%, or in individuals with high levels of haemoglobin F. However, neither of the two tests can distinguish between SCA and SCT.

[0017] The tests for distinguishing SCA from SCT are expensive and sophisticated in that they require specialized instruments and trained manpower. Such tests include HB electrophoresis, Isoelectric focusing (IEF) and high pressure liquid chromatography (HPLC). These tests have close to 100% sensitivity and specificity, and can also detect other variant haemoglobins. All the three test methods use the biochemical characteristics of the haemoglobin protein for identification of disease [Tubman et al. and Clarke, Gwendolyn M., and Trefor N. Higgins. “Laboratory investigation of hemoglobinopathies and thalassemias: review and update.” Clinical chemistry 46, no. 8 (2000): 1284-1290]. Thus, there is an urgent need for a screening method that is inexpensive, simple and rapid enough to enable universal screening for SCT and SCA in resourcelimited settings lacking established infrastructure, specialized equipment or trained personnel.

[0018] OBJECT OF THE INVENTION

[0019] Accordingly, an object of the present invention is to provide a compact automatic sickle-cell anaemia detection tool, which is economical, and easy to manufacture and maintain, and useful for resource constrained areas.

[0020] Another object of the present invention is to provide a bench-top, in vitro blood smearing method involving measuring the length of smear which provides the concentration of HbS. The method is economical, and easy to perform in resource constrained areas.

[0021] Another object of the present invention is to provide a bench-top blood smearing method for screening of sickle cell trait which employs a smearing device that utilizes only a few microlitres of blood to be tested.

[0022] Another object of the present invention is to provide a bench-top blood smearing method which employs a smearing device that utilizes only a few microlitres of blood to be tested, especially for sickle-cell individuals with compromised haemoglobin count.

[0023] Another object is to provide the viscosity of the blood sample from the speed of the blade and the length of the smear.

[0024] Yet another objective of the invention is to develop the technique as a point of care screening device for detection of sickle-cell anaemia in resource constrained areas where basic diagnostic facilities are limited. SUMMARY OF THE INVENTION

[0025] In one aspect, the present invention provides a method of screening for sickle cell trait, the method comprising steps of: i) Pre-treating a sample with a reducing agent; ii) drawing a smear of the sample by a moving blade at a speed of about 10-

[0026] 200 mm / s; and iii) determining the concentration of HbS in the sample.

[0027] In a second aspect, the present invention provides a method of screening for sickle cell anaemia, the method comprising steps of: i) pre-treating the sample with a reducing agent; ii) smearing the sample at a speed of about 10-200 mm / s; and iii) calculating the concentration of HbS in the sample.

[0028] In a third aspect, the present invention provides a smearing technique employing a smearing device for determining the concentration of HbS in blood, the method comprising the steps of: i) placing a drop of sample on a glass slide at the indicated position; ii) drawing the smear on the slide by a moving blade held at an angle of between 20° to 45° and at a speed of about 10-200 mm / s; and iii) calculating the concentration of HbS from the smear length.

[0029] In a fourth aspect, the present invention provides a method to measure the concentration of HbS comprising measuring the length of a smear drawn by a moving blade at a speed of about 10-200 mm / s and at a blade angle between 20-45°.

[0030] In a fifth aspect the present invention provides a rapid quantification of HbS levels for monitoring individuals who are on treatment or transfusion therapies for sickle cell diseases. In a further aspect the present invention provides a smear card having graduations to measure the smear length and the corresponding viscosity (in cP) and a device comprising such smear card; reducing agent and substrate.

[0031] BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0032] The above and other aspects, features and advantages of the embodiments of the present disclosure will be more apparent in the following description taken in conjunction with the accompanying drawings, in which:

[0033] Figure 1 depicts a drawing of the smearing technique. The smearing device is used for the viscosity measurement technique.

[0034] Figure 2 depicts two drawings of glass slides with normal viscosity markings and with smears of different lengths at the same speed, based on viscosity before and after sickling.

[0035] Figure 3 shows photographs of smears from two donors (sickle-cell anaemia individuals) and the variation of smear lengths with increasing speeds. The photographs (a and b) represent sickle-cell trait and diseased (SCT and SCA) before and after the sickling treatment.

[0036] Figure 4 (a and b) shows a graph between the speed of the blade and the length of the smear obtained for both SCT and SCA before and after sickling treatment. The intercept in used in extracting viscosity of the blood sample.

[0037] Figure 5 shows a graph depicting a relation between the amount of HbS content in the blood samples (for both SCT and SCA) and the percentage change in the viscosity before and after the sickling treatment.

[0038] Figure 6(a) shows a card (also called a SMEAR CARD®) with graduations to measure the smear length and the corresponding viscosity (in cP). An example of a glass slide with blood smear placed on the SMEAR CARD® is shown in Figure 6(b). The smear was obtained at a blade speed of 109.3mm / s. The measured viscosity is 3.0 cP (indicated by red arrow).

[0039] DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0040] The following description with reference to the accompanying drawing and figures is provided for better comprehension and understanding of various embodiments of the present invention and the viability and accuracy of the technique in serving as a screening test for sickle-cell anaemia.

[0041] Described herein is a method of measuring the concentration of HbS employing the present technique of drawing the smear, the method comprising steps of: i) pre-treating a sample with a reducing agent; ii) drawing a smear of the sample by a moving blade at a speed of about 10-

[0042] 200 mm / s; and iii) calculating the concentration of HbS in the sample.

[0043] The present technique is capable of measuring a concentration of HbS upwards of 10%.

[0044] The present invention is based on the blood smear technique where a drop of sample is placed on a glass slide and a blade / second glass slide is brought in contact with the sample and the bottom substrate. The blood sample spreads along the line of contact to form a meniscus between the blade and the substrate. The blade is next moved along the substrate to spread / smear the blood onto the substrate. The theory relating the thickness of the smear film and the capillary number (a function of speed of the blade, viscosity and surface tension of the sample) as given by Landau-Levich (L Landau and B Levich, Dragging of a liquid by a moving plate, Acta Physicochimica U.R.S.S, Vol XVII, No. 1-2, 1942) is the following:

[0045] Here, R is the radius of curvature of the meniscus, U is the velocity of the blade, p is the viscosity of sample, and c is the surface tension of sample. C is a constant and is equal to 1.34 for the film-drag out problem (Kenneth J Ruschak. Coating flows. Annual Review of Fluid Mechanics, 17(1): 65-89, 1985).

[0046] IN 446889 describes a smearing device operating at constant speed that generates a film with the film thickness varying along the length of the blade. The thickness profile is given by the equation, where x is the distance from starting position of the blade. The total length of the final film, % is obtained by setting, = 0 at %=% and using equation (1) ,

[0047] The surface tension of blood is assumed to be consistent with the reported value of 0.0559 N / m [E Hmcir and J Rosina. Surface tension of blood. Physiol Res, 46(4):319-321, 1997]. To determine the viscosity, we take the logarithm of equation (3). We fit a straight line of slope fixed at -2 / 3. On substituting the values of other parameters in equation (3), the intercept of the equation yields the viscosity.

[0048] Even though previous studies have reported an increase in the viscosity of sickle cell blood when subjected to de-oxygenated conditions and the aforementioned technique can be used to relate the extent of viscosity increase to the concentration of HbS, there is no method known hitherto which can accurately measure the concentration of HbS based on the smear length.

[0049] The said technique is advantageous in developing inexpensive, simple and rapid screening for sickle-cell diseased individuals in resource-limited settings lacking established infrastructure, specialized equipment or trained personnel.

[0050] The present technique can be employed by an automatic smearing device. One such smearing device has been disclosed in Indian Patent no: 446889, which uses an automated motorised shaft and has a compact and sturdy metallic body 1.

[0051] Each set of smears (obtained from 3 to 8 different blade speeds) takes not more than 3 minutes for a reliable viscosity measurement. In Figure 1, a drawing depicts the smearing technique involved. The smear film generated (for a blood drop 3) by a moving blade 1 held at an angle 9 over a substrate 2 has a thickness of Aoo 4 that varies along the length of the smear. The smear length is denoted by Xf and is dependent on the speed of the blade.

[0052] In order to test the feasibility of the method in determining the viscosity of blood samples, smears of blood samples at 8 different speeds were prepared. The smear lengths at the corresponding speeds shorten as the speed increases. The viscosity was extracted by the intercept from equation (3). The same method can be applied in determining the change in viscosity as the deformability in sickled RBC changes upon sickling treatment.

[0053] The sickling tests were conducted using a reducing agent, Sodium metabisulphite. Sodium metabisulphite induces a hypoxic condition by reducing the oxygen in blood thereby inducing polymerization of HbS in red blood cells. The polymerization process hardens cells containing HbS leading to a higher bulk viscosity. Cells containing a high fraction of HbS, transform into a sickle-shape. The present test uses fresh blood sample but it is possible to used stored blood samples also. The purpose is to use minimal amount of blood and mix some amount of SMBS powder to induce sickling. SMBS powder was added to blood to make a 1% SMBS solution in the blood sample and left for 30 minutes for SMBS to deoxygenate the solution. Subsequently, smears were prepared to check for change in smear length, which is indicative of the viscosity change. The smears of the blood samples before and after the sickling treatment were analysed for viscosity measurement.

[0054] Figure 2 shows the drawings of smears prepared before and after the sickling treatment in sickle cell individuals. Figure 2 (a) shows a glass slide 5 with markings denoting a normal range of smear length tail endings. The smearing device is operated at a fixed speed, for example between 10 - 200 mm / s. The smears 6 prepared before sickling result in smear lengths within the normal range. Figure 2 (b) shows a glass slide 7 with similar markings for normal range, however, after sickling treatment, the blood smear of the diseased / trait individuals is shorter 8 on account of increase in viscosity.

[0055] This occurs because of the increased rigidity and therefore lower deformability of the RBCs in the SCA individuals upon treatment with 1% SMBS in blood. The confirmatory test based on the viscosity changes is used to detect the concentration of HbS protein.

[0056] These results show that the concentration of the HbS can be directly determined based on the length of the smear. High concentration of HbS, such as in the sickle-cell anaemia blood sample, would lead to a greater stiffening of the red blood cell in the deoxygenated state. Consequently, stiffer cells lead to a higher viscosity and therefore a significant decrease in smear length. Therefore, the smear technique enables a direct correlation between the change in the smear length upon deoxygenation and the concentration of the HbS in the blood sample. In an embodiment, the present invention provides a smear card having graduations to measure the smear length and the corresponding viscosity (in centipoise, cP). An example of a glass slide with blood smear placed on the SMEAR CARD® is shown in Figure 6(b).

[0057] In another embodiment, the present invention provides a device comprising a substrate (for example a glass slide), a reducing agent and a smear card.

[0058] The present invention enables rapid quantification of HbS levels, which is useful for monitoring individuals who are on treatment or transfusion therapies for sickle cell diseases. Further, it finds application in the field of screening and development of new drugs for the treatment of sickle cell disease.

[0059] Examples

[0060] To demonstrate the accuracy of the screening test, a detailed comparison of viscosity changes in sickle-cell blood with normal donor blood samples was made. Samples from 46 individuals - 6 healthy individuals along with those with sickle-cell trait (20 individuals) and sickle-cell anaemia (20 individuals) were used to measure the change in viscosity upon deoxygenation. The smearing experiments were approved by the Institute Ethics Committee at IIT Bombay. First, normal blood smears prior to sickling treatment were prepared at 5 different speeds and smears lengths were measured and plotted against their corresponding speeds to extract viscosity.

[0061] A similar 1% SMBS treatment for 30-35 minutes was conducted to monitor a change in viscosity based on smear length variation. Figure 3 shows photographs of the blood smears of two sickle-cell individuals before (a) and after (b) sickling. Red dotted markings denote the tail end of the smears up to which width of the smear remains constant. The measurement of smear length begins at the top (below the blood blob) and ends at the red mark. It is clear from the images that upon SMBS treatment, the oxygen deficit condition reduces the length of the smear at a fixed smearing speed.

[0062] Figure 4 shows graphs of smears of two sickle cell individuals (SCT and SCA) relating the speed of the blade with the length of the smear. The graph is based on equation 3 and the slope, when fitted at -2 / 3, gives an intercept which is used to calculate the viscosity. There is a good correlation (R2> 0.94) between the speed of the blade and the smear length.

[0063] It was found that the increase in viscosity in SCT is less than that in SCA, because of the difference in proportion of HbS in the red blood cells. The HbS concentration is higher in SCA individuals compared to SCT individuals, resulting in higher degree of HbS polymerisation, hence, a larger increase in viscosity in sickle-cell anaemia individuals compared to individuals with sicklecell trait.

[0064] The percentage increase in viscosity,

[0065] %— = 100 can be calculated by measuring the smear length before and after deoxygenation, and using equation (3). Here, is the blood viscosity measured in the deoxygenated condition (i.e. after sickling treatment) while 'sthe blood viscosity measured in its original state. A blood sample is considered to be SCT when the percentage increase in viscosity is between 10-40% while the sample is SCA when the percentage increase in viscosity is above 75%. Intermediate viscosity increase, between 40-75%, correspond to samples that have other sickle-cell diseases such as sickle cell P-thalassaemia, sickle -haemoglobin C disease, and mild sickle cell P- thalassaemia. Further tests using HPLC would be needed to diagnose these cases. To distinguish samples of SCA from SCT and normal blood, Figure 5 plots a graph relating the % change in viscosity against the HbS content in sickle cell individuals (both SCT and SCA) and normal blood (0 HbS). The HbS content was determined independently using a commercial, high pressure liquid chromotagraphy (HPLC) machine. The star symbols denote the normal individuals, the diamond symbols denote the trait and the circles denote the sickle-cell anaemia individuals. It is observed that for normal samples, there is little to no change in viscosity upon deoxygenation. However, in both SCT and SCA samples, the percentage change in viscosity is high, with a larger increase observed for SCA blood compared to SCT blood. The concomitant increase in viscosity with HbS content demonstrates that careful measurement of viscosity using the smearing technique can be used to determine the concentration of HbS protein. The method utilises only a few microlitres of blood to reliably measure the change in viscosity upon treatment with a reducing agent that induces sickling in sickle cell blood. The methodology utilizes far less sample volume compared to the HPLC technique to distinguish SCA from SCT and normal blood samples. This methodology is easily implemented on an economical and point-of-care device such as that described in Indian Patent no: 446889 to reliably measure the concentration of HbS protein.

[0066] The table below lists the smear length and the corresponding blood viscosity determined using equation (3) for <5=0.056 N / m, C=1.34, U=109.2 mm / s, Ro=O.38mm and f(0)=2.42.

[0067] Similar tables may be generated for other blade speeds from equation (3). An example of a smear card that allows easy reading of viscosity is shown in Figure 6.

Claims

CLAIMS:

1. A method of screening for sickle cell trait, the method comprising steps of: i) pre-treating a sample with a reducing agent; ii) drawing a smear of the sample by a moving blade at a speed of about 10-200 mm / s; and iii) calculating the concentration of HbS in the sample.

2. A method of screening for sickle cell anaemia, the method comprising steps of: i) pre-treating the sample with a reducing agent; ii) smearing the sample at a speed of about 10-200 mm / s; and iii) calculating the concentration of HbS in the sample.

3. The method as claimed in claim 1 and claim 2, wherein the step of pretreatment comprises treating the sample with a reducing agent for about 30 minutes.

4. The method as claimed in claim 3 wherein the reducing agent is sodium metabisulphite.

5. The method as claimed in claim 1 and claim 2„ wherein the method utilises about 5-20 pl of sample.

6. The method as claimed in claim 1 and claim 2, wherein the drawing of the smear is by a moving blade held at an angle between 20° to 45°.

7. The method as claimed in claim 1 and claim 2, wherein the length of the smear is a function of the concentration of the HbS.

8. A smearing technique employing a smearing device for determining the concentration of HbS in blood, the method comprising the steps of: i) placing a drop of sample on a glass slide at the indicated position; ii) drawing the smear on the slide by a moving blade held at an angle of between 20° to 45° and at a speed of about 10-200 mm / s ; and iii) calculating the concentration of HbS from the smear length.

9. A method to measure the concentration of HbS comprising measuring the length of a smear drawn by a moving blade speed at a speed of about 10-200 mm / s and at a blade angle between 20-45°.

10. A rapid quantification of HbS level for monitoring individuals who are on treatment or transfusion therapies for sickle cell diseases.

11. A smear card having graduations to measure the smear length and the corresponding viscosity (in cP).

12. A device comprising: i. a substrate; ii. a reducing agent; and iii. a smear card.

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