Sickle cell disease is a hereditary blood disorder with an autosomal recessive inheritance pattern and leads to abnormal shaped red blood cells. This disease is associated with chronic hemolytic anemia and various complications secondary to Vaso-occlusive phenomena. The molecular change in sickle cell disease (SCD) is base substitution of valine for glutamate at the sixth position of beta globulin gene leading to production of abnormal hemoglobin S.

First described in 1910 by Sir James Herrick, peculiar elongated sickle-shaped red cells in a severely anemic medical student from Grenada. Linus Pauling and his colleagues showed that sickle hemoglobin (HbS) had altered electrophoretic mobility and they were the first to define it as a molecular disease in 1949. A few years later in 1957, Vernon Ingram discovered that sickle hemoglobin resulted from a single amino acid substitution in the hemoglobin molecule. The disease results from a single base Adenine>Thiamine mutation in the triplet encoding the sixth residue of the β-globin chain, leading to a substitution of valine for glutamic acid and formation of abnormal hemoglobin S (HbS).

In sickle cell anemia, once the hemoglobin S form is deoxygenated it has a tendency to polymerize that causes changes in red cell membrane structure and function leading to sickling of red cells. The other mechanisms that play a role independent of polymerization are on vascular endothelial factors and environmental factors including dehydration, and hypoxemia.

Normal red blood cells have a life expectancy of 110-120 days. However, the red cells in SCD are destroyed at higher rates and have a life expectancy of 15-20 days. Clinical presentation of sickle cell disease is variable, with some patients having a normal life; however, some patients show increased morbidity and mortality due to Vaso-occlusive, severe thrombotic, aplastic and sequestration crisis.

 In developed countries, the life expectancy of SCD patients has been improved by early diagnosis, comprehensive treatment, and general medical care. Therefore, early detection supports the effective management of the disease. Detection of hemoglobin S and diagnosis of sickle cell disease depend mainly on the clinical laboratory, where a combination of biochemical and molecular tests is used in the detection and confirmation of the diagnosis. The most popular methods for detecting these diseases are the Hb electrophoresis, and high-performance liquid chromatography (HPLC). However, there are other affordable methods also available with of varying reliability, ease of applicability and cost effective for early screening of SCD in low income countries. Solubility test is one of the screening methods use to screen sickling, easily applicable and cost effective. So, in this study we will analyse sensitivity 

The present study was carried out in a tertiary care hospital, P.D.U Medical College and Hospital, Rajkot, Gujarat, Western India. Our study population consists primarily of tribal population from Central Gujarat and Southern Madhya Pradesh mostly. A total of 2500 patients suspected to have sickle cell hemoglobinopathies were examined for sickling screening test. All positive cases were evaluated by HPLC to separate constituent haemoglobins. The study is conducted in patients with six months period data from January 2025 to June 2025. We used 2 ml of venous blood sample collected in EDTA vacuette tube for analysis.

SOLUBILITY TEST

The principle of solubility method was based on solubility difference between sickle haemoglobin (HbS) and adult haemoglobin (HbA) in concentrated phosphate buffer solution. A volume of 2 ml of 0.02% sodium dithionite working phosphate buffer was taken in a test tube and 20μl of blood sample was added to it. The test tube was kept for 5 min before reading the test. A piece of white paper was taken and dark bold black lines were drawn on it for interpretation of the test. The test is read as positive, if turbidity is present. To confirm turbidity the paper with bold black line is kept at a distance of 1 inch from the test tube against a bright light. If turbidity is present, it impairs the visibility of dark bold lines. A negative test is indicated by a clear pinkish-violet solution. Then mixed solution of reagent and blood sample (100 µL in this case) was subjected to centrifugation at 1200 rpm for 5 minutes. Precipitated haemoglobin (HbS) forms a red precipitate on top layer leaving the lower solution clear and colourless. The soluble haemoglobin (HbA) gives a clear red lower solution with a red precipitate ring on top layer with a light to pink colour lower solution.

High-Performance Liquid Chromatography (HPLC):

 The machine used for estimating HPLC was Bio-Rad ‘Variant II’ (beta thalassemia short program) Program analyzer which is an automated cation exchange HPLC instrument. It operates on the principle of HPLC and the column comprises a small cation exchange cartridge, with a requirement of only 2ml of the blood sample, and each sample taking only 6.5 minutes for analysis. The samples are injected into the analysis stream and separated by the cation exchange cartridge using a phosphate ion gradient generated by mixing 2 buffers of different ionic strengths to elute the different hemoglobins. It is based on the time required for gradient elution of the different hemoglobin fractions. This is called the retention time (RT). The data is processed and the separated hemoglobin fractions were analysed based on their retention times, and the software provided a printed report with chromatograms showing the hemoglobin fractions eluted.

The printed chromatogram of HPLC shows all the hemoglobin fractions eluted, the retention times, the areas of the peaks and the values (%) of different hemoglobin components.

Table 1: Proportion of Different Haemoglobins in Normal individuals and in Haemoglobin disorders.

Table 1 shows reference ranges in which common variants have been observed to elute using the extended program.

Table 2: Proportion of Different Haemoglobins in Normal Individuals according to Retention time in minutes

A total of 2500 blood samples were collected, over a period of 6 months from January 2025 to June 2025. Of these 2500 samples, 251 were positive for solubility test [Table 3].

Table 3: Month-wise distribution of collection of samples and solubility test positivity from total samples collected

Table 4: The summary of the Haemoglobin AS, HbS-beta Thalassemia, SS detected by the sickling solubility test and by High Performance Liquid Chromatography (HPLC)

Table 5:  Comparison of Solubility Test as a screening method against HPLC

Table-6: HPLC Interpretation with the distribution of the total number of cases percentage-wise.

More than 50% of the total global sickle cell anaemia (SCA) cases are in India. It was found that sickle cell gene is widely spread in southern districts of Gujarat, heavily influenced by tribal communities in southern districts. The state’s proactive measures—including widespread screening is critical to addressing this burden.

The combined use of the solubility test and high-performance liquid chromatography (HPLC) for detecting sickle cell disorder (SCD) offers a balanced approach of cost-effectiveness and diagnostic accuracy, particularly in tertiary care settings. The solubility test, which is rapid and inexpensive, serves as an initial screening tool by detecting the presence of sickle hemoglobin (HbS) based on its reduced solubility in a deoxygenated state. However, while it is sensitive in identifying HbS, it lacks specificity and cannot distinguish between sickle cell trait (HbAS) and sickle cell disease (HbSS), nor can it detect other hemoglobinopathies.

To overcome these limitations, HPLC is employed as a confirmatory test. HPLC is a highly reliable and sensitive method that enables precise quantification and differentiation of various hemoglobin fractions, including HbS, HbA, HbF, and HbA2. In tertiary care centers where comprehensive diagnostic services are essential, HPLC adds significant value by confirming the diagnosis, detecting compound heterozygous conditions, and facilitating accurate phenotype classification.

Sickle solubility tests identify hemoglobin S with high sensitivity and specificity. However, False-negatives are seen in patients with Coinheritance of alpha-thalassemia trait, hereditary persistence of fetal hemoglobin, with severe anemia or in patients with a hemoglobin S fraction <10%.

Besides the solubility test could lead to high false positive rate, which is suggestive of low diagnostic accuracy of test. In a similar type of study conducted by Robert et al.  noted that factors such as Erythrocytosis, highly marked Leucocytosis and Hyperlipidemia were possibly be linked to false positivity by this method.

In present study, the sensitivity and specificity of the solubility test were 95.4% and 96.4% respectively for the samples. Solubility test cannot differentiate between Sickle cell trait and Sickle cell disease hence, confirmation is needed by HPLC.

Solubility test is reliable for mass screening, because it is rapid (takes just about 5 min), reliable with minimal observer variation, does not need any microscope and requires very small blood sample. It is also a cost-effective test used widely for screening test in tertiary care centres in Gujarat. The sensitivity is 95.4% while specificity is 96.4%, whereas the Sickling test is cumbersome and time consuming and, thus, it is inconvenient for large sample screening. Also, improper sealing by coverslip may lead to drying of sample especially in hot season. Sickle solubility test would therefore be the recommended screening test, using HPLC as confirmatory method.

1. Herrick JB. Peculiar elongated and sickle-shaped red blood corpuscles in a case of severe anemia. Arch Intern Med. 1910;VI(5)517-521. doi: [Article:https://doi.org/10.1001/archinte.1910.00050 330050003]
2. Serjeant GR, Serjeant BE. Sickle cell disease. 3rd ed, Oxford Univ Press. 2001.
3. Frank F, Chesterman C, Penington D, Rush B. de Gruchy’s Clinical Haematology in Medical Practice, 5th edition. Oxford University Press. 1990;171–87.
4. Stuart MJ, Nagel RL. Sickle cell disease. Lancet. 2004;364(9442)1343-1360. doi: [Article:https://doi.org/10.1016/s0140 6736(04)17192-4]
5. Rees DC, Williams TM, Gladwin MT. Sickle cell disease. Lancet. 2010;376(9757)2018-2031. doi: [Article:https://doi.org/10.1016/s0140 6736(10)61029-x]
6. Bunn HF. Pathogenesis and treatment of sickle cell disease. N Engl J Med. 1997;337(11)762 769. doi: [Article:https://doi.org/10.1056/nejm199709113371 107]
7. Odièvre MH, Verger E, Silva-Pinto AC, Elion J. Pathophysiological insights in sickle cell disease. Indian J Med Res. 2011;134;532-537.
8. Kumar Vinay, Abbas KA, Aster CJ. Red blood cell and Bleeding disorders, In- Kumar, Abbas, Aster, Robbins and Cotran Pathologic Basis of Disease. Haryana: Elsevier, Thomson Press India Ltd, 9th ed. 2014; p-629-39.
9. Surve RR, Mukherjee MB, Kate SL, Nagtilak SB, Wadia M, Tamankar AA, et al. Detection of the beta S gene- an evaluation of the solubility test against automated chromatography and haemoglobin electrophoresis. Br J Biomed Sci. 2000;57(4)292-294.
10. Robert, M.D. Schmidt, M. W. Sylvia. Standardization in detection of abnormal hemoglobins. Solubility tests for hemoglobins.S.JAMA,225(1973),1225-1230
11. Kawthalkar SM. Anaemias due to Excessive Red Cell Destruction. In: Essentials of Hematology. 1st edition. New Delhi: JAYPEE; 2006; 136–137.