Sickle cell disease is a hemoglobinopathy caused by a point mutation in the ß globin gene that leads to the production of hemoglobin S, which polymerizes under deoxygenated conditions and causes red blood cells (RBCs) to form a sickle shape. ^{11 – 13} Sickle RBCs result in hemolysis, vaso-occlusive pain crises, acute chest syndrome, post-operative infections, congestive heart failure, cerebrovascular accident and endothelial damage. ¹⁴ Of all the screening tests used for screening cases of SCD, the sickling test is the most commonly used. However, these basic screening tests are plagued by a lot of disadvantages which may range from technical to human errors. The changes in the edge, location, shape, and size of the cells make blood film analysis extremely difficult. A computerized approach has been created as a result to provide a simple technique for identifying the type of anemia. The peripheral blood smear is an imperfect test, It is a formative hematological test that depends on the pathologist’s skills, and there aren’t many skilled pathologists to choose from. Haemoglobin electrophoresis, on the other hand, does not suffer from all these disadvantages and gives accurate results. Hemoglobin electrophoresis is the gold standard for the diagnosis of SCD. The routine protocol followed in the diagnosis of SCD is to do a screening test first and then go for a confirmatory test thus delaying the final diagnosis. The present study aims to employ both these tests for the screening cases of SCD and reliability with each other. Employing a superior diagnostic test will greatly reduce delays in diagnosing cases of SCD to a considerable extent. ¹⁵

Recently, many studies have been done on techniques for the detection of SCD. Wjdan A. Arish et al.in (2021) studied the various prospective approaches that could be used to aid in the early identification of SCD, as well as the present and new methods for sickle cell disease detection. ¹⁶

Emmanuel Gbenga Dada et al.’s (2022) study reported that traditional methods for diagnosing SCD are expensive, error-prone, time-consuming, and necessitate the help of medical professionals. The development of affordable and controllable methods for the early identification and diagnosis of SCD is thus required immediately. ¹⁷

To evaluate the reliability of the sickling test and peripheral blood film method for screening of sickle cell disease.

1.

To evaluate the sensitivity and specificity of the sickling test, peripheral blood smear, and hemoglobin electrophoresis for SCD screening.

This will be a descriptive, laboratory-based, observational and comparable study.

Ethical approval for the study was obtained from the institutional ethics committee of Datta Meghe Institute of Higher Education and Research (Deemed to be University) (ref. No: DMIHER (DU)/IEC/2023/24), date of approval: 21/07/2023. A written participant information sheet will be given regarding the details of the study, and it will be explained to participants before enrolment in the study. Their involvement benefits and harms will be explained to the participants. Written informed consent from the participants will be obtained before involving them in the study.

The following study will be conducted in the Department of Pathology (Central Clinical Laboratory), AVB Rural Hospital, Sawangi Meghe (Wardha), India.

Inclusion criteria 1.

Patients aged 6 months – 60 years who present with anemia.

Exclusion criteria 1.

Patients aged <6 months ad >60 years.

Potential participants will be selected on the basis of their chief complaints such as clinical anemia, any bone pain, family history along with complete blood count report and peripheral blood examinations suggesting hemolytic anemia. After that, we will do the sickling test and the correlation with peripheral blood examination will be done. If the sickling test is positive, then we will do Hb electrophoresis which will confirm type of hemolytic anemia on the basis of presence of abnormal hemoglobin such as HbS. This will further confirm the type of hemolytic anemia as sickle cell anemia (SCA). Informed consent will be obtained prior to participation in the study. The patient will be comfortably seated and a blood sample will be taken under aseptic precautions. 5ml blood will be drawn into a dipotassium ethylenediaminetetraacetic acid (K2EDTA) tube through a standard central cubital vein, for testing of SCD.

All patients will be examined further for 1.

Sickling test

Sickling test

The sickling test’s basic method depends on microscopic observations of red blood cells (RBC) that are exposed to low oxygen tension or start to swell. An equal volume of EDTA blood will be mixed with 2% sodium metabisulfite. A drop of blood and a mixture of reagents will be taken on a glass slide. A cover slip will be placed over it, and the four corners will be sealed with wax and the slide will be observed under a microscope for presence of any sickle shaped red blood cells if any (40x) after 2, 4, and 24 hrs.

Peripheral blood smear

The peripheral blood smear (PBF) examines the blood cell’s morphology and evaluates any microscopic alterations, which can offer useful information that helps in the diagnosis of SCD.

The method is as follows: the smear will form a thin film of blood. Allow it to air dry, and then place it in a staining rack. Leishman’s stain will be placed dropwise on the slide and left for 2 minutes. This allows the determination of PBF in methyl alcohol. Add a double volume of buffered water dropwise to the slide and let stand for 8 minutes and then rinse in water for 1 to 2 minutes. Air dry and examine under the oil immersion lens of a microscope.

Hemoglobin electrophoresis

Equal parts of distilled water and packed cells will be combined and then the mixture will be centrifuged. As a sample, this hemolysate will be used. Medium 2% agarose gel pH 8.6 with Tris/EDTA/borate (TEB) buffer will be used. The basic idea behind this method is based on the fact that proteins can be positively or negatively charged based on the amino acids that are normally charged.

The positively charged proteins will move to the cathode when an electric field is applied to a solution containing protein molecules, and vice versa. Different hemoglobin will separate and migrate at different rates depending on their charges, size, and shape. Following this, bands are stained and their color concentration is scanned in comparison to known controls.

All necessary measures to control bias at all levels will be taken. As it is an observational study, all the samples which are routinely received in the section of hematology will be taken into consideration for comparison between the Hb electrophoresis technique analogous to the routine sickling test. Institutional supply of EDTA samples and chemical constituents present in a buffer from different companies make minor modifications, essential part of Hb electrophoresis protocol. Diagnostic search influences the finding of electrophoresis sickling band.

Daniel’s formula for sample size n = Z 2 p 1 − p d 2

Where,

Z∝2 statistics for a level of confidence (for the level of confidence of 95%, which is conventional, the Z value is 1.96) = 1.960

p = prevalence of sickle cell anemia = 5 % = 0.05

D = desired error of margin = 6% = 0.06

n is the no. of patients is 72.99 n = 1.96 2 ∗ 0.05 ∗ 1 − 0.05 0.05 2

n = 72.99

= 75 patients needed in the study .

Formula reference: Daniel et al. (1977). ¹⁸

Primary outcomes

We will observe any sickle shaped red blood cells under the microscope which can be easily seen in case of sickle cell disease (SCD) patients, but gold standard for diagnosis of SCD is always hemoglobin electrophoresis which shows the percentage of HbS hemoglobin on its report.

Expected outcome

To evaluate the sensitivity and specificity of the sickling test, peripheral blood smear, and hemoglobin electrophoresis for SCD screening.

SPSS version 23.0 will be used to conduct the statistical analysis. A p value of 0.05 will be regarded as significant. The mean distribution of inhibition zones will be documented as part of the descriptive analysis. Patient and repeated measures will be analysed using ANOVA.

The results will be published in an indexed journal.

The study is set to start in September 2023.

False-positive results occur when the test indicates the presence of SCD when it is not actually present, leading to unnecessary concern and follow-up testing. False-negative results occur when the test fails to identify individuals with SCD, delaying necessary interventions and treatment.

The reliability of the sickling test and peripheral blood film method may vary with the age of the individual being tested. Some infants with SCD may not show symptoms or positive test results immediately after birth, leading to potential delays in diagnosis. The accuracy of both methods is highly dependent on the technical expertise of the personnel performing the tests. Improper sample collection, preparation, or interpretation can lead to inaccurate results. Distinguishing between sickle cell trait (SCT) and SCD is essential, as individuals with SCT are carriers of the gene but typically do not experience symptoms or complications. Both screening methods may not always differentiate between SCT and SCD.

The quality of the blood sample used for testing can impact the accuracy of the results. Factors such as hemolysis or contamination can affect the reliability of the tests.

Sickling method confirms abnormal hemoglobin presence, while peripheral blood smear reveals sickle-shaped red blood cells, aiding the diagnosis of sickle cell disease and assessing disease severity.

Peripheral smear and sickling evaluation offer a reliable and widely applicable method for diagnosing hemoglobin disorders in the general population.