Assessment and comparison of the sensitivity and specificity of sickling and haemoglobin electrophoresis in haemolytic anaemia patients: A study protocol

Introduction

Hereditary causes of haemolytic anaemia include defects in haemoglobin (Hb) protein chains, leading to a reduced production rate of one or more of the beta-globin chain of Hb. Almost 200 million people, or 3% of the world’s population, are thought to be affected, in addition to the about 150,000 affected people born per year.¹

Sickle cell anaemia caused due to mutation of Hb beta chains. These cells transform the typical biconcave disc shape to a crescent, holly wreath, and other forms when the oxygen pressure is reduced. This process is known as sickling.²

The pathophysiology of sickle cell disease (SCD) depends on haemoglobin sickle (HbS) polymerization, which is caused by a reduced oxygen affinity. The formation of permanently sickle cells results from repeated polymerization and sickling of cells. Haemolytic anaemia is the outcome of this, which accelerates cell death and shortens the lifespan of cells by greater than 75%.³

The heterogeneous presentation of sickle cell disease has been classified into trait (normal haemoglobin and reticulocytes, unconjugated bilirubin<1 mg/dL), mild (Hb>11 g/dL, reticulocytes 3–6%, unconjugated bilirubin 1–2 mg/dL), moderate (Hb 8–12 g/dL, reticulocytes 6–10%, unconjugated bilirubin>2 mg/dL) and severe (Hb>8 g/dL, reticulocytes>10%, unconjugated bilirubin>3 mg/dL). In such a case, iron deficiency is seen as uncommon, and clinical practice typically avoids prescribing iron supplements to these patients. Worldwide, anaemia due to primarily caused by iron deficiency, particularly in children and adult women.⁴

In the reticuloendothelial system, haemolysis may occur intravascularly or extravascularly, or both. Poor deformability, which leads to capture and phagocytosis, antibody-mediated destruction through phagocytosis, or direct complement activation, fragmentation caused by the microthrombi, physical injury, oxidation, or cellular apoptosis are a few mechanisms of haemolysis. Acute anaemia, jaundice, haematuria, dyspnoea, tiredness, tachycardia, and potentially hypotension are all possible symptoms of haemolysis in patients. Laboratory test results indicate haemolysis when reticulocytosis, increased lactate dehydrogenase (LDH), increased unconjugated bilirubin and decreased haptoglobin levels are present.⁵

Youderian et al. noted that several proteins exist whose amino acid composition is entirely known, whose three-dimensional structure is known or determined by crystallography and for which a significant number of known amino acid substitutions are available for investigation. The vast amount of electrophoretic data currently available from surveys using the same electrophoretic approach may be calibrated using the findings.⁶

Peripheral blood smear helps diagnose haemolytic anaemia. Peripheral smear cells show polychromatic neutrophils, micro spherocytes, high reticulocyte count, nucleated and fragmented RBC.⁷ Identifying “subclinical” haemolysis early is crucial since it can progress abruptly and quickly.⁸ The early effects in haemolytic anaemia patients are protein deficiency and dysfunction of the muscle skeleton of protein is seen in sickling tests.⁹ In a previous study, the sodium dithionite, sodium metabisulfite and moist seal methods were employed. The tests were repeated on specimens harmful to sickling.¹⁰

When separating abnormal haemoglobins, cellulose acetate is a particularly good medium for routine use.¹¹ The most prevalent haemolytic disorder includes HbS, foetal haemoglobin (HbF), and sickle cell trait (HbAS) which is not a haemolytic disorder but its carrier forms found via Hb electrophoresis; these are crucial for family counselling, which aims to address physiological support, behavioural and emotional issues associated with the condition.¹² Hb electrophoresis is a special technique used to identify certain haemolytic anaemias due to thalassemia syndromes and abnormal structural HbS.¹³

Ali et al.,¹ in 2011, conducted a study on Hb electrophoresis to diagnose hemoglobinopathies in hypochromic, microcytic, and sickle cell blood films. The study used Hb electrophoresis to analyse 80 blood samples from patients with suspected hemoglobinopathies. Seven participants (8.8%) had SCD (HbS), eight (10%) had sickle cell minor carrier status (HbAS), and 12 (15%) had sickle cell major (HbF) and thalassaemic significant (HbAF) types, respectively. Type 7 (8.8%) haemoglobin (HbA2) indicates mild thalassemia. However, 22 patients also had Hb electrophoresis results that were normal. Hb gel electrophoresis is an easy and suitable technique with alkaline pH (4.8 to 6.8) for the study of the inherited hemoglobinopathies.

Maroufa et al.,¹³ in 2002, conducted research on hemoglobinopathies and Hb electrophoresis in Kuwait. The research comprised 2386 Hb electrophoresis tests performed on persons of various ages. The study found that 561 patients had abnormal Hb genes. Patients with haematological and clinical characteristics suggestive of hemoglobinopathies or those with a positive family history should be the only ones eligible for the test.

Kohn conducted a study on the HbS separate on cellulose acetate paper. The study applying a continuous current of 0-2 to 3 milli ampere/cm width established a bridge gap of 5 to 6 cm and a potential gradient of 200 to 240 V. Depending on the type of separation needed, the electrophoretic run time ranges from 30 to 90 minutes; for HbF, for example, it takes around 90 minutes. However, exploratory studies using marker HbS are the most effective way to determine the ideal circumstances. On standard laboratory equipment, “high voltage electrophoresis” conditions, such as 100 V/cm, can be easily reached by delivering 300 V over a 3 cm bridge gap. With this method, a completely adequate separation can be attained in eight to 15 minutes. The small opening prevents nearly all overheating, which results in an abundant supply of buffer, a relatively short running period, and these factors. Iterative electrophoresis is used to separate the applied samples until it is successful. The ideal way to monitor the electrophoretic run is to observe the movement of the heterozygous marker haemoglobins, such as AS or AC. The cellulose acetate paper is dried or fixed when the separation is finished, much like with regular protein electrophoresis.¹¹

Protocol

Results

Appropriate results will be obtained after the sensitivity, specificity, positive predictive value and negative predictive value of sickling test and haemoglobin electrophoresis are compared.

Discussion

Haemolytic anaemia is the premature destruction of RBC while paroxysmal nocturnal haemoglobinuria (PNH) is chronic and life threatening. Most cases take place in less developed countries where there are no reliable testing campaigns available. Some of the risk factors include acquired immunodeficiency syndrome, acute anaemia, jaundice, hematuria, dyspnea, tiredness, tachycardia, and potentially hypotension. Physical examination of blood count and peripheral blood analyses will be carried out in all cases. Haemoglobin electrophoresis is a confirmative test of haemolytic anaemia and has higher sensitivity and specificity than screening test for sickling. According to the outcomes of randomized clinical research, blood transfusion and nutrition (calories, total fat, saturated fat, trans fat, cholesterol, sodium (salt), total carbohydrate) are considered standard care for haemolytic anaemia patients.

In 2020, Bee et al. researched iron deficiency anaemia, and found abnormal haemoglobin disorder was the most common cause of microcytic hypochromic anaemia. It is possible to make a differentiating diagnosis with a full hemogram and a peripheral smear. Still, other tests, such as a serum iron profile and Hb electrophoresis, are necessary to make the diagnosis. The structural and functional abnormalities of Hb, or hemoglobinopathies, are a significant global health issue. Thalassemia and sickle cell anaemia are examples of these single-gene, autosomal, recessive monogenic illnesses. Microcytic hypochromic anaemia is a symptom of hemoglobinopathies. Hemoglobinopathies, which do not require iron, are often misdiagnosed and treated as iron deficiency anaemia. The body becomes poisonous when exposed to excess iron that is not needed. Microcytic hypochromic anaemia can be divided into various subtypes using electrophoresis. Correct diagnosis is made with the aid of electrophoresis.¹⁷

In 2006, Huang et al. undertook research T-antigen activation to predict haemolytic anaemia and pneumococcus-induced haemolytic uremic syndrome. The outcome for positive TA initiation for P-HA was 57% explicit and 86% sensitive. Overall, 76% of expectations were exact. There was no intergroup struggle contrast in the pneumococcal serotype 14 that causes P-HUS most often (5/10) and is sensitive to anti-microbial. A total of 13 of the 36 patients needed packed RBC transfusions, three passed away, and two needed extracorporeal membrane oxygenation. Three of the patients also required transfusions. None experienced long-haul kidney impacts.¹⁸

In 2019, Garden observed the value of immune-mediated haemolytic anaemia diagnostic tests. The study compared saline and coombs test, which can be utilised to help to determine immune mediated haemolytic anaemia when examined.¹⁹

In 2016, Englum et al.’s results suggested that total splenectomy may require more serious consideration in children with the most severe haemolytic diseases. Future studies must look at these results in the context of the clinical advantages for children with various conditions. A total of 130 children were included in the analysis, and 81 of them (62.3%) had total splenectomy. All hematologic measurements in children with HS improved following TS, including an increase in Hb of 4.1 g/dl. Although the reaction was less strong, hematologic indicators similarly improved after peripheral smear (haemoglobin increase of 2.4 g/dL, p 0.001). In children with SCD, Hb levels were unchanged. Laparoscopy did not affect hematologic outcomes when compared to open surgery. Shorter lengths of stay were associated with total splenectomy and laparoscopy.²⁰

In 2015, Sipol et al. conducted a study on the correlation among iterative labs for the distinguishing proof of paroxysmal nocturnal haemoglobinuria (PNH) copies utilizing high-responsiveness bloom cytometry in a Russian cohort. Seven PNH patients, five of whom were female and two of whom were male, had their copy sizes measured. Mean patient age was 37 years); three patients (one with aplastic weakness and two without it) had extreme haemolytic PNH and high plasma lactate dehydrogenase. Five of the patients had a history of aplastic frailty. PNH copy sizes at discovery ranged from 0.41 to 9.7% of granulocytes in patients with milder side effects, to 58 to almost 100% in patients with severe side effects. There were only negligible contrasts in the subsequent copy size estimation for every patient between the six research centres, especially in those with high qualities at determination.²¹