Assessing the efficacy of hemoglobin electrophoresis as a diagnostic tool in moderate to severe anemia: a study protocol

Introduction

The branch of medicine known as haematology deals with a variety of physiological, pathological, clinical, and disorders that impact the platelets, white blood cells, and red blood cells that make up blood. The most prevalent haematological disorder, anaemia is defined as a reduction in haemoglobin or red blood cell (RBC) as a result of several causes. Anaemia in varied degrees can have a variety of clinical repercussions.¹

The essential oxygen-transporting metaloprotein found in erythrocytes is called haemoglobin (Hb). An iron-containing porphyrin ring, often known as a heme group and a tetramer of the globin polypeptide chain make up the two primary parts of the haemoglobin molecule. The globin chains protect and make the heme soluble while the heme serves as an oxygen-carrying group. Porphyrias are uncommon inherited abnormalities of the porphyrin ring^1,2

In developing countries, anaemia is the most prevalent issue among the rising age group. Iron deficiency anaemia (IDA), which has detrimental effects on health, especially in children and adolescents, results from a decrease in haemoglobin (Hb) concentration in the blood when iron deficiency is sufficiently severe. The different forms of haemoglobins present are measured using a procedure called haemoglobin electrophoresis.²

Two dimers of globin chains that are similar to one another make up the globin tetramer. The - and -chains, which are encoded on chromosome 16, are the globins that resemble globins. The -, and -chains, which are encoded on chromosome 11, are the globins that resemble globins. The embryonic Hbs, which are present in healthy embryos and foetuses less than three months gestation, are made up of the - and -chains. Hb F (22) predominates during foetal life. The correct maternal-fetal oxygen exchange in pregnancy depends on foetal red blood cells (RBCs) with Hb F having a higher oxygen affinity than adult RBCs. Before 20 weeks of pregnancy, adult Hb, Hb A (22), is only minimally created; production starts as soon as -chain synthesis is initiated.^2,3

Hb A makes about 15–40% of the total Hb at birth. At six months of age, Hb F makes up about 8% of the total Hb due to the ongoing increase in the ratio of Hb A to Hb F. Adults typically have 97% Hb A, 1% Hb F, and 1.5-3.5% Hb A levels. Haemoglobin (Hb) is a crucial metaloprotein that transports oxygen and is present in erythrocytes. The two main components of the haemoglobin molecule are a tetramer of the globin polypeptide chain and an iron-containing porphyrin ring, commonly referred to as a heme group. Heme functions as an oxygen-carrying group and is shielded and made soluble by the globin chains. Rare inherited anomalies of the porphyrin ring are known as porphyrias.³

The most common condition affecting children in underdeveloped nations is anaemia. Over the past few decades, the diagnosis of haemoglobin disorders has changed, moving from being predominately driven by symptomatic patients, aberrant lab results, or peripheral blood smears, to the present emphasis on universal screening in the majority of developed healthcare systems. This evolution has kept pace with our growing understanding of these illnesses and the improvement of diagnostic techniques.^3,4

Dr. Linus Pauling made the first molecular analysis of disease in 1949 by connecting sickle cell disease to a molecular abnormality. Since his groundbreaking study, more than 400 thalassemias and 1,000 Hb variations have been discovered. Haemoglobin (Hb), hematocrit (HCT), or RBC count reductions are all indicators of anaemia. It can be classified as macrocytic, microcytic, or normocytic and is a manifestation of an underlying ailment. Patients with anaemia often exhibit nebulous symptoms such lethargicness, weakness, and fatigue. Syncope, shortness of breath, and a decreased ability to tolerate physical activity are symptoms of severe anaemia. This exercise describes anaemia assessment and management, as well as the function of the interprofessional team in treating individuals with this condition. Over 2 billion individuals worldwide suffer from anaemia, which is the most prevalent haematological condition and a significant health burden. It is a symptom of a problem that is underlying and is indicated by a decrease in the percentage of red blood cells (RBCs). Anaemia can be caused by a wide range of conditions, including nutritional deficiencies, chronic illnesses, inflammatory conditions, medicines, cancer, renal dysfunction, genetic diseases, and bone marrow problems. Although cation-exchange HPLC is rapidly becoming the method of choice for the identification and quantification of Hb variants, Hb electrophoresis has been used since the 1960s and continues to be a viable tool for diagnosing Hb abnormalities. The Bio-Rad Variant (Bio-Rad Laboratories) is the most widely used commercial cation-exchange HPLC system. By using cellulose acetate and zone electrophoresis at an alkaline pH, the naturally occurring Hb A and Hb A2 are separated from the often found variations, Hb S, Hb F, and Hb C. Using citrate agar electrophoresis, the co-migrating Hbs (Hb S from Hb D and Hb G. Densitometric scanning is used to quantify Hb in electrophoresis, however it can be unreliable when variations are present in low concentrations. Isoelectric focusing (IEF), a relatively recent technique for high-resolution electrophoresis, divides haemoglobin variations according to their electric charge in a pH gradient produced by carrier ampholytes present in the medium. Capillary electrophoresis, a technique that uses haemoglobin electrophoresis in a negatively charged capillary system, has also gained favour recently.⁴

Another significant factor contributing to anaemia globally is infection-related anaemia, which is thought to account for around 12% of all cases in 2019-12 and is closely correlated with the geographic distribution of infection. Infections can affect how nutrients are metabolised and absorbed, or they might result in nutritional loss. The immune-driven anaemia known as anaemia of inflammation can also be brought on by prolonged illness and inflammation. Pro-inflammatory cytokines boost the production of the hormone hepcidin, which regulates iron absorption. Hepcidin then causes iron to be sequestered as ferritin, preventing iron from being transferred to erythrocyte precursor cells in the bone marrow and reducing erythropoiesis as well as erythrocyte survival.^4,5

A blood test called a haemoglobin electrophoresis is performed to assess and distinguish between the various forms of haemoglobin in the bloodstream. Disease diagnosis is no exception to the automation that already permeates nearly every part of life, including medicine. It is now easier, quicker, and less expensive to diagnose diseases because to computer-aided disease diagnosis technologies. By saving time and effort, they make decision-making easier. A further advantage of automated diagnosis is that it is less susceptible to variations in physician preferences. Computers may also be useful in the detection and forecasting of anemia. It is estimated that nearly every second child under the age of five has anaemia, which is a serious ailment that primarily affects women and children. Although there are several procedures, the Hb electrophoresis test technique is straightforward. In a nutshell, haemoglobin is released from red blood cells and dissolved into a solution. This lysate is then used to perform electrophoresis on a suitable electrophoresis medium, such as a cellulose acetate strip. The different Hb variants move across different distances because of differences in ionic charge. Blood samples from many patients (up to eight patients) are included in the Hb electrophoresis test photographs on a single cellulose sheet.⁶

Methods

The following study is a diagnostic investigative study that will be conducted in the hematology division of the Dept. of Pathology, Jawaharlal Nehru Medical College (JNMC), Sawangi (Meghe), Wardha, in collaboration with the Acharya Vinoba Bhave Rural Hospital (AVBRH), Sawangi (Meghe), Wardha, from 2022 to 2024.

Discussion

R Hafeez et al.(2022) did a study to concluded that the most frequent causes of severe anaemia in local kids were found to be 89 cases of malaria, 37 cases of septicemia, 7 cases of sickle cell anaemia, and 232 cases of iron deficiency (63.6%%). It must be remembered that these factors may eventually lead to severe anaemia because anaemia is a global health issue and can negatively affect children’s cognitive and physical development. To reduce the risk of severe anaemia in these kids, preventative and therapeutic measures must be taken.⁷ S Krishnamurthy et al.(2022) did a study to concluded that identifying anaemia in the elderly is important, as is classifying the kind and severity of the anaemia, looking for the underlying reason, and then treating the aetiology that has been found. In senior people with iron deficiency anaemia, it’s crucial to convince them of this and test their GIT with bilateral scopes. Despite having received the best workup possible, some individuals may appear with anaemia that is undiagnosed or without a known cause. It is satisfying to notice that we can greatly contribute to the general improvement in the quality of life of the elderly and their ability to carry out their daily activities wherever a reversible cause of anaemia is found and treated. Although natural and inevitable, remaining healthy as you age is a fundamental human right.⁸ LM Rogers et al. (2023) did a study to concluded that Nutritional, physiological, social, and environmental factors should all be taken into account when determining anemia’s occurrence. In order to address and combat anaemia and the related cause(s), a diagnosis of anaemia and the determination of its causes are necessary. The optimum method for determining haemoglobin (Hb) is to use venous blood that has undergone automated haematology analysis and undergone high-quality control procedures. Actions toward anemia diagnosis and control at public health levels will require global, regional, and country actions, It should include both general and context-specific qualities. New instruments to diagnose anaemia and its determinants in a single, more cost, practical, and quick test could be developed as a result of research into the biology, epidemiology, diagnosis, and treatment of anaemia. The introduction of sandwich ELISAs and other technologies that can assess numerous markers simultaneously has made it easier to identify anaemia risk factors, while commutability studies are still needed. For other indicators to assess causes of anemia, relatively large volumes of blood are needed, the costs are higher, and specialized laboratories are required. The WHO Comprehensive framework for integrated action on the prevention, diagnosis, and management of anaemia seeks to provide strategic, effective, and implementable actions to reduce anaemia, accelerate progress towards the global target on anaemia, and optimise health and well-being. Establishing a correct diagnosis of anaemia and its causes would also support this framework. Stakeholders must comprehend and agree that a multispectral, coordinated response is necessary for anaemia prevention and management.⁹ AK Shrestha et al.(2022) did a study to concluded that Haemoglobin Hb J is a rare variety, and research on it is still going strong and quickly; 71 new variants have been discovered so far. Although the Hb J is typically clinically asymptomatic, hemolysis and severe anaemia may occasionally be present. Where Hb J manifests as a P3 peak, CE-HPLC is the gold standard for differentiating these haemoglobin types. When Hb J is present, anaemia as such should cause a search for a coexisting hemoglobinopathy or the presence of an unstable form. When analysing differentials for any case of anaemia, this case prompts us to consider Hb J as a probable cause.¹⁰ T Milovanovic et al.(2021) did a study to concluded that GPs are a patient’s initial point of contact with the healthcare system. In addition to the inadequate research data from primary care units, the scarcity of anaemia recommendations may be motivated by worries about overburdened endoscopy services due to patient referrals or local priorities, as well as opinions about the costs and advantages of diagnostic testing.¹¹ V Geetha et al. (2022) did a study to concluded that The hazards to the mother and foetus are reportedly increased when anaemia is prevalent in pregnant women. It is advised that primary healthcare be reinforced and that treating anaemia in pregnancy should be prioritised in order to prevent it from occurring in the first place. There are five key factors that affect the elimination of iron deficient anaemia. The main contributing factor is low iron supplementation compliance because to its negative effects. Some of the other factors are as follows: due to the difficulties of obtaining and getting to healthcare facilities, people with parasite and worm-related infections, symptoms that go undiagnosed, a shortage of micronutrients, and insufficient monitoring or evaluation of the supplementing plan. Additionally, it is necessary to enhance the healthcare programme, including the dietary intake and the health environment.¹² M Salman Khan et al. (2022) did a study to conclude that for the automated assessment of thalassaemia, a novel deep-learning-based screening method has been investigated. The main goal was to identify whether strips of electrophoresis pictures of human beings were normal or abnormal in relation to thalassaemia by automatically extracting the lanes from the strips and classifying the subjects. When employing the InceptionV3 network, the proposed technique has a thalassaemia classification accuracy of 95.8% when tested on data from 524 cases. This was the first attempt to categorise electrophoresis test photos using a deep learning classifier model, and the technique produced passable accuracy. In the pipeline for thalassaemia screening, the suggested technique replaces manual assessment with automated assessment and detection. The findings also demonstrated that electrophoresis pictures combined with CNNs can provide superior results for thalassaemia screening when compared to more costly tests and methods. We plan to eventually add more electrophoresis picture data to the created database. In addition, we want to create gear that would enable automated thalassaemia screening.¹³ Kamran DS et al. (2023) did a study to concluded that peripheral smear can be used in high-risk locations and, although though it is not a very sensitive tool for screening for the Sickle trait, it can be supported by more advanced methods in ambiguous circumstances.¹⁴ Kharche K et al.(2023) did a study to concluded that in order to treat microcytic hypochromic anaemia with a hemolytic component that is resistant to treatment, a systematic review of 14 research from four regions and across India was conducted. The rare haemoglobin variations that the electrophoresis was unable to identify are likely best discovered by the HPLC. This review also comes to the conclusion that anaemia in children can result from both extremely unusual hemoglobinopathies like Hb J and common hemoglobinopathies like TM. The evaluation of haemoglobin abnormalities in the form of characteristics and diseases is advised for all paediatric patients with refractory anaemia.¹⁵ F Afshin et al. (2021) did a study to concluded that the examination of Hb Bart’s and Hb H are direct approaches for the diagnosis of thalassemia, however only a few cases of thalassemia were found in this study, and the most of them were missed. Because of this, -thalassemia must be confirmed in Hb Bart’s newborns, and molecular investigation is required to count the number of defective genes. In fact, lower MCV and MCH along with normal or low HbA2 levels appear to be a good opportunity to identify -thalassemia carriers. The CE result, whether generated from established genotypes or novel mutant genotypes, may be used as proof of Hb Hb and Hart Bart’s illness. Capillary electrophoresis is an effective method for identifying Hb H and Hb Bart’s disease due to the specificity of anaemia in blood analysis.¹⁶ VS Hariharan et al. (2023) did a study to concluded that the current research offers significant background data on PHT epidemiology among children and adolescents with SCA globally. PHT is one of the significant problems among SCA patients, regardless of their age, according to the findings of this systematic research. This study also emphasises how little research have looked into the degree of PHT among SCA patients in low- and middle-income areas. Clinicians in high-risk environments should create and implement diagnostic and intervention packages that are specifically aimed at these patients. It is important to conduct more research on the causes of the high prevalence of PHT in SCA patients since doing so will enable clinicians to better grasp the process, act decisively, and develop patient-specific therapies.¹⁷ N Kim et al. (2023) did a study to concluded that a specific Ampliseq-based panel was created for the genetic screening of HHA people, and it found 10 pathogenic mutations and 1 VUS. Although just a small number of people were chosen for our cohort, the data give a general idea of the molecular traits found in HHA. While we cannot rule out the possibility that genetic factors are not the primary causes of disease in these people, mutation-negative individuals from our cohort will need additional testing to identify the probable molecular flaws underlying their HHA.¹⁸ SR Menakuru et al. (2022) did a study to concluded that due to the description of the rare phenomena of HHS and the procedures necessary to identify the disease, this case is crucial to the body of current literature. As additional blood transfusions could result in a life-threatening anaemia, doctors need to have a high level of clinical suspicion when treating HHS patients. Usually, IVIG and steroids are used in the treatment to reduce macrophage activation; but, in a few, very uncommon cases, such as this one, HHS may be resistant to these methods. Given that macrophages are the cause of the syndrome and that SCD is a pro-inflammatory state, using tocilizumab, an IL-6 inhibitor, in SCD-induced acute HHS may be justified. More investigation into the relationship between cytokine levels and HHS is advised by the authors since it may be possible to administer tailored therapy once testing is more widely accessible.¹⁹ H Luo et al.(2022) did a study to concluded that presented the most thorough mutation spectrum of thalassemia and aberrant Hb variants in the HBB gene among Jiangxi Province people of reproductive age to date. Additionally, we offered comprehensive genetic counselling and prenatal testing to couples who had reasons to delay the conception of foetuses with ß-thal intermediate or significant. Our work may serve as a foundation for genetic counselling, public awareness campaigns, and as a guide to enhance ß-thal prevention.²⁰ ME Fields et al.(2022) did a study to concluded that children with non-SCAs had higher OEF than unaffected children, indicating that anaemia is the main cause of this compensatory mechanism. However, even adjusting for the effect of anaemia, children with SCA have considerably higher OEF than kids without SCAs, indicating that there are other pathophysiologic mechanisms in SCA than anaemia that affect cerebral metabolic demand. Future treatment options for neuroprotection in this vulnerable population may be revealed by a better knowledge of the variables causing this rise in cerebral metabolic stress in SCA.²¹

Conclusion

Conclusion will be drawn on the basis of observation, result and interpretation of result pertaining to the study.