Laboratory Evaluation of Alpha Thalassemia

Etiology and Epidemiology

α-globin chain genes are located on chromosome 16. There are normally four genes in total (αα/αα), two inherited from each parent. When there is a deletion in any number of the α globin gene, α-thalassemia manifests. The most common presentation is anemia of varying degrees, which is dependent on the number of deleted genes, affecting the amount of α globin chain production.[4]

Types of Alpha-thalassemia

α-Thalassemia Silent Carrier (αα/α-): When one α gene is deleted, it is known as a silent carrier of α-thalassemia. There is still enough production of α-chains to ensure a normal hemoglobin synthesis. The patient remains asymptomatic, and the mutation is benign. But mild anemia can occur in some instances, like increased stress or pregnancy. These patients can pass on the mutation to their future progeny, which may or may not manifest as the thalassemia disease state.[5][6]

α-Thalassemia Minor (αα/–) or (α-/α-): Occurs when two α genes are deleted. So now normal α globin chain production is reduced to half. In adults, an increased rate of production of red blood cells can compensate, up to some extent,  for the decrease in α chain production, thus balancing the amount of α and β globin chains. Patients are usually asymptomatic, and any anemia, if present, is mild.[5][6]

Hemoglobin H Disease (α-/–): The state of a compound heterogeneity for α-thalassemia silent carrier and α-thalassemia minor results in Hb H disease, which varies in severity from mild anemia, which is non-transfusion dependent thalassemia (NTDT) to the more severe end where it may be a transfusion-dependent thalassemia (TDT). The excess of β globin chains aggregates to form tetramers known as Hemoglobin H (Hb H). Hb H often precipitates within RBCs, causing damage to the RBC membrane and resulting in hemolytic anemia.[5][6]

This disease has a broad phenotypic spectrum. HbH disease may not be diagnosed until adulthood. The patients may suffer from splenomegaly, mild jaundice, and sometimes characteristic thalassemia-like bone changes due to extramedullary erythropoiesis. Sometimes the patients may also develop gallstones and suffer acute episodes of hemolysis following infections or exposure to oxidant drugs.[6]

α-Thalassemia Major (–/–): In homozygous states, the most severe and fatal form of thalassemia results, namely, Hb Bart hydrops fetalis. In newborns, there occurs an excess production of γ globin chains. These γ globin chains form tetramers resulting in hemoglobin Barts (Hb Barts). Hb Barts has increased oxygen affinity and is inefficient in oxygen unloading at the tissue level of the developing fetus, resulting in hydrops fetalis, rendering the fetus non-viable.[5][6]  

The clinical characteristics seen here are the prenatal onset of generalized edema accompanied by severe pleural and pericardial effusions. These are a result of congestive cardiac failure resulting from severe anemia. Extramedullary erythropoiesis, hepatosplenomegaly, and a large placenta are also seen.[6]

Thalassemias are known to be extremely heterogeneous at the molecular level. Over 200 different mutations of α-thalassemias have been found. The thalassemias are most frequently seen in southeastern and southern Asia, the Middle East, Mediterranean countries, and North and Central Africa; however, due to increased global movement and the mixing of various ethnicities, thalassemias have now become increasingly common in Northern Europe and North America.[2]

Pathophysiology

The major pathophysiological change seen in thalassemia is imbalanced globin chain production. As a result, Red Blood Cell (RBC) precursors are destroyed in the bone marrow or peripheral blood. This causes chronic anemia, splenomegaly, and typical skeletal deformities due to the expansion of the bone marrow.[7]

The blood picture is often similar to that of iron deficiency anemia showing RBCs that are mildly microcytic or even normal in the case of alpha-thalassemia silent carrier. α-thalassemia 1 usually presents with mild anemia, a slight decrease in RBC indices (MCV, MCH), hypochromia, microcytosis, and anisopoikilocytosis. HbA2 level is in the low to low-normal range (1.5 to 2.5%). During the neonatal period, moderate amounts of Hb Bart (3 to 8%) can be seen on the blood film. A few (1:1000/10 000 RBC) Heinz inclusion bodies (intracellular Hb precipitates) may also be detected. Hematological parameters should be reevaluated after iron supplementation.[5]

HbH disease, which is generally NTDT, occurs when α-globin synthesis is decreased by about one-fourth of the normal level. The presence of HbH (homotetramers of β-globin chains) can be detected by HPLC or electrophoresis. HbH amount is between 3 and 30% and is associated with mild-to-severe microcytic/normocytic anemia and elevated bilirubin level due to a moderate hemolytic component. Both parents, in such cases, are carriers of the α-thalassemia trait.[8][9] 

The most severe form of α-thalassemia disease is the homozygous state for α°-thalassemia, which is Hb Bart hydrops fetalis syndrome. The fetus is unable to synthesize any α-globin chains to make HbF or HbA. Fetal blood shows the presence of only Hb Bart (γ4) and some amount of embryonic Hb Portland. In such cases, prenatal diagnosis is very important, for which the diagnosis of the parents, who are thalassemia carriers, is vital.[8][9] 

TDT patients develop various complications due to iron overload in their bodies. Iron gets deposited in the RBC membrane due to the denaturation of hemoglobin, which causes the weakening of the membrane and hemolysis. The concomitant effects of ineffective erythropoiesis, chronic anemia, and hypoxia lead to increased GI absorption of iron. All these factors also cause increased iron deposition in tissues resulting in hemosiderosis. Free iron species generate Reactive Oxygen Species (ROS), leading to free radical-mediated tissue damage and, eventually, organ dysfunction and failure. TDT patients require regular iron chelation therapy and monitoring of iron levels to avoid such complications.[10]

Specimen Requirements and Procedure

Specimen collection depends on the type of test chosen for diagnosis. To identify the exact type of α-thalassemia affecting a patient, a battery of various laboratory tests is required, including CBC and Hb analysis for identifying and quantifying various types of Hb like HbF, HbA2, HbH, HbBarts, HbCS (constant spring), etc. EDTA vials are used for whole blood collection for CBC, Hb analysis, and molecular testing.[11]

Cytogenetic analysis on chorionic villi samples or cells isolated from the amniotic fluid sample is the procedure of choice for prenatal diagnosis of α-thalassemia in a suspected carrier or thalassemia minor parents. The intact chorionic villi can be preserved in a culture medium for up to 7 days.[12][13]

Diagnostic Tests

Complete Blood Count (CBC) - can be done using automated hematology analyzers in the laboratory. However, Hb, mean corpuscular volume (MCV), and mean corpuscular Hb concentration (MCHC) cannot differentiate between the thalassemia trait and iron deficiency or between α- and β-thalassemia. Thalassemia, however, has no direct effect on the platelet count and WBC count.[11] RBC counts are raised in the case of thalassemia, which can be a differentiating feature from iron deficiency anemia which has a low RBC count.[14]

Iron Studies - Serum ferritin levels, if normal or slightly increased, and almost normal transferrin levels, indicate thalassemia, which will be decreased and increased, respectively, in case of iron deficiency anemia.[11]

Peripheral Smear - Peripheral smear studies show microcytic hypochromic anemia, target cells, teardrop cells, and cells with basophilic stippling. However, these findings are in common with iron deficiency anemia. The presence of ‘golf ball-like Hb inclusions indicates HbH disease.[11]

Hemoglobin Analysis - Quantifying different types of Hb present in a patient’s blood can be done using HPLC or Electrophoresis techniques. These techniques can be applied to diagnose the type of thalassemia disease or carrier. The precision and reproducibility are also good with these techniques.[15]

Molecular Testing – Alpha thalassemias are mainly caused by deletions of different lengths or point mutations. Point mutations are single-base substitutions or small insertions or deletions. These different types of mutations can be detected by allele-specific PCR, reverse dot blot (RDB) analysis, Gap-PCR, real-time PCR with melting curve analysis, and DNA sequencing. Primer sequences are published to diagnose several α+ or α° deletional mutations.[16][17]

Testing Procedures

The use of new and high-throughput techniques, such as HPLC and capillary electrophoresis for hemoglobin quantification and PCR, next-generation sequencing (NGS), etc., for screening and accurate diagnosis of various types of thalassemias, is feasible.

High-Performance Liquid Chromatography - The HPLC system is a cation resin exchange system. It has a silica gel column. Different types of hemoglobin molecules are separated due to their interaction with a hydrophobic matrix, largely based on Hb polarity. Elution is done using inorganic phosphate buffers, and the sequence of elution is from low to high polarity. The chromatograms show the retention time (RT), peak, and percentage of different fractions.[18]

Capillary Electrophoresis system - Hb components are separated using silica capillaries in an alkaline buffer medium. Hb being a negatively charged molecule at an alkaline pH, migrates to the anode. The structural variants of Hb, having different charges on the surface, will separate. It is then read using the photometry technique at a wavelength of 415 nm. This can be used for both pre-and post-natal diagnosis of hemoglobinopathies. As an added advantage, these two systems can also detect Hb H, Hb Bart’s, and Hb CS in Hb H and Hb H-CS diseases.[19][20]

Allele-specific PCR - This technique utilizes two primers identical in sequence except at the 3′-terminus base. One of the primer 3’base is complementary to the wild-type, and the other is for the mutant base. A common primer for the opposite strand is used as well. Taq polymerase is used, which needs perfect matching of the primer 3′-end with the DNA template. In a normal individual, PCR product is observed only in the reaction where the wild-type primer set is present. A heterozygote will produce a band using both mutant and wild-type primer sets. An individual having a homozygous mutation will be negative with the wild-type and positive with the mutant primer set.[21]

Reverse Dot Blot analysis - For better identification of a suspected mutation, the PCR product is transferred on a membrane filter sheet as a dot. This PCR product can then hybridize with an allele-specific oligomer (ASO) DNA probe. This probe can be radiolabeled with P for autoradiography or other reporter groups like biotin or an enzyme such as horseradish peroxidase) can be attached. This can be visualized using a chemiluminescent or colorimetric reaction. A normal person will show positive dots with each wild-type probe but not with any of the mutant probes. Heterozygotes show a mixed pattern by showing a positive hybridization with one mutation dot and the normal dots. On the other hand,  homozygotes will show a positive dot with the mutant probe only and not with its corresponding normal sequence in addition to the positive spots for the remaining normal probes.[22]

Real-Time PCR with melting curve analysis - Real-time PCR can be considered better than conventional PCRs as they are less time-consuming and less labor-intensive. It is now widely used to detect, characterize, and quantify nucleic acids. It also has a low risk of post-PCR contamination. Currently, its application for thalassemia diagnosis is mainly based on two approaches; A) Intercalating dye assays and B) Probe-based assays. A fluorescent signal is obtained from the synthesis of the product in PCR. Dyes like SYBR-Green are used here. The melting curve analysis is useful in distinguishing α-thalassemia 1 heterozygotes, α-thalassemia 2 homozygotes, Hb H disease, and α-thalassemia 1 homozygotes (Hb Barts).[23]

Direct DNA sequencing - The sequencing of the product of PCR can help in identifying the specific gene mutations in alpha-thalassemia. The method usually employed is Sanger’s dideoxy termination method.[24]

Multiplex Ligation-dependent probe amplification - It is a multiplex PCR method that detects any duplications or deletions in the screened region. The extra advantage of this technique is that it can find known and unknown deletions in unsolved cases after the conventional techniques fail. It is easy to use, and only a thermocycler and electrophoresis equipment are needed.[25][26]

Next Generation Sequencing - NGS technology is a big step toward advancing sequencing technology. It can sequence the entire human genome in an ultra-high throughput manner at high speed. The target NGS approach can be used to analyze entire globin genes coding regions, the key regulatory regions, and modifier genes. NGS may be more accurate than conventional thalassemia diagnostic methods like CBC, Hb analysis, and Hb typing. It increases the capacity of gene sequencing from a few hundred bp to several thousand in a single analysis.[27]

Interfering Factors

Many factors can interfere with various methods of diagnosis of alpha-thalassemia. They can be broadly categorized as pre-analytical, analytical, and post-analytical factors. Any lapse in following standard protocols during collecting, transporting, or storing various samples like blood or chorionic villi samples can interfere with the method and affect the results. During analysis, the buffers, reagents, or stains used, if not properly stored at their optimum temperature, can yield false results. The levels of various Hb variants can decrease in the blood sample if kept for long storage, especially at high temperatures, and can affect the results of HPLC or CE analysis.[28] 

Techniques like NGS, allele-specific PCR, and multiplex ligation-dependent probe amplification are not feasible for widespread use in diverse populations due to their high cost, need for greater maintenance, etc.[26][27] When analyzing a chorionic villi sample, removing any maternal tissue is an important prerequisite as it may result in diagnostic error.[13]

Results, Reporting, and Critical Findings

Usually, adults have the maximum amount of Hemoglobin A (HbA), which makes up 95 to 98% of the total hemoglobin. Other variants like hemoglobin A2 (HbA2) make up 2 to 3%, and hemoglobin F (HbF) makes up <2% of total adult hemoglobin. All alpha-thalassemia-affected individuals show a variable degree of anemia (low Hb), decreased mean corpuscular hemoglobin (MCH), decreased mean corpuscular volume (MCV), and a normal to slightly decreased level of HbA2.[11]

Alpha-Thalassemia Carriers

α trait is an asymptomatic carrier state. A slight microcytosis can be seen sometimes, but the RBC can be normal too. HbA and HbF levels are normal. Notably, in the neonatal period, a minor amount (1 to 3%)  of Hb Bart's (γ) may sometimes be detected. α°-trait is usually marked by slight anemia, slightly reduced MCV and MCH, RBC microcytosis, hypochromia, and anisopoikilocytosis. During the neonatal period, Hb Bart's can be detected in moderate amounts (3–8%). Some Heinz inclusion bodies (intracellular Hb precipitates) may also be detected. The carriers have increased RBC count, which helps differentiate it from iron deficiency anemia.[11] Clinically asymptomatic cases are often diagnosed during a routine health check-up or antenatal screening. 

Alpha-Thalassemia Minor

The patients have mild-moderate anemia with Hb, not going below 7 g/dL, ranging from 7 to 10 g/dL, even in severe forms. HbA2 from 3 to 3.5%, and HbF ranges between 10 to 50%. Molecular testing is a more accurate diagnostic tool.[29]

HbH Disease

The predominant characteristic of this disease is anemia with varying amounts of HbH (0.8 to 40%). The type of mutation (deletional/non-deletional) affects the clinical severity. The patients present early in life with severe anemia that is normocytic/microcytic. The Hb levels can go below  7 g/dL. The MCH is low (<20 pg), and the peripheral blood film shows poikilocytosis with tear-drop cells, increased erythroblasts, and target cells.[11] 

In adult life, due to decreased alpha-globin chains, the excess β globin chains form β tetramers of HbH (0.8 to 40%), known as Inclusion bodies, can be identified by using 1% brilliant cresyl blue stain (BCB), or when this HbH is present in large quantities, it can be detected by routine Hb analysis.[11][30]

The laboratory findings in the deletional type of HbH disease show microcytic hypochromic anemia and inclusion bodies. On performing electrophoresis or HPLC in the sample of these patients, HbH and Hb Bart appear as fast-moving hemoglobin. The more severe form is the non-deletional type of HbH disease. The erythropoiesis is largely ineffective. The Hb is quite low (average 2 g/dL).[11][30]

Alpha-thalassemia Major (Hb Barts Hydrops Fetalis Syndrome)

The Hb Bart hydrops fetalis syndrome shows the presence of Hb Bart and the complete absence of HbF. When α globin synthesis falls lower than ~70% of normal, in the fetal period, the relatively excess γ globin chains form Hb Bart's, which can be identified on routine Hb analysis. The non-functional homotetramers γ and β constitute most of the Hb in the affected fetus' erythrocytes. The only functional Hb in these infants is found to be Hb Portland (ζγ) which acts as the carrier of oxygen.[11][29][30]

Clinical Significance

Alpha thalassemia has a broad spectrum of clinical presentations. Thus, an accurate and precise diagnosis is of utmost importance for appropriate patient management. A prenatal diagnosis of alpha thalassemia in such pregnancies where the fetus is diagnosed with Bart hydrops fetalis syndrome can be terminated, or timely initiation of intra-uterine transfusions can result in the birth of non-hydropic infants without significant neurological or congenital abnormalities in some of them.[31] 

Parents with undetected silent carriers or alpha-thalassemia minors can be given proper counseling. Techniques like molecular analysis can be used in hematological and clinically difficult cases, even with family hematological evaluations. This may be due to the occurrence of mild mutations or α- and β-interactions. The laboratory evaluation of alpha thalassemia thus holds paramount importance in improving the quality of life of such patients by timely initiation of therapy, counseling sessions, blood transfusions, if required, and prevention of future complications.[11]

Quality Control and Lab Safety

The factors that are involved in analytical quality in laboratories correlate to the definition, creation, and control of quality. Several errors can arise from external and internal sources, along with certain permanent and variable factors. Internal Quality Control (IQC) systems and External Quality Assurance systems (EQAS) need to be established for every individual laboratory. In-house standard operating procedures (SOPs) must be established and strictly adhered to by every lab personnel. Even a slight change in any of the QC parameters can make a significant difference, resulting in erroneous results.[32] 

The use of reference standards and control materials is important in each analysis. Controls and standards are important pre-requisite and are specific for each analytical method, and these can be prepared in-house or obtained commercially. All laboratories should establish a reference range for all the parameters for their particular methods, but the range obtained should not be significantly different from the published data.[32]

Monitoring is required at each step to ensure that the established protocols are followed and to check for any errors or deviations from the normal. Levey Jennings chart can be of great assistance for monitoring. It is a graphic representation of daily controls or averages of the results. Thus, even a single-day error can be identified, and corrective measures can be taken on time.[32][33]

Enhancing Healthcare Team Outcomes

It is essential that the clinician knows and understands a detailed workup of a case of suspected alpha-thalassemia. Every step in diagnosis requires joint efforts by various personnel who are a part of an interprofessional healthcare team for these patients and their families.

Good communication and coordination are the responsibility of the staff involved. Each team member needs to remain updated about diagnostic criteria, laboratory techniques, and interpretations of results obtained. It is also mandatory to follow the proper procedures and protocols right from the entry of a suspected patient of alpha-thalassemia in a clinic to its lifelong management. All laboratory team members must be aware of the universal safety precautions required in the lab.

The interprofessional team approach to diagnosis and management will result in the best patient outcomes. [Level 5]