Laboratory Evaluation of Alpha Thalassemia

Etiology and Epidemiology

α-globin genes are located on chromosome 16. Humans typically inherit 4 α genes (αα/αα), 2 from each parent. α-thalassemia manifests when α-globin gene deletion in any number occurs. Anemia of varying degrees results from the mutation, as the number of deleted genes affects the amount of α-globin chain produced.[12]

Thalassemia is known to be extremely heterogeneous at the molecular level. Over 200 different mutations are associated with α-thalassemia. The condition is most frequently seen in southeastern and southern Asia, the Middle East, Mediterranean countries, and North and Central Africa. However, increased global movement and mixing of various ethnicities have resulted in the rising thalassemia incidence in Northern Europe and North America.[13] Hb Bart's occurs mainly in Southeast Asia and Southern China.

Pathophysiology

The significant pathophysiological change in thalassemia is imbalanced globin chain production, resulting in RBC fragility. RBC precursors are thus easily destroyed in the bone marrow or peripheral blood. Symptoms include chronic anemia, splenomegaly, and skeletal deformities.[14]

The blood picture is often similar to IDA, with slightly microcytic RBCs. Erythrocytes may be normal in the α-thalassemia silent carrier type. α-thalassemia 1 usually presents with mild anemia, slight RBC index decreases (mean corpuscular volume or MCV and mean corpuscular hemoglobin or MCH), hypochromia, microcytosis, and anisopoikilocytosis. HbA2 level is in the low to low-normal range (1.5%-2.5%).

During the neonatal period, moderate amounts of Hb Bart's (3% to 8%) can be seen on blood films. A few (1:1,000-10,000 RBCs) intracellular hemoglobin precipitates forming Heinz inclusion bodies may also be detected. Hematological parameters should be reevaluated after iron supplementation.

HbH disease generally occurs as NTDT, with α-globin synthesis reduced by about a quarter of the expected level. The presence of HbH, which contains β-globin chain homotetramers, can be detected by HPLC or electrophoresis.[15] HbH amount is between 3% and 30% and is associated with mild-to-severe microcytic or normocytic anemia. Elevated bilirubin levels arise due to a moderate hemolytic component.[16]

The most severe α-thalassemia form is the homozygous α°-thalassemia state or Hb Bart's hydrops fetalis syndrome. The fetus cannot synthesize any α-globin chains to make HbF or HbA. Fetal blood shows the presence of only Hb Bart's (γ4) and some amount of embryonic Hemoglobin Portland (Hb Portland). Prenatal diagnosis is crucial for parental counseling in such cases.[17] 

TDT patients develop various complications due to systemic iron overload. Hemoglobin denaturation causes iron deposition in RBC membranes, resulting in membrane weakening and hemolysis. The concomitant effects of ineffective erythropoiesis, chronic anemia, and hypoxia increase gastrointestinal iron absorption. All these factors also cause increased iron deposition in tissues, resulting in hemosiderosis. Free iron generates reactive oxygen species, leading to free radical-mediated tissue damage, organ dysfunction, and subsequent organ failure. TDT patients require regular iron chelation therapy and iron level monitoring to avoid such complications.[18]

Specimen Requirements and Procedure

Specimen collection depends on the choice of diagnostic test. α-thalassemia type identification requires a battery of various laboratory tests, including CBC. Hemoglobin analysis identifies and quantifies various hemoglobin types, including HbH, Hb Bart's, and non-gene deletion mutants associated with more severe symptoms like Hb Constant Spring (HbCS). EDTA vials are used for whole-blood collection for CBC, hemoglobin analysis, and molecular testing.[19]

Cytogenetic analysis on chorionic villi samples or amniotic fluid cells is the procedure of choice for prenatal α-thalassemia diagnosis in suspected carriers or thalassemia minor parents. The intact chorionic villi can be preserved in a culture medium for up to 7 days.[20][21]

Diagnostic Tests

The initial diagnostic tests for α-thalassemia should include the following:

• CBC: This lab testing may be done using automated laboratory hematology analyzers. Differentiating the thalassemia trait from IDA or α- from β-thalassemia cannot be done by only determining the hemoglobin level, MCV, and MCH concentration (MCHC). However, RBC count elevation can distinguish α-thalassemia from IDA, which often has a low RBC count.[22] Thalassemia does not directly affect the platelet and WBC counts.
• Iron studies: Serum ferritin and transferrin levels can differentiate thalassemia from IDA. A normal or slightly increased ferritin level with nearly normal transferrin is more consistent with thalassemia than IDA, which often manifests with low ferritin and elevated transferrin.
• Peripheral smear: This lab result shows microcytic, hypochromic anemia, target cells, teardrop cells, and cells with basophilic stippling in thalassemia cases, though these findings are also common in IDA. The presence of golf-ball-like hemoglobin inclusions indicates HbH disease.
• Hemoglobin analysis: HPLC or electrophoresis can quantify the different hemoglobin types in the patient’s blood (see Image. α-Thalassemia Major on Hemoglobin Electrophoresis). These techniques can also help differentiate different thalassemia types with good precision and reproducibility.[23]
• Molecular testing: allele-specific PCR, RDB analysis, gap-PCR, real-time PCR with melting curve analysis, and DNA sequencing can help identify the mutation causing α-thalassemia. Molecular testing is essential to genetic counseling and therapeutic research.[24][25]

Test results should be interpreted in conjunction with clinical findings to guide treatment decisions.

Testing Procedures

High-throughput techniques offer the advantages of speed, efficiency, and scalability, making them valuable tools in both research and clinical settings for detecting and characterizing α-thalassemia mutations. The choice of technique depends on factors such as the desired resolution level and available resources.

High-Performance Liquid Chromatography 

HPLC separates, identifies, and quantifies different hemoglobin molecules. The specimen is passed through a slightly negatively charged silica gel column. Highly positively charged hemoglobin variants have more affinity for the column and thus elute after less positively charged hemoglobin types. Inorganic phosphate buffers help during elution.[26] The chromatograms generated by HPLC show the retention time, peak, and percentage of different hemoglobin fractions, allowing for the accurate diagnosis of hemoglobinopathies and thalassemia.[27]

Using inorganic phosphate buffers in the HPLC system is essential for maintaining mobile phase pH and effective separation of hemoglobin variants. The buffer and its pH must be carefully selected to ensure optimal chromatographic conditions and prevent damage to the column hardware and bed.[28]

Capillary Electrophoresis System 

Hemoglobin components are separated using silica capillaries in an alkaline buffer medium. Hemoglobin is negatively charged in alkaline solutions and thus migrates to the anode. The structural hemoglobin variants, having different surface charges, separate. The photometry technique then determines the amounts of each variant at a wavelength of 415 nm. Capillary electrophoresis can be used for both prenatal and postnatal hemoglobinopathy diagnosis.[29] Both HPLC and capillary electrophoresis can detect HbH, Hb Bart’s, and HbCS.[30]

Allele-Specific Polymerase Chain Reaction

PCR utilizes 2 primers with identical sequences except at the 3′-terminus base. One of the primer 3’ bases is complementary to the wild-type, and the other complements the mutant base. A common primer for the opposite strand is also used. Taq polymerase amplifies the genes but needs perfect matching of the primer 3′-end with the DNA template.

In healthy individuals, the PCR product is observed only in the reaction where the wild-type primer set is present. In contrast, a heterozygote produces a band in both mutant and wild-type primer sets. Individuals with homozygous mutations are negative in the wild-type and positive in the mutant primer set.[31]

Reverse Dot Blot Analysis

The PCR product may be transferred on a membrane filter sheet as a dot to identify a suspected mutation more easily. This PCR product can then hybridize with an allele-specific oligomeric DNA probe. The probe can be radiolabeled with phosphorus for autoradiography or other reporter groups like biotin. Alternatively, an enzyme such as horseradish peroxidase may be attached. The product may be visualized using a chemiluminescent or colorimetric reaction.

A healthy individual normally shows positive dots with each wild-type probe but not with any of the mutant probes. Meanwhile, heterozygotes often have a mixed pattern, demonstrating hybridization with both mutant and normal dots. Homozygotes typically demonstrate hybridization with the mutant probe only and not with normal probes.

Real-Time PCR with Melting Curve Analysis

Real-time PCR is often less time-consuming and labor-intensive than conventional PCRs. Real-time PCR is widely used to detect, characterize, and quantify nucleic acids. This technology also has a low risk of post-PCR contamination. Real-time PCR for thalassemia diagnosis uses 2 approaches: intercalating dye and probe-based assays. A fluorescent signal is obtained from PCR synthesis of the product. Dyes like SYBR® Green are used. The melting curve analysis is useful in distinguishing α-thalassemia 1 and α-thalassemia 2 heterozygotes, HbH disease, and α-thalassemia 1 homozygotes (Hb Bart's).[32]

Direct DNA Sequencing

PCR product sequencing can help in identifying the specific gene mutations in α-thalassemia. Sanger’s dideoxy termination method is the most widely used.[33]

Multiplex Ligation-Dependent Probe Amplification

This method uses multiplex PCR to detect duplications or deletions in the screened region. One advantage of this technique is that it can find known and unknown deletions in unsolved cases when conventional techniques fail. Multiplex ligation-dependent probe amplification is easy to use, and only a thermocycler and electrophoresis equipment are needed.[34][35]

Next-Generation Sequencing

Next-generation sequencing (NGS) technology can sequence the entire human genome in an ultra-high throughput manner at high speed. The target NGS approach may be used to analyze entire globin gene-coding regions, key regulatory regions, and modifier genes.[36] NGS may be more accurate than conventional thalassemia diagnostic methods like CBC and hemoglobin analysis and typing. This technology increases gene sequencing capacity from a few hundred to several thousand base pairs in a single analysis. The additional advantages of NGS include lower sample input requirements, higher accuracy, and the ability to detect variants at lower allele frequencies than Sanger sequencing.[37]

Interfering Factors

Elements that can compromise accuracy can reduce the utility of the above diagnostic tests. Sources of errors are broadly categorized as preanalytical, analytical, and postanalytical. Preanalytical factors consist of lapses following the standard protocols during collecting, transporting, or storing specimens like blood or chorionic villi samples. Such lapses can introduce errors in the method, rendering the results inaccurate. For example, hemoglobin can denature if stored for long periods or at high temperatures. Poor sample preparation, as in failing to remove maternal tissue from chorionic villi specimens, can also produce measurement errors.

During analysis, using improperly stored or prepared buffers, reagents, or stains can contribute to inaccurate analysis. Rounding off reported figures is an example of a postanalytical factor that can introduce diagnostic errors.[38]

The accessibility of these diagnostic tests is another factor that can reduce their utility. 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 and laborious maintenance.

Results, Reporting, and Critical Findings

Healthy adults usually have the maximum amount of hemoglobin A (HbA), comprising 95% to 98% of their total blood hemoglobin. Variants like hemoglobin A₂ (HbA₂) constitute 2% to 3%, while hemoglobin F (HbF) consists of less than 2% of total adult hemoglobin. All α-thalassemia-affected individuals show varying degrees of anemia that may manifest as low hemoglobin, MCH, and MCV and normal to slightly decreased HbA₂ levels.

α-Thalassemia Carriers

α trait is an asymptomatic carrier state. RBC levels may be normal, though with slight microcytosis. A minor amount (1% to 3%) of Hb Bart's may sometimes be detected in the neonatal period.

α° trait is usually marked by slight anemia, slightly reduced MCV and MCH, RBC microcytosis, hypochromia, and anisopoikilocytosis. Hb Bart's may be detected in moderate amounts (3% to 8%) during the neonatal period. Some Heinz inclusion bodies may also be noted.

α-thalassemia carriers often have increased RBC count, which helps differentiate it from IDA (see Image. Thalassemia and Iron-Deficiency Anemia Laboratory Differentiation). Clinically asymptomatic cases are usually diagnosed during a routine health checkup or antenatal screening. 

α-Thalassemia Minor

α-Thalassemia minor results from 2 α-chain gene deletions. Patients have mild-to-moderate anemia, with hemoglobin levels usually ranging from 7 to 10 g/dL. HbA₂ levels range from 3% to 3.5%, while HbF levels range between 10% and 50%. Molecular testing is a more accurate diagnostic tool.[39]

α-Thalassemia Intermedia (HbH Disease)

This condition's predominant characteristic is anemia with varying HbH amounts (0.8% to 40%). The mutation type, whether deletional or nondeletional, affects clinical severity. Patients often present with severe microcytic or normocytic anemia early in life. Hemoglobin levels below 7 g/dL may be reported. MCH is also often low at less than 20 pg, and the peripheral blood film typically shows poikilocytosis with tear-drop cells, increased erythroblasts, and target cells.[19]

In adults, the low α-globin chain levels cause excess β-globin chains to form the β tetramers comprising HbH (0.8% to 40%), detected as inclusion bodies on microscopy. These inclusion bodies may be identified by 1% brilliant cresyl blue staining. Routine hemoglobin analysis can also detect HbH if present in large quantities.[40]

Meanwhile, the laboratory findings in HbH deletional types demonstrate microcytic, hypochromic anemia and inclusion bodies.[19] HbH and Hb Bart's appear as fast-moving hemoglobin on electrophoresis or HPLC in these patients. Nondeletional HbH types like HbCS have more severe presentations, as erythropoiesis is largely ineffective. Hemoglobin is often severely low, about 2 g/dL on average.[40]

α-Thalassemia Major (Hb Bart's Hydrops Fetalis Syndrome)

Hb Bart's hydrops fetalis syndrome is notable for the presence of Hb Bart's and the absence of HbF. α-globin synthesis lower than 70% of the normal in the fetal period induces tetramerization of excess γ-globin chains to form Hb Bart's, which may be identified on routine Hb analysis. Nonfunctional γ and β homotetramers constitute most of the hemoglobin in the fetal erythrocytes. The only functional hemoglobin in these infants is Hb Portland (ζγ), which acts as the oxygen carrier.[40]

Mothers pregnant with a fetus with Hb Bart's hydrops fetalis usually present clinically between 20 and 26 weeks of gestation with pregnancy-induced hypertension and polyhydramnios. Fetal ultrasound should reveal hydrops. On Doppler measurement, fetal anemia is suggested by an increase in the middle cerebral artery's peak systolic velocity compared to the gestation-specific reference interval. Fetal blood sampling by cordocentesis typically demonstrates severe fetal anemia, with hemoglobin less than 80 g/L.[41] Other possible causes of hydrops fetalis must also be ruled out, such as toxoplasmosis, rubella, cytomegalovirus, herpes simplex, parvovirus B19 infection, and hemolytic disease of the fetus due to red cell alloantibodies.[42]

HPLC of a cordocentesis blood sample shows 1 or 2 very sharp and narrow peaks at the chromatogram injection point.[43] The major band is Hb Bart's, while the smaller band is attributable to Hb Portland. The complete absence of HbF should be noted. Alkaline electrophoresis shows the Hb Bart's band migrating to the anodal position and the Hb Portland band moving to the HbA position.[11]

Hb Bart's hydrops fetalis is almost invariably fatal if left untreated, with some fetuses dying in utero and others surviving a few hours after birth. Treatment with intrauterine transfusion may be able to salvage the fetus. However, the risk of complications like growth retardation and severe brain damage remains, which may be related to hypoxemia following long-standing intrauterine anemia.[41]

Laboratory investigation of the parents of fetuses with Hb Bart's hydrops fetalis shows a typical HPLC pattern with normal HbF and HbA₂ quantification.[44] Parental analysis typically shows a decreased hemoglobin concentration, MCH, and MCV. The blood smear also typically shows hypochromic, microcytic red cells (see Image. Peripheral Blood Smear for a Case of Hydrops Fetalis). The HbH inclusion body test is usually positive in the parents.[43]

Clinical Significance

α-thalassemia has a broad spectrum of clinical presentations. Accurate diagnosis is thus crucial to management decision-making, as in determining whether a fetus with Hb Bart's should be aborted or given intrauterine transfusions.[45] 

Parents with the silent carrier or α-thalassemia minor type may be properly counseled if diagnosed. Techniques like molecular analysis may be used in clinically difficult cases, as in families with mild mutations or α and β interactions. The laboratory evaluation of α-thalassemia thus holds paramount importance in improving patients' quality of life by timely initiation of therapy, counseling sessions, blood transfusions if required, and prevention of future complications.

Quality Control and Lab Safety

Laboratory analytical quality factors should be guided by a facility's definition and criteria for quality. Several errors can arise from external and internal sources and permanent and variable factors.[46] Establishing internal quality control and external quality assurance systems (EQAS) is imperative for laboratories to uphold testing methodology precision, reliability, and quality standards.[47] EQAS serves as a crucial mechanism for evaluating a laboratory's performance and fostering continuous improvement through external assessment and comparison with other laboratories.[48]

In-house standard operating procedures must be established, to which laboratory personnel should strictly comply. Even a slight change in quality parameters can make a significant difference and render erroneous results.[49] Reference standards and control materials must be meticulously noted during analysis. Method-specific controls and standards may be prepared in-house or obtained commercially. Laboratories should establish a reference range for all specific parameters analyzed, but such references should not be significantly different from published data.[50]   

Monitoring is required at each step to ensure compliance with established protocols and check for deviations from the normal. The Levey Jennings chart may aid in the monitoring process. This chart is a graphic representation of daily controls or averages of the results. Thus, the method can help identify and correct single-day errors.[51]

Every clinical laboratory should implement a formal safety program as its top priority to ensure patient and laboratory staff safety. Such a program should be the foundation for recognizing, reducing, and controlling potential laboratory hazards. Adhering to strict safety protocols significantly diminishes the chances of errors, incidents, and exposure to unsafe materials.[52]

Protective gear such as gloves, masks, protective eyewear, and gowns must be employed when collecting patient specimens. Disposable gloves made from nonsterile latex or alternative materials offering adequate barrier protection should be used. Phlebotomists should change gloves when moving between blood draws from different patients. Hand hygiene protocols are essential when changing gloves.[53]

Facial protection is necessary for lab technicians when the risk of blood or bodily fluid contamination is considerable. Minimizing syringe use and disposing of needles in rigid containers is advised. Wearing protective clothing is recommended to safeguard against potentially infectious substances. Removing protective gear is essential before leaving the laboratory.[54]

Enhancing Healthcare Team Outcomes

α-thalassemia diagnosis and management involve a collaborative effort from an interprofessional healthcare team. Members of the team may include the following:

• Hematologist: responsible for diagnosing and managing α-thalassemia, interpreting laboratory results, and overseeing treatment plans.
• Genetic counselor: provides information and counseling to individuals and families about the genetic aspects of α-thalassemia. This professional also helps assess the risk of transmitting the condition to the offspring, explains genetic test results, and assists with family planning decisions.
• Laboratory technologist: conducts various laboratory tests, including CBC, hemoglobin electrophoresis, molecular genetic testing, and other specialized assays, ensuring analytical accuracy and report timeliness. 
• Pediatrician or internal medicine physician: involved in the overall care and management of individuals with α-thalassemia.
• Obstetrician/gynecologist: monitors high-risk pregnancies and addresses potential complications, such as fetal hydrops.
• Nurse practitioner: helps provide ongoing care, education, and support to individuals with α-thalassemia and their families.
• Pharmacist: collaborates with the healthcare team to manage medications, such as iron chelators or other supportive therapies.
• Transfusion specialist: may be involved in managing blood transfusions for individuals with more severe forms of α-thalassemia.
• Clinical geneticist: consulted for complex genetic cases or when additional expertise is required to interpret genetic testing results.

Effective communication and collaboration among these professionals are crucial for providing comprehensive and coordinated care for individuals with α-thalassemia. A holistic, patient-centered approach helps ensure the best possible patient outcomes.