Advances in testing technologies are improving the diagnosis of hemoglobinopathies. Yet testing for these blood disorders remains highly complex, and quality largely relies on the skill and experience of laboratory professionals as well as the sophistication of lab techniques. Medical Laboratory Observer recently sought answers to common questions about testing for hemoglobinopathies from Thomas E. Burgess, PhD, DABCC, FACB, Technical Director for Quest Diagnostics’ Southeast Business based in Atlanta, Georgia. Dr. Burgess specializes in hemoglobinopathy testing and lectures widely on the topic. He moderated and spoke at a full-day interactive workshop, “191001 Red Cells in the Sunset: The Laboratory Identification of Hemoglobinopathies,” presented at the AACC annual conference last summer.
What is a hemoglobinopathy?
A hemoglobinopathy is a hematologic disorder caused by an alteration in the genetically determined molecular structure of hemoglobin (alpha, beta, gamma and/or delta chain alterations). These alterations often cause anemia. Hemoglobinopathies are typically inherited (autosomal). The most common and best known hemoglobinopathy is sickle cell disease. In addition, the thalassemias are globin-chain production abnormalities. The most common ones are alpha thalassemia and beta thalassemia. Hemoglobinopathies are found in all areas of the world, and thalassemias are most common in populations in the Mediterranean and Southeast Asia.
Testing for hemoglobinopathies and thalassemias is generally performed either to diagnose a disorder or to determine if an individual is a carrier. The carrier status of certain hemoglobinopathies and thalassemias is especially critical in the prenatal period where the hemoglobin phenotype of the partner may be of importance if a homozygous hemoglobinopathy or severe thalassemia in the resulting fetus is a possibility.
However, not all hemoglobin variants are pathological; in fact, most hemoglobinopathies are benign in nature and of no clinical significance. For example, while homozygous hemoglobin S (sickle cell disease) is an extremely significant hemoglobinopathy, the corresponding “pure” AS trait (not associated with alpha thalassemia) is essentially benign. The traits, such as sickle trait (AS), beta thalassemia trait, and alpha thalassemia trait, are particularly important in the prenatal patient, where it is vital to understand the likelihood of severe versus mild disease.
The most common variants/traits found in the clinical laboratory are Hb S, Hb C, Hb E, Hb D-Punjab (also known as D-Los Angeles) and Hb O-Arab. In addition, beta thalassemia and alpha thalassemia are very prevalent in the population. Alpha thalassemia is insidious in that the hemoglobin phenotype in an alpha-thalassemia trait patient is completely normal. In both alpha and beta thalassemia, significant microcytosis (decreased MCV and/or MCH) is usually present, and in beta thalassemia, elevations in hemoglobin A2 are also usually detected.
What technologies are used today to detect hemoglobinopathy? What are their advantages and limitations?
Currently, most laboratories specializing in hemoglobinopathy detection are screening with either high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). The time-honored alkaline and acid electrophoretic techniques are still in use but are rapidly being replaced by HPLC or CZE as the primary screening technology. Ancillary testing protocols such as isoelectric focusing (IFE), globin chain electrophoresis, dithionite solubility testing (sickle cell solubility testing), and molecular assays for alpha and beta chain globin mutations should be available to the hemoglobinopathy laboratory when and where needed. However, the single most valuable “ancillary” piece of data required to properly identify hemoglobinopathies is a well-performed hemogram consisting of at least RBC, Hgb, MCV, MCH, and RDW. Without these data, the laboratory will be significantly hampered in its ability to identify the important hemoglobinopathies and thalassemias.
In terms of limitations, HPLC, while generating a very good separation pattern, can be initially intimidating to the bench technologist. Rather than a “simple” 2-3 band pattern found normally on electrophoresis, patterns containing 20-25 bands are the norm on HPLC. While initially daunting, the specificity and differentiation capabilities inherent in HPLC provide superior identification capabilities to the laboratory. Also, techniques such as isoelectric focusing are very technique-dependant.
In my opinion, there is no useless test in hemoglobinopathy detection. Even with the advent of HPLC and CZE there are occasions when the migration patterns of a properly performed alkaline and acid electrophoresis provide the piece of data that allows positive identification of the abnormal hemoglobin.
What big trends are you seeing in hemoglobinopathy testing?
From my perspective, the next big development will hinge on two main areas: HPLC tandem mass spectrometric (LC MS/MS) identification of hemoglobin variants and molecular identification of alpha and beta chain mutation analyses.
The first is a logical progression from the HPLC separations now being achieved by several manufacturers and will result in “positive” identification from a mass perspective rather than relative migration times. While providing a significant increase in specificity, the current instrumentation cost and expertise required for LC MS/MS may initially restrict its use to high-volume hemoglobinopathy centers or reference laboratories until the cost structure of these methods is reduced and the operation of said instruments becomes “cookie cutter” in nature.
The second technique, molecular identification of the alpha and beta chain mutations, is also potentially a cost-prohibitive technology that awaits a hands-off, fully automated solution to allow prime-time implementation. That being said, the power of a molecular level identification system for hemoglobin variants is indeed the holy grail of hemoglobinopathy detection and is, in my opinion, the future of this field.
What are the major potential impediments to quality testing for hemoglobinopathy?
Given the complexity of hemoglobinopathy testing, labs require a high degree of advanced technology, technical proficiency, and quality control to perform clinically appropriate testing.
It is important to consider the ethnicity of the patient. Hemoglobinapothies are subject to ethnic variants, and identification of a hemoglobinopathy can, in some cases, require knowledge of the patient’s ethnicity (country of origin or maternal/paternal homeland). In certain cases, this ethnicity can allow differentiation between two or more possible hemoglobins, the only difference being the country of origin/ethnicity of the patient. This knowledge is of special importance in the prenatal patient.
In female patients of childbearing age, a determination of pregnancy status in patients with elevated hemoglobin F levels may help in differentiating pregnancy-associated hemoglobin F increases from hereditary persistence of fetal hemoglobin (HPFH). Fortunately, a simple phone call to the patient’s physician can produce the needed information.
Special patients, such as those with sickle cell disease and a transfusion history or those who have had bone marrow transplants, require special consideration. Sickle cell disease patients who undergo routine transfusions exhibit a pattern that on the surface phenotypically mimics S-beta+ thalassemia, the combination of sickle disease and beta thalassemia. The differentiating factor is in the MCV, MCH, and RDW (reflecting the value of a hemogram =). S-beta+ thalassemia will exhibit suppressed MCV and MCH, while a sickle disease patient following a transfusion will show normal or elevated MCVs and MCHs. Sickle cell disease transplant patients will exhibit “non-sense” patterns in the early stages of transplant, often showing several banding patterns not consistent with “normal” hematopoietic function. Again, a phone call to the physician will provide the answer to this problem.
One of the impediments to quality is improper collection and preservation of a specimen. A specimen that is too old (greater than 7 days after draw) can generate “degradation” peaks that will confound even experienced technologists.
Finally, technologists should be mindful that as HPLC/capillary columns age, the quality of the separation degrades, making proper identification of the hemoglobin variants more challenging. Routine column replacement following manufacturer’s guidelines is highly recommended.
What steps would you suggest hospital and regional commercial laboratories consider when testing for hemoglobinopathy?
Testing for hemoglobinopathy is not for the faint of heart, and should only be practiced in labs with the necessary technological sophistication, controls, and proficiency. Labs that insource testing should ensure that their separation technology vendor has a competent consultation service. It is wise to explore the vendor’s level of expertise—and its access to resources, such as an academic center, which can augment its knowledge as needed. It is also important to hire and retain excellent scientists to perform and initially interpret the testing. This is easier said than done, but it is vital to quality testing.
Finally, if your lab does insource testing, make sure a quality reference lab is available to aid in interpreting complex cases. You will learn from a proficient reference lab’s interpretations, which may reduce the need to send them repeat specimens on the same hemoglobinopathy.
Thomas E. Burgess, PhD., DABCC, FACB, is Technical Director for Quest Diagnostics’ Southeast business based in Atlanta, Georgia. Dr. Burgess specializes in hemoglobinopathy testing and lectures widely on the topic.