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Impact of hemoglobin variants on Hb [A.sub.1c] interpretation: do we assume to much?

Approximately 7% of the world's population carries a hemoglobin (Hb) variant, making these variants one of the most common monogeneic diseases and a major health issue worldwide. (1) Originating in the Mediterranean, Asia, and Africa, these mutations have spread throughout the world via international migration, and in many countries they are classified as endemic diseases. (1) The effects of these Hb variants range from clinically silent to life-threatening. (1) Some Hb variants can lead to decreased red blood cell (RBC) survival. However, given that such effects on RBCs often have minimal or no clinical impact, a complete characterization of the extent to which a Hb variant may alter RBC survival is sometimes lacking. Acknowledging this gap in our understanding has become increasingly important as the recommended test to diagnose diabetes and monitor estimated average glucose (eAG) in people with diabetes relies on the ability to interpret results of glycated Hb, or Hb [A.sub.1c]. In cases where Hb variants may preclude the interpretation of Hb [A.sub.1c], results, the fundamental component of Hb [A.sub.1c] to manage or diagnose diabetic patients may prove useless. Factors known to affect the interpretation of Hb [A.sub.1c] results include not only analytical interferences resulting from the most common of Hb variants, which have been well characterized, but also the effect of altered RBC turnover, which is less understood. Given the global occurrence of both Hb variants and diabetes, it is important to understand the potential impact of Hb variants on RBC survival and ultimately how the most common Hb variants may affect the clinical interpretation of Hb [A.sub.1c] test results used in the diagnosis and management of diabetes.

Hemoglobin

Hemoglobin is comprised of four globin chains. The most abundant adult form of Hb, [A.sub.0], consists of two [alpha] and two [beta]-chains ([[alpha].sub.2] [beta]2) and accounts for approximately 90% of Hb in hematologically normal adults and children over 6 months of age; (2) Hb [A.sub.2], ([[alpha].sub.2], [[delta].sub.2]) and Hb F ([[alpha].sub.2] [[gamma].sub.2]) constitute the other two minor components of Hb. (3) Post-translational glycation of Hb [A.sub.0] leads to the formation of Hb [A.sub.1c]. (3) the most abundant minor component of the Hb protein. Hb [A.sub.1c] is formed when the N-terminal of the [beta]-chain of Hb A is a non-enzymatically glycated under physiological conditions in the presence of circulating free sugars. (2)

Hemoglobin variants

Mutations and deletions in the genes encoding for the [alpha] - and [beta]-chains that result in amino acid changes are responsible for the production of Hb variants. (2) While almost a thousand Hb variants have been identified, (4) the four most common worldwide are Hb 5, E, C, and D, in order of descending prevalence (Figure 1), while in the U.S. the order is Hb 5, C, E, and D. (5) When a Hb variant is present in the homozygous state, an individual is said to have Hb X disease, and when present in the heterozygous form, an Hb X trait exists. Through international migration, these common Hb variants are now present throughout most of the world. For example, in the U.S., people of African, Mediterranean, or Southeast Asian descent are particularly likely to have a Hb variant (Table 1). While these populations are known to be likely carriers of a Hb variant, little is known about the geographic distribution of the different Hb variants within the U.S. Reasons for this knowledge gap are two-fold, given the lack of data from patients born before newborn hemoglobinopathy screening programs began and the lack of data from those who have immigrated to the U.S. (6) Consequently, the National Heart, Lung, and Blood Institute of the National Institutes of Health created the pilot program RuSH (Registry and Surveillance System in Hemoglobinopathies) in 2010. (6) Under this program, the Centers for Disease Control and Prevention (CDC), together with six state health departments (California, Florida, Georgia, Michigan, North Carolina, and Pennsylvania), will gather data with the goal of determining the prevalence of various hemoglobinopathies (including the four most common Hb variants) in the general population, as well as their geographic distribution. (6) RuSH may prove to be an invaluable database in the U.S., especially in populations where there is a high incidence of both Hb variants and diabetes.

Hb Variant  Prevalence in the U.S.

Hb S        8.3% of African Americans

Hb E        30% of Southeast Asians

Hb C        3% of African Americans

Hb D        2% of North Indians and descendants

Table 1. Prevalence of the most common Hb variants in the U.S.


Diabetes and Hb [A.sub.1c]

Diabetes is classified as a worldwide epidemic. The International Diabetes Federation (IDF) has reported the prevalence of the disease in 2011 as 366 million. (7) Current estimates are that this number will increase to 552 million by 2030. (8) Assessing glycemic control in diabetics is crucial for management of the disease and for the prevention of long-term complications. (9) Hb [A.sub.1c], is currently the most widely used index of average glycemia. (9)

After the Diabetes Control and Complications Trial (DCCT) and UK Prospective Diabetes Study (UKPDS), it was clear there was a direct relationship between Hb [A.sub.1c] and mean blood glucose. These studies demonstrated that a person with diabetes can delay the onset and slow the progression of diabetic complications by maintaining Hb [A.sub.1c], levels near normal.

Complementary to these studies was one sponsored by the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD), which established the relationship between Hb [A.sub.1c], and cAG using reliable regression equations available to calculate eAG. (10) Of note, however, was the exclusion of individuals with "any form of hemoglobinopathy or hemolytic process which interferes with reliable assessment of diabetic control with conventional assays for glycosylated hemoglobin (e.g., sickle trait)" making these studies not entirely representative of the population. (10-12)

Effects of Hb variants on Hb [A.sub.1c] interpretation. Factors affecting the clinical utility of Hb A ic in the presence of Hb variants include both analytical interferences and the clinical interpretation of the result. Based on recent College of American Pathology (CAP) proficiency testing data, close to 30 commercially available methods are available to determine glycated Hb levels. These include measurements based on charge (ion exchange chromatography) and structure (affinity chromatography and immunoassay). (13) Descriptions of the method-specific analytical interferences of the four most common Hb variants on Hb Ale measurement are available (www.ngsp.org), and will not be reviewed here. However, it is important to note that in areas where Hb variants are present, Hb [A.sub.1c] methods must be carefully selected by reviewing product instructions from the manufacturer to reduce the chance of inaccuracy of these measurements.

Hb [A.sub.1c] concentration is dependent on a variety of factors, including the concepts that RBC survival is constant, that RBCs are freely permeable to glucose, and that the nonenzymatic glycation of Hb occurs at a rate directly proportional to the ambient glucose concentration. (14) If each of these requirements are met, then Hb [A.sub.1c] should accurately reflect an average glycemic history of approximately the previous 120 days in a hematologically normal individual. (14) For persons affected by conditions proven to shorten RBC survival such as Hb S, C, or D disease, the use of alternate tests are recommended to estimate a patient's past average glycemia. (5), (15) The ADA states that Hb [A.sub.1c] can be used to assess glycemic control in patients with Hb S trait; (16) other groups have expanded this recommendation to include Hb C and Hb D trait. (5), (15), (17) The vast majority of studies evaluating the utility of the various Hb [A.sub.1c] assays in patients with Hb variant traits, however, evaluate only analytical interferences and fail to address other potential limitations that may affect Hb [A.sub.1c] interpretation, such as altered RBC survival. An extensive review of the literature suggests that current data and references are lacking to support the assumption that common Hb variant traits do not significantly affect RBC survival (Table 2), and ultimately the interpretation of Hb [A.sub.1c] as it relates to eAG.

Hb    Average RBC Lifespan in Days (n)

Hb A                               120

Hb S                       93 (3) (20)

Hb E                           No Data

Hb C                       87 (6) (18)

Hb D                      115 (3) (19)

Table 2. Average lifespan of RBCs from hematologically
normal or Hb trait individual


Heterogeneity of red blood cell lifespan. The lifespan of RBCs is a known determinant of Hb [A.sub.1c] concentration. Because there is some evidence to suggest that Hb variants may affect RBC lifespan even in Hb trait patients who are asymptomatic, (18-20) it is worth considering how the presence of common Hb variants may affect Hb [A.sub.1c] interpretation, and whether additional studies are needed to determine the correlation of Hb [A.sub.1c] and average glucose specifically in these populations.

In 2004, there were approximately 23.4 million non-Hispanic African Americans living in the U.S. (5) Of these, 10% carried either the Hb C or Hb S allele. (5) When combined with an estimated prevalence rate of diabetes in this population of 13.0% to 16.3%, it was estimated that between 305,000 and 383,000 non-Hispanic African American individuals have both diabetes and either Hb C or Hb S trait. (5) The prevalence of diabetes in areas where Hb variants are most widespread worldwide is shown in Figure 2 and summarized in Table 3.

Region             Number of individuals with  Prevalence
                   diabetes (millions)          (%)

Southeast Asia                           71.4         9.2

European                                 52.8         6.7

North America and                        37.7        10.7
Caribbean

Middle East and                          32.6        11.0
North Africa

Africa                                   14.7         4.5

Table 3. Prevalence of diabefes by selected regions (7)


The number of individuals affected by both diabetes and one of the most common Hb traits is even more significant in other areas of the world. For example, the current population of India is estimated at 1.2 billion (21) with a Hb S prevalence of 4.3%. (22) Approximately 9.2% of the population of India is affected by diabetes, (7) making it the country with the world's largest population of diabetic patients. Together, these numbers estimate that approximately 4.7 million people living in India have both diabetes and Hb S. A similar review of data available from Thailand estimates that almost one million people in that Southeast Asian country are affected by both Hb E and diabetes.

While the analytical interference of Hb S and Hb C has been well characterized for many Hb [A.sub.1c] methods, less is known about the RBC lifespan in these patients and whether the relationship between Hb [A.sub.1c] and estimated average glucose may be significantly different from that seen in hematologically normal individuals, thereby affecting the interpretation of the Hb [A.sub.1c] result. The diabetes epidemic that now affects much of the world, combined with the globalization of Hb variants, reinforce the importance of understanding the clinical implications of Hb variants on the correlation of Hb [A.sub.1c] and eAG.

Red blood cell lifespan in common hemoglobin variant traits: what is known?

Hemoglobin S. Hb S results from a substitution of valine for glutamic acid in the sixth position of the [beta]-chain of Hb A. (23) The Hb S variant is most common in West Africa, where it affects approximately 25% of the population. Hb S is also found in people of Hispanic and Mediterranean descent, and is also common in the U.S., affecting primarily people of African descent. (24)

Hb S disorders are divided into sickle-cell trait, which denotes all heterozygous genotypes, and sickle-cell disease, which is the homozygous condition. The mechanism for increased RBC turnover in Hb S disease is well known. (25) It is widely accepted that persons with Hb S trait have normal RBC survival. However, studies that have measured Hb S RBC survival suggest otherwise, (20), (26) demon-strating that the lifespan of RBCs in Hb S trait is approximately 93 days compared to 120 days in hematologically normal persons. (20) The small number of studies that have determined RBC survival in patients with Hb S trait indicates a need to better characterize RBC lifespan in this genetic background.

Hemoglobin E. The Hb E variant results from a mutation in the Hb [beta]-chain, and is extremely common in Southeast Asia--equaling Hb A in frequency in some areas. (27), (28) In recent years, emigration to North America from areas where Hb E mutations are prevalent has led to Hb E disorders becoming a health concern in North American (27) (Table 1). While the mutation giving rise to the Hb E variant can also activate a cryptic mRNA splice site that causes decreased synthesis of the [beta]-E chain and a thalassemic phenotype, Hb E trait is reported to have no clinical significance? (28) However, in a literature search, there have been no studies that have examined RBC survival in Hb E trait patients.

Hemoglobin C. The lib C variant arises from a single amino acid substitution in the normal Hb A f3-chain, where the glutamic acid at position 6 is replaced by a lysine, (29) and is found at the highest frequencies in West Africa, where approximately 40% to 50% of the population in the nations of Burkino Faso, Cote d' Ivoire, and Ghana are carriers of the gene (Figure 1). (30) Through migration, the Hb C variant has spread from West Africa, and it is present in approximately 3.5% of West African descendants in the Caribbean, in Southern Europe, and in the U.S. (30) (Table 1).

Persons affected by Hb C trait are reported to enjoy complete clinical health, while those with Hb C disease most commonly present with mild hemolytic anemia. (1) While some reviews indicate without references that RBC survival in those with Hb C trait is normal, (5) other studies suggest that RBC survival is reduced in Hb C trait patients (18), (31), (32) (Table 2). The discordance between these two conclusions indicates the need for additional studies.

Hemoglobin D. Hb D was first identified in 1951 in a family of mixed British and American origin, and arises as the result of a single amino acid change at position 121 on the [beta]-chain, changing a glutamic acid into a glutamine. (33) While there are several Hb D variants, the most common is Hb D-Punjab, also known as Hb D-Los Angeles. (33) The Hb D allele is mainly found in people from northwest India (1% in Fujeratis, 2% to 3% in Sikhs), Pakistan, and Iran (33) (Figure 1).

While some groups suggest that the clinical impact of this Hb variant trait is minima1 (34), (35) and that RBC survival does not differ significantly from norma1. (36), (37) there is a lack of published data to support the latter assumption. Thus, further studies are needed before the conclusion can be drawn that the correlation of Hb [A.sub.1c] and average glucose in patients with Hb D trait is not significantly impacted.

Estimation of average glucose levels over approximately the preceding 120 days, as measured by Hb [A.sub.1c], should not be used in patients with abnormal RBC turnover. (16) The idea that RBC survival in Hb S trait patients is normal can be found throughout the literature, without reference to a particular study. (16) A review of the literature, however, provides some evidence that Hb S trait shortens RBC lifespan. This discordance may indicate the need for additional studies to characterize in detail the effect of this common Hb variant trait on RBC survival and to determine the impact, if any, on the correlation of Hb [A.sub.1c] and eAG. Data is also lacking for the remaining three most common Hb variants despite the widely accepted practice of using Fib [A.sub.1c] to interpret average glycemia in diabetic patients with fib C, Hb D, or Hb E trait. While the incidence of diabetes is expected to rise, and the distribution of Hb variants continues to widen, the need to understand how the most common Hb variant traits influence Hb [A.sub.1c] interpretation is becoming increasingly important.

References

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(2.) Chandalia HB, Krishnaswamy PR. Glycated hemoglobin. Current Science. 2002;83(12):1522-1532.

(3.) Bunn H, Gabbay KH, Gallop PM. The glycosylation of hemoglobin: relevance to diabetes mellitus. Science. 1978;200(4337):21-27.

(4.) Takara be D, et al. Hemoglobin variant HbE found in two South Asian diabetic patients. Intern Med. 2009;48(16):1397-1401.

(5.) Little RR, Roberts WL.A review of variant hemoglobins interfering with hemoglobin A1c measurement. J Diabetes Sci Technol 2009;3(3):446-451.

(6.) National Heart, Lung, arid Blood Institute. NHLBI, CDC launch surveillance and research program for inherited blood disorders. 2010. http://www.nih.govinews/health/feb2010/nhibi-18.htm. Accessed March 28, 2012.

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(10.) Nathan DM, et al. Translating the A1c assay into estimated average glucose values. Diabetes Care. 2008;31(8)1 473-1478.

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(16.) Standards of medical care in diabetes-2012. Diabetes Care. 2012;35(Suppl 1):S111163.

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(24.) Piel FB, et al. Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis. Nat Commun. 1:104.

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(28.) Vichinsky E. Hemoglobin e syndromes. Hematology Am Soc Hematol Educ Program. 2007;79-83.

(29.) Stamatoyannopoulos G. The molecular basis of hemoglobin disease. Anna Rev Genet. 1972;6:47-70.

(30.) Bachir D, Galacteros F. Hemoglobin C. Orphanet Encyclopedia, 2004.

(31.) Martina WV, et al. Beta-N-terminal glycohemoglobins in subjects with common hemoglobinopathies: relation with fructosamine and mean erythrocyte age. Clin Chem. 1993;39(11 Pt1):111259-2265.

(32.) Derebail VK, et al. Variant hemoglobin phenotypes may account for differential erythropoiesis-stimulating agent dosing in African-American hemodialysis patients. Kidney Int 2011;80(9):992-999.

(33.) Taghavi Basmanj M, et al. Co-inheritance of hemoglobin D and beta-thalassemia traits in three Iranian families: clinical relevance. Arch Iran Med. 2011;14(1):61-63.

(34.) Clarke GM, Higgins, TN. Laboratory investigation of hemoglobinopathies and thalassemias: review and update. Clin Chem. 2000;46(8 Pt 2):1284-1290.

(35.) Girisha KM, et al. Compound heterozygosity for HbD Punjab and polyadenylation signal mutation causes clinically asymptomatic mild hypochromia and microcytosis. Ann Hematol. 2010;89(6):625-626.

(36.) Little RR, et al. Effects of hemoglobin (Hb) E and Hb0 traits on measurements of glycated Hb (HbA1c) by 23 methods. Clin Chem. 2008;54(8):1277-1282.

(37.) Higgins TN. QA aspects for HbA1c measurements. Clinical Biochemistry 2008;41(1-2):88-90.

By Jeanne M. Rhea, PhD, Tiffany K. Roberts-Wilson. PhD. and Ross J. Molinaro, PhD, MT(ASCP). DABCC, FACB

Jeanne M. Rhea, PhD, is a Post-Doctoral Fellow working with Ross J. Molinaro, PhD, MT(ASCP), DABCC, FACE, within the Department of Pathology and Laboratory Medicine at Emory University School of Medicine in Atlanta, GA. Tiffany K. Roberts-Wilson, PhD, is a Clinical Chemistry Post-Doctoral Fellow working in the Department of Pathology and Laboratory Medicine at Emory University School of Medicine in Atlanta, GA.
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Title Annotation:Cover story
Author:Rhea, Jeanne M.; Roberts-Wilson, Tiffany K.; Molinaro, Ross J.
Publication:Medical Laboratory Observer
Date:Jun 1, 2012
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