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Hemoglobinopathies and clinical laboratory testing.

Hemoglobin (Hb) is the oxygen-carrying protein of erythrocytes. It is an iron-containing, tetrameric metalloprotein that consists of two pairs of unlike globin chains (i.e., two [alpha]-type and two [beta]-type globin chains). The globin chains form a shell around a central cavity containing four heme prosthetic groups, each covalently linked to a single chain. The heme found in Hb is a porphyrin ring bound to a central iron atom, which serves as the site of oxygen binding. The [alpha]-type globins are encoded by a gene cluster on chromosome 16 that contains the embryonic [xi] gene and two adult [alpha] genes in series, oriented in the 5-prime (5') to the 3-prime (3') direction (see Figure 1A). The [beta]-type globin genes are clustered on chromosome 11 (see Figure 1A), and include the embryonic [epsilon], two tandem fetal [gamma] genes, and the adult [delta] and [beta] genes oriented in the 5' to 3' direction. The globin genes are activated from 5' to 3' during embryonic and fetal development. In the healthy neonate, fetal Hb, or Hb F ([[alpha].sub.2][[gamma].sub.2]), is the major species (~70%) (see Figure 1B). Hb F is replaced by the major and minor adult Hbs, Hb A ([[alpha].sub.2][[beta].sub.2]) and Hb A2 ([[alpha].sub.2][[delta].sub.2]), during the first 6 to 12 months of life (see Figure 2). Thus, in healthy adults, Hb is composed of Hb A (~95%) and Hb A2 (~3.5%), with only trace amounts of Hb F. The normal diploid cell produces Hb A from four [alpha] and two [beta]-globin genes. The [alpha]- and [beta]-globin chains consist of 141 and 146 amino-acid residues, respectively.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Hemoglobin Expression in Human Development

Species           Chains      Embryo  Neonate  Adult

Gower1       [xi]2[epsilon]2    50%      0%       0%
Gower 2   [alpha]2[epsilon]2    25%      0%       0%
Portland       [xi]2[gamma]2    25%      0%       0%
HbF         [alpha]2[gamma]2     0%     75%      <1%
Hb A         [alpha]2[beta]2     0%     25%      97%
Hb A2       [alpha]2[delta]2     0%     <1%       3%


Hemoglobinopathies are inherited single-gene disorders that affect Hb production and function. It is estimated that around 7% of the world population carries a globin-gene mutation; and in the majority of cases, it is inherited as an autosomal recessive trait. (1) Hemoglobinopathies can be classified broadly as qualitative and quantitative disorders. Qualitative hemoglobinopathies result from [alpha]- or [beta]-globin gene mutations that cause structural alterations in the Hb molecule. Quantitative hemoglobinopathies, or thalassemias, arise from mutations that cause decreased synthesis of otherwise normal [alpha]- or [beta]-globin chains, resulting in stoichiometric imbalance of the subunits. More than 700 structural Hb variants and thalassemias have been described in published scientific journal articles (2); and, as of June 19, 2009, 1,361 entries have been posted on Hb-Var, a database on human Hb variants and thalassemias available online at http://globin.cse.psu.edu/hbvar/menu.html.

Most hemoglobinopathies are clinically benign; and in most cases, not enough scientific evidence has been demonstrated to prove or disprove any pathological effect. The convention used by Hb Var to characterize structural variants includes the affected globin gene, altered residue number, protein domain, and amino-acid substitution. Thus, for example, Hb S is characterized as [beta] 6(A3) Glu>Val to indicate that a [beta]-globin mutation at residue 6, in domain A3, causes an amino-acid change from glutamic acid to valine. This change results in Hb tetramers that aggregate into arrays upon deoxygenation in the tissues, leading to deformation of red blood cells (RBCs) into sickle-like shapes, making them relatively inflexible and unable to traverse the capillary beds. Among the structural variants, a high gene frequency and significant clinical or hematological effects are observed with Hb S, Hb C ([beta] 6(A3) Glu>Lys) and Hb E ([beta] 26(B8) Glu>Lys). In the case of Hb C, the glutamic-acid to-lysine substitution leads to an unstable Hb that precipitates in red blood cells to form crystals, thus decreasing the deformability of red blood cells. Affected erythrocytes are removed by the spleen. In the case of Hb E, the mutation at codon 26 of the [beta]-globin gene causes a substitution of glutamic acid by lysine and also activates a cryptic mRNA splice site, resulting in the reduced synthesis of the [beta]-E chain, leading to a thalassemia pheno-type. (3)

Hb S is the variant that causes sickle-cell disease (SCD) (OMIM database No. 603903). SCD includes a group of genetic disorders characterized by chronic hemolysis and episodic vascular occlusion. (1) The disorders arc found primarily in people of African, Mediterranean, and Southeast Asian ancestry. (4) Vascular occlusion results in episodes of severe pain and tissue infarction, while the consequences of hemolysis include chronic anemia, jaundice, predisposition to aplastic crisis, cholelithiasis, and delayed growth and sexual maturation. The condition can result in acute and chronic injury to most of the organs, in particular, the spleen, brain, lungs, and kidneys. (5) Sickle-cell anemia (Hb SS) accounts for 60% to 70% of sickle-cell disease in the United States. (6) Other forms result from coinheritance of Hb S with other abnormal [beta]-globin-chain variants. The most common of these forms includes sickle-Hb C disease (Hb SC), (1) a condition that occurs in about one in 835 African-American births and in about one in every 25,000 births in the general population, as well as two types of sickle [beta]-thalassemia, Hb S [beta]/zero-thalassemia and Hb S [beta]/plus-thalassemia, which together occurs in about one in every 50.000 births. Those with Hb S [beta]/zero-thalassemia usually have a severe form of the disease. People with Hb S [beta]/plus-thalassemia lend to have a milder form of the disease. Sickle-cell trait refers to situations where the individual is a sickle-cell carrier and is asymptomatic.

Reduction in the amount of the normal globin chain produced is characteristic of thalassemias. The clinical manifestations range from mild anemia with microcytosis (thalassemia trait) to fatal severe anemia (Hb Bart's hydrops fetalis (7) or [beta]-thalassemia major). The two main types of thalassemia are named according to which adult globin gene is dysfunctional: [alpha]- and [beta]-thalassemia. (8) The decreased globin-chain synthesis may result from gene deletion or from mutations that adversely affect the transcription or stability of mRNA products. Disease is caused by insufficient functional hemoglobin, as well as tetramer formation of the unaffected globin chain. In [alpha]-thalassemia, tetramers of adult [beta] chain {named Hb H) and fetal [gamma] chain (named Hb Bart's) are unstable in erythrocytes and cause hemolytic anemia. In [beta]-thalassemia, tetramers of [alpha] globin are unstable in erythrocytes and bone-marrow erythroblasts, resulting in hemolytic anemia and intramedullary ceil death. In severe cases, massive erythroid hyperplasia in the marrow causes bone deformities. The great majority of [alpha]-thalassemia cases are caused by large deletions of one or both [alpha]-globin genes (HBA1 and HBA2) on chromosome 16(see Figure 1A). Single-gene deletions of HBA1 or HBA2 are prevalent in many areas of the developing world. In contrast, large deletions encompassing both HBA1 and HBA2 are most common in Southeast Asia, and are very rare in patients of African ancestry. The [alpha]-globin deletions can be inherited as homozygous or heterozygous defects, resulting in loss of one to four [alpha] globin genes. Severity of disease is dependent on the extent of gene deletion:

* Loss of one [alpha]-globin gene is clinically and hematologically silent.

* Loss of two genes results in [alpha]-thalassemia trail, characterized by microcytosis with little or no anemia. Two-gene deletion can occur when both genes on a single chromosome 16 are lost in the patient's genome ("deletion in cis"), or when a single gene is lost on each version of choromosome 16 in the patient's genome ("deletion in trans").

* Loss of three genes results in Hb H disease, a moderate hemolytic anemia.

* Loss of all four genes is incompatible with independent life.

* Death occurs in utero from Hb Bart's hydrops fetalis.

In contrast to [alpha]-thalassemia, [beta]-thalassemia is characterized by small missense or nonsense mutations in the [beta]-globin gene (HBB), which reduce or completely abrogate gene expression. Mutations that eliminate expression are labeled [beta]-zero, while those that reduce expression are labeled [beta]-plus. Severity of this condition is dependent on the extent of [beta]-globin chain underexpression. Heterozygous inheritance of a [beta]-thalassemia mutation results in a trait condition, sometimes termed [beta]-thalassemia minor. Like [alpha]-thalassemia trail, this condition is characterized by microcytosis with little or no anemia, although it is distinguished by increased Hb A2 production. Homozygous inheritance results in [beta]-thalassemia major (absent Hb A production) or [beta]-thalassemia intermedia (severely reduced Hb A production), which are associated with moderate to severe hemolytic anemia

Diagnostic recommendations regarding the laboratory investigation of these conditions were first made in 1975 by the International Committee for Standardization in Hematology expert panel on abnormal Hbs and thalassemias. The recommended initial testing included a complete blood count, or CBC, electrophoresis at pH 9.2, tests for solubility and sickling, and quantification of Hb A2 and Hb F. (2) The identification of an abnormal Hb required further testing, using addition techniques such as electrophoresis at pH 6.0 to 6.2. globin-chain separation, and isoelectric focusing (IEF). Heat and isopropanol stability tests were recommended for detection of unstable Hbs or Hbs with altered oxygen affinity. (2) Although electrophoresis at alkaline and acid pH has been widely used for many years, the emergence of cation-exchange high-performance liquid chromatography, or HPLC, as the method of choice for quantification of HbA2 and Hb Hand identification of Hb variants, (9) streamlined the recommended preliminary and follow-up tests for the identification of hemoglobinopathies and thalassemias, and provided rapid and complete diagnostic work-up in a majority of eases. The elements of choice include a CBC, Hb H test, ferritin, HPLC for Hb A2 and F quantification, and detection of any Hb variants followed by electrophoresis at both alkaline and acid pH.

[FIGURE 3 OMITTED]

Measurement of hematological indices

The hematological profile consists of measurements of the RBC indices and includes Hb concentration, hematocrit, RBC number, mean corpuscular Hb (MCH), mean cell volume (MCV), and red-cell distribution width (RDW. an indicator of RBC size variation). Routinely, a blood film accompanies the RBC indices. Structural hemoglobinopathies may have an impact on the red-cell indices, and red-cell indices are critical to the differential diagnosis of thalassemias. The general classification of thalassemias is hypochromic and microcytic anemias; therefore, the MCV can be considered as a key diagnostic indicator. Virtually all automated hematology analyzers now provide a measurement of MCV that is both precise and accurate.

Thalassemic individuals have a reduced MCV, and an MCV of 72 femtoliter (fL or [10.sup.-12] liters) is maximally sensitive and specific for presumptive diagnosis of thalassemia syndromes. (10) The RDW can be used to differentiate between microcytic anemias, most notably iron deficiency (increase in RDW), and the thalassemias, which--in contrast--tend to produce a uniform microcytic red-cell population without a concomitant increase in RDW. (2) RDW may provide complementary information but is not useful as a lone indicator. The RBC count is also useful as a differential tool because the thalassemias produce a microcytic anemia with an associated increase in the RBC number, while the other causes of microcytic anemia, including iron deficiency and anemia of chronic disease, are more typically associated with a decrease in the RBC number that is proportional to the degree of decrease in Hb concentration. The Hb concentration can provide complementary information since it is typically decreased in thalassemia. The thalassemia-minor conditions produce minimal decrements in the Hb concentration; whereas, thalassemia intermedia and thalassemia major may be associated with moderate to severe decreases in Hb concentration. (2)

Hb H inclusions

Hb H, an insoluble tetramer comprising four [beta]-globin chains, arises in the setting of [alpha]-thalassemia where the decreased production of [alpha]-globin chains leads to [beta]-globin excess. These Hb H tetramers, when oxidized in vitro, precipitate and. hence, can be visualized microscopically. Staining unfixed cells with an oxidative dye such as new methylene blue or brilliant cresyl blue generated Hb H inclusions. Because batch-to-batch variability in the dye occurs, positive and negative control slides are critical. (11) The Hb H stain is non-specific in that other nucleic-acid or protein precipitates are also stained. Hb H inclusions might be confused with reticulin and Howell-Jolly bodies. In the case of Hb H disease, a disorder in which three of four [alpha]-globin chain genes are non-expressed, 30% to 100% of red cells contain typical inclusions; in contrast, [alpha]-thalassemia minor may be associated with as few as one inclusion-containing cell in 1,000 to 10,000 cells. The absence of Hb H inclusions, therefore, does not exclude thalassemia trait, but the presence of typical inclusions may be helpful in confirming a presumptive diagnosis.
Figure 4. CAP survey results for Hb methods. Adapted CAP 2009 HB-A
Proficiency Survey.

2009

Capillary electrophoresis   7%
Cation-exchange HPLC       93%

Note: Table make from pie chart.


[FIGURE 4 OMITTED]

Electrophoretic analytical methods

Electrophoresis has been the method of choice in traditional hematological laboratories for qualitative and quantitative analyses of the various Hb fractions. Cellulose-acetate electrophoresis at alkaline pH (8.2 to 8.6), and citrate-agar or agar or-gel electrophoresis at acid pH (6.0 to 6.2) provide a clear background, allowing for the separation of the major Hbs (i.e., Hb A, Hb F, Hb S/D, Hb C/E/O-Arab) and a number of less common Hb variants by densitometry scanning. (12) Visualization of the Hb bands is made possible by staining with amido black and acid violet (or similar stains). The electrophoretic migration of Hb C, Hb E, Hb A2, and Hb O is similar at alkaline pH. Hb S, Hb D, and Hb G also co-migrate. The electrophoretic separation of Hb C from Hb E, and Hb O and Hb S from Hb D and Hb G is accomplished at acidic pH. It is not possible to differentiate between Hb E and Hb O, and Hb D and Hb G using electrophoretic methods. (2) Because of its simplicity, cellulose-acetate electrophoresis remains one of the most popular methods for Hb screening. In addition to being laborious, however, electrophoretic techniques have the disadvantage of inaccurate quantification of low-concentration Hb variants (e.g., Hb A2) or in the detection of fast Hb variants (Hb H, Hb Bart's). The precision and accuracy of HbA2 measurements using densitometric scanning of electrophoretic gels is poor, especially with the emergence of various HPLC techniques. (13)

Capillary electrophoresis is also used for Hb analysis. This methodology, offered by Sebia, utilizes liquid-flow electrophoresis for applications in the clinical-diagnostic setting to separate, detect, and quantify normal Hbs and Hb variants. Red cell hemolysates can be automatically prepared on the instrument. Samples are then loaded onto the capillary, migration occurs, and relative quantification and presumptive identification of the Hb fractions can take place. According to the College of American Pathology (CAP) 2009 HG-A proficiency surveys, 7% of clinical laboratories reporting hemoglobinopathies use capillary electrophoresis.

Isoelectric focusing

Isoelectric focusing (IEF) on agarose gels is an electrophoretic technique with excellent resolution and can be used to separate different Hb fractions and variants and globin chains. (14), (15) IEF is frequently the first analytical test used for the diagnosis of Hb fractions. If a better resolution is required, polyacrylamide gels can be used instead. IEF allows the separation of Hb variants with isoelectric points that differ by as little as 0.02 pH units. IEF allows for more precise and accurate quantification than standard electrophoresis due to the narrow bands obtained. IEF is an equilibrium process in which Hb migrates in a pH gradient to a position of 0 net charge. The Hb migration order of IEF is the same as that of alkaline electrophoresis with resolution of Hb C from Hb E and Hb O, and Hb S from Hb D and Hb G. In addition, Hb A and Hb F are clearly resolved.

Capillary isoelectric focusing

Capillary IEF (CIEF) (16-18) is a technique that combines the sensitivity of capillary electrophoresis with the automated sampling and data acquisition of HPLC. Many published works have described the utility of CIEF in the detection and quantification of Hb variants; however, its use in the clinical laboratory is limited. Separation of the Hb in this method is related to the isoelectric point of the Hb. CIEF has been used to quantify Hb variants Hb A2 and Hb F. (16)

Chromatographic analysis of Hbs and globin chains

Chromatographic methods are also widely used for Hb quantitation and initial screening of Hb variants. Cation-exchange HPLC has become the method of choice for the initial screening of Hb variants (19) (including neonatal screening where this is mandated) and for quantification of Hb A2 and Hb F concentrations, and detection of several abnormal Hbs. This method replaces electrophoretic techniques for primary screening of Hbs of clinical significance, and is at least a complementary tool for the presumptive identification of Hb variants. (20) Bio-Rad Laboratories offers automated cation-exchange HPLC instrumentation that has been widely used to quantify Hb A2, Hb F, Hb A, Hb S, and Hb C. This HPLC technique suffers, however, from intrinsic interpretive problems due to the fact that some Hb variants may co-elute with Hb A2, hence, making quantification of Hb A2 impossible in those cases. (16), (21) In individuals with Hb S, the presence of Hb S adducts falsely increases Hb A2, thus complicating the quantification of Hb A2 using cation-exchange HPLC. Recent advances in the cation-exchange HPLC technology have resulted in the addition of columns and buffers that alleviate this problem. In addition, using capillary-zone electrophoretic method (16) as well as micro anion-exchange column methodologies, has been described (22) that eliminates this interference. According to 2009 CAP HB-A proficiency surveys, ion-exchange HPLC, in which Hb species are separated based on charge differences, accounted for the majority (93%) of the methods used for the measurement of Hb and detection of hemoglobinopathies.

The quantification of Hb F is important in the diagnosis of hereditary persistence of fetal Hb, juvenile chronic myelogenous leukemia, and monosomy-7 syndrome, as well as for therapeutic monitoring in patients with sickle-cell anemia. Immunodiffusion techniques are laborious and relatively insensitive, and the densitometric scans of an alkaline-electrophoretic gel cannot detect Hb F in healthy adults or in those with marginally increased Hb F. Hb F quantities obtained by HPLC techniques tend to be lower than from published Hb F quantities from standard texts often are the result of alkali denaturation/spectrophotometry methods.

In recent years, reversed-phase HPLC (RP-HPLC) of globin chains has become an important tool for the study of Hb abnormalities. HPLC has been used to diagnose thalassemia and hemoglobinopathies, including detection of [alpha]-thalassemic genotypes in cord blood. (21) It has been used mostly to measure the [gamma]-globin chain ratios in various Hb disorders, and. in addition, liquid chromatography/mass spectrometry, or LC-MS, has been used to characterize other variant Hbs. (23)

Molecular diagnosis of hemoglobinopathies

After presumptive identification of hemoglobinopathies and thalassemia syndromes--and particularly for purposes of genetic counseling--defining the mutation or deletion present may be required. With the advent of polymerase chain reaction (PCR), the array of diagnostic tools has been expanded. As with many other genetic disorders, DNA amplification is coupled to a variety of methodologies for detecting known mutations or screening for unknown sequence alterations inside the human globin loci.

DNA is extracted from white blood cells for the molecular diagnosis of thalassemias from chorionic villus samples and from amniotic-fluid cells for prenatal diagnosis. Various low- (<20 to 30 samples/day), medium- (<100 samples/day), and high- (4,200 samples/day) throughput molecular-diagnostic techniques are available for genetic testing of Hb disorders. (12) Southern-blot hybridization of particular restriction enzyme digests to labeled complementary gene probes is typically used for the diagnosis of deletional mutations causing [alpha]-thalassemia syndromes and some rare [beta]-thalassemias. In addition, molecular methods for detecting and typing the [alpha]-thalassemia deletions typically have required the use of Southern-blot analysis. In general, laboratory diagnosis of [alpha]-/zero-thalassemia carriers is performed by the HbH-inclusion body test (HbH prep). (24) This test is laborious, observer-dependent, and reported to have poor sensitivity. Multiplex PCR has been shown to be more effective for the diagnosis of [alpha]-thalassemia than the HbH prep. It substantially increases the sensitivity of the HbH prep for the detection of [alpha]-/zero-thalassemia. The HbH prep, when used in conjunction with a low MCV, continues to have value for the diagnosis of [alpha]-/zero-thalassemia in laboratories where PCR methods are not available. (24) Previous work has also demonstrated the successful application of a gold nanoparticle-filled CE multiplex PCR method for the diagnosis of [alpha]-thalassemia deletions. DNA containing [alpha]-thalassemia deletions showed good agreement with results obtained by gel electrophoresis. (25) The identification of known globin-chain mutations/deletions, including those for Hb S, E, D, and O. and several [beta]-thalassemias arc achieved by PCR techniques using allele-specific probes after globin-gene amplification, allele-specific primers, or deletion-dependent amplification with flanking primers. (26) When a deletion mutation is not identified and there is high suspicion for a hemoglobinopathy, then sequence analysis can be used to identify point mutations or sequence variation. In addition, several PCR-based methods, including denaturing gradient-gel electrophoresis and single-strand conformation polymorphism analysis, as well as sequencing of the amplified globin gene DNA may be used for identifying unknown mutations; however, DNA sequencing remains the ultimate method for definitively identifying unknown sequence alterations. (12) A recent study utilized multiplex ligation-dependent probe amplification (MLPA) to analyze [alpha]-thalassemia patients from Southern China and concluded that MLPA was a rapid and reliable method to determine the cause of both deletional and non-deletional [alpha]-thalassemia. (27)

Neonatal screening for sickle-cell disease and other hemoglobinopathies

Neonatal screening for sickle-cell disease receives the highest recommendation from the U.S. Preventive Services Task Force (grade "A"), indicating high certainty of substantial net benefit based on evidence from well-designed, well-conducted studies in representative primary-care populations. (28) Neonatal screening is beneficial because presymptomatic diagnosis of sickle-cell disease, followed by prophylactic penicillin therapy, has been shown to reduce the incidence of pneumococcal sepsis by more than 80%. (28), (29) Ideally, prophylactic penicillin is started in infancy and is implemented with pneumococcal and other vaccines, urgent management of febrile illness, and family education about signs and symptoms of splenic sequestration. (30) Overall, the combination of newborn screening and appropriate clinical follow-up has markedly decreased childhood mortality from sickle-cell disease. For example, comparing rates for 1999 to 2002 versus those for 1983 to 1986, mortality from sickle-cell disease decreased by 68% at age 0 to 3 years (95% confidence interval 58% to 75%) and by 39% at age 4 to 9 years (95% confidence interval 16% to 56%). (31)

Because of this significant reduction of morbidity and mortality, neonatal screening for sickle-cell disease and other hemoglobinopathies has become standard practice in the United Stales. Some statewide programs initially implemented targeted screening for high-risk racial and ethnic groups, in particular infants of African, Mediterranean, Middle Eastern, (East) Indian, Caribbean, and South and Central American descent. These selective programs, however, experienced rates of missed cases as high as 30%. as well as increased administrative costs and litigation risk. Therefore, universal screening is recommended as most efficacious and cost-effective by the American Academy of Family Physicians, the American Academy of Pediatrics, and the American College of Medical Genetics, and is currently implemented in all 50 United Stales, the District of Columbia, and the U.S. Virgin Islands. (28), (29), (32)

Statewide screening programs analyze an eluate from the dried blood spot that is obtained for tests of other congenital disorders. The most common Hb testing methods are IEF and HPLC. Most programs retest abnormal specimens with a complementary electrophoretic technique, HPLC, immunologic tests, or DNA-based assays. (33), (34) Solubility tests are inappropriate for confirming sickle Hb in newborns, because high levels of fetal Hb give false-negative results. The sensitivity and specificity of current statewide screening systems are extremely high, approaching 100%. (28) Still, rare cases of hemoglobinopathy remain undetected at birth--even in states with universal screening--mainly due to preanalytical errors that include failure to offer testing, requisition or specimen mislabeling, extreme prematurity with lack of adult Hb, and blood transfusion prior to screening. (29), (33)

By convention, Hbs identified by neonatal screening are reported in order of expression level. At birth, normal infants (homozygous AA) express a majority of fetal Hb (Hb F) and a minority of adult Hb (Hb A) and. thus, would be characterized as Hb FA. By analogy, infants with a homozygous hemoglobinopathy would be characterized as Hb F-Variant (e.g., Hb FS for homozygous sickle-cell disease). Those with a heterozygous hemoglobinopathy trail would generally be characterized as Hb FA-Variant (e.g., Hb FAS for sickle-cell trait).

A range of sickle-cell disease variants can be detected and differentiated by neonatal screening (29), (33) (see Table 1). Hb FS in infancy is compatible with several genotypes, implying a wide range of future clinical severity. Most infants with FS-screening results have homozygous SS, but other genotypes including sickle/[beta]-zero thalassemia, sickle/[beta]-plus thalassemia, and sickle/delta-[beta] thalassemia are possible. In addition, compound heterozygosity for sickle Hb and hereditary persistence of fetal Hb (S/HPFH) produces an FS phenotype at birth. Although relatively uncommon, S/HPFH is very important to distinguish from sickle-cell disease, since it is a benign trait condition. It can be suspected by an absence of clinical and hematological effects, and by follow-up Hb analysis showing persistent Hb F beyond 6 to 12 months of age. Newborn-screening algorithms are capable of identifying sickle-cell-disease variations caused by compound heterozygosity of Hb S and other mutant Hbs (e.g., FSC, FSC-Harlem. FSD-Punjab, FSO-Arab), or by sickle/[beta]-plus thalassemia with Hb S predominant over Hb A (i.e., FSA).
Table 1. Sickle-cell disease variants: typical screening and diagnostic
test results.

Disorder     % of   Hemoglobins  Hemoglobins  Vasoocclusive
             cases  at neonatal    at two       disease and
               in     screen       months        hemolytic
             U.S.                                 anemia

SS             65     FS              FS      Severe (by one year) *

SC             25     FSC             FSC     Mild-Moderate (by one
                                                 years)

S               8     FSA or FS       FSA        Mild-Moderate
[beta]-plus                                      (by two
thalassemia                                      years)

S               2     FS              FS         Severe (by one
[beta]-zero                                      year)
thalassemia

S HPFH          1     FS              FS         None

             Hematologic studies by two years
Disorder     MCV         Hemoglobin  Hemoglobin
                         [A.sub.2]      F %
                           (%)

SS           Normal or      <3.5       <25
             Increased

SC           Normal or      <3.5       <15
             Decreased

S            Decreased      >3.5       <25
[beta]-plus
thalassemia

S            Decreased      >3.5       <25
[beta]-zero
thalassemia

S HPFH       Normal or      <2.5       May be
             Decreased                 >25


All infants with Hb S detected by newborn screening should have confirmatory tests of a second blood sample prior to 2 months of age, to allow early detection of sickle-cell disease. Infants with Hb FS and other significant sickle hemoglobinopathies should begin penicillin prophylaxis by 2 months of age, and parents should be educated about the importance of urgent medical evaluation and treatment for febrile illness, and for signs and symptoms of splenic sequestration. (29), (33) Clinical and laboratory evidence of sickle-cell disease is rare before 2 months of age due to continued expression of fetal Hb, which inhibits polymerization of sickle Hb. Postnatal suppression of Hb F occurs at a variable rate in infants with sickle-cell disease. Therefore, some cases may be difficult to distinguish from S/HPFH, in which case parental testing and/or DNA analysis may be helpful if clinically indicated. (29)

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Screening for other hemoglobinopathies and [beta]-thalassemias

Neonatal screening can also identify infants with non-sickle hemoglobinopathies, some of which may be severe and require transfusion therapy (29), (33) (see Table 2), For example, infants expressing only Hb F at birth may have the disabling condition [beta]-thalassemia major. This neonatal phenotype, however, may also be compatible with a normal Hb genotype in premature infants with a lack of Hb A production. Therefore, premature infants without Hb A need repeal testing to confirm eventual production of adult Hb. Neonatal screening does not detect most infants with milder [beta]-thalassemia syndromes (i.e., [beta]-thalassemia minor and [beta]-thalassemia intermedia), since some Hb A is produced. Infants with FE require family studies. DNA analysis, or repeated hematologic evaluation during the first one to two years of life to differentiate homozygous Hb E. which is asymptomatic, from Hb E [beta]-zero-thalassemia, which is a variably severe hemolytic anemia. (35-37)
Table 2. Non-sickle hemoglobinopathies identified by neonatal screening.

Hb atl           Possible conditions      Clinical manifestations
neonatal
screen

F only    * [beta]-zero thalassemia    * Severe microcytic hemolytic
            major                        anemia

          * Premature Infant           * Repent screenung is indicated

FE        * EE                         * Mierocytosis with mild or no
                                         anemia

          * E [beta]-zero thalassemia  * Mild to severe anemia

FC        * CC                         * Mild microcytic hemolytic
                                         aneima

          * C [beta]-zero thalassemia  * Mild microcytic hemolytic
                                         anemia

FCA       * C [beta]-plus thalassemia  * Mild microcytic anemia


Screening for [alpha]-thalassemias

The red cells of newborns with [alpha]-thalassemia syndromes contain Hb Ban's, a tetramer of gamma globin that is detected and reported in most neonalal-screcning programs (33), (34), (38), (39) (see Table 3). Infants with Hb Bart's at birth may have a "silent carrier" state (deletion of one [alpha].-globin gene), [alpha]-thalassemia trail (deletion of two genes), or Hb H disease (loss of three genes). Loss of all four [alpha]-globin genes (hydrops fetalis) is incompatible with intrauterine survival and, thus, is not seen in subjects of newborn screening. Silent carriers are the largest group with Hb Ban's at birth. They have small amounts of Hb Bart's and do not develop any clinical or laboratory manifestations. Persons with [alpha]-thalassemia trait generally show a decreased MCV with mild or no anemia. Newborns with >10% Hb Bart's by IEF. or >30% Hb Barfs by HPLC. and/or who develop more severe anemia may have Hb H disease. (33), (34), (38), (39) The identification of Hb Bart's in Asian infants can have important genetic implications for couples, since the cis deletion of both a genes on a single chromosome 16 is common in this ethnic group. Thus, couples may be at risk for subsequent pregnancies complicated by hydrops fetalis. (34).
Table 3. [alpha]-thalassemias identified by neonatal screening

Screening  Possible condition     Clinical
results                           manifestations

FA+Barts   * [alpha]-thalassemia  * Normal CBC
           "silent carrier"

           * [alpha]-thalassemia  * Microcytosis with
           trait                  muld or no anemia

           * Hemoglobin H         * Mild-moderate
           disease                microcytic hemolytic
                                  anemia

FAS+Barts  * [alpha]-thalassemia  * Clinical
           with structural        manifestations
           hemoglobin variant     depend on structural
                                  variant and severity
                                  of [alpha]-thalassemia

FAC+Barts

FAE+Barts

FE+Barts


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(3.) Vichinsky E. Hemoglobin e syndromes. 2007;79-83.

(4.) Rubin, LP, Hansen K Testing for hematologic disorders and complications. Clin Lab Med. 2003; 23(2):317-343.

(5.) Steinberg MH. Pathophysiology of sickle-cell disease. Baillieres Clin Haematol. 1998; 11(1):163-184.

(6.) Lane PA. Sickle-cell disease. Pediatr Clin North Am. 1996;43(3):639-664.

(7.) Chui DH, Waye JS. Hydrops fetalis caused by alpha-thalassemia: an emerging health care problem. Blood. 1998;91171:2213-2222.

(8.) Weatherall DJ, Clegg JB Thalassemia--a global public health problem. Nat Med. 1996;2(8):847-849,

(9.) Colah RB, et al. HPLC studies in hemoglobinopathies. Indian J Pediatr. 2007;74(7):657-662.

(10.) Lafferty JD, et al. The evaluation of various mathematical RBC indices and their efficacy in discriminating between thalassemic and non-thalassemic microcytosis. Am J Clin Pathol. 1996; 106(2):201-205.

(11.) Hall RBH. Guerra JA, Castleberry CG, et al. Optimizing the detection of hemoglobin H disease. Lab Med. 1995;26:736-741.

(12.) Patrinos GP, Kollia P. Papadakis, MN. Molecular diagnosis of inherited disorders: lessons from hemoglobinopathies. Hum Mutat. 2005;26(5):399-412.

(13.) Papadea C, Gate JC. Identification and quantification of hemoglobins A, F, S, and C by automated chromatography. Clin Chem 1996;42(1):57-63.

(14.) Campbell M, Henthorn JS, Davies SC. Evaluation of cation-exchange HPLC compared with isoelectric focusing for neonatal hemoglobinopathy screening. Clin Chem 1999;45(7): 969-975.

(15.) Turpeinen U, et al. Two alpha-chain hemoglobin variants, Hb Broussais and Hb Cemenelum, characterized by cation-exchange HPLC, isoelectric focusing, and peptide sequencing. Clin Chem. 1995;41(4):532-536.

(16.) Cotton F, et al. Evaluation of a capillary electrophoresis method for routine determination of hemoglobins A2 and F. Clin Chem. 1999;45(2):237-243.

(17.) Hempe JM, Graver RD. Quantification of hemoglobin variants by capillary isoelectric focusing. Clin Chem. 1994:40(12):2288-2295.

(18.) Mario N, et at. Capillary isoelectric focusing and high-periormance cation-exchange chromatography compared for qualitative and quantitative analysis of hemoglobin variants. Clin Chem. 1997:43(11):2137-2142.

(19.) Eastman JW, et al. Automated HPLC screening of newborns for sickle-cell anemia and other hemoglobinopathies. Clin Chem. 1996;42(5):704-710.

(20.) Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. Clin Chem. 2004;50(10):1736-1747.

(21.) Fucharoen S, et al. Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC. Clin Chem. 1998;44(4):740-748.

(22.) Suh DD, Krauss JS, Bures K. Influence of hemoglobin S adducts on hemoglobin A2 quantification by HPLC. Clin Chem. 1996;42(7)-113-1114.

(23.) Shackleton CH, Witkowska HE. Characterizing abnormal hemoglobin by MS. Anal Chem. 1996; 68(1):29A-33A.

(24.) Bergstrome JA, Poon A. Evaluation of a single-tube multiplex polymerase chain reaction screen for detection of common alpha-thalassemia genotypes in a clinical laboratory. Am J Clin Pathol. 2002; H8(1):18-24.

(25.) Chen YL, et al. Genotyping of alpha-thalassemia deletions using multiplex polymerase chain reactions and gold nanoparticle-filled capillary electrophoresis. J Chromatatogr A 2009;1216(7):1206-1212.

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(27.) Liu JZ, et al. Detection of alpha-thalassemia in China by using multiplex ligation-dependent probe amplification. Hemoglobin. 2008;32(6):561-571.

(28.) U.S. Department of Health and Human Services. Agency for Healthcare Research and Duality. Screening for Sickle-cell disease in Newborns: Recommendation Statement U.S.PS.T.F. (USPSTF) Editor. 2007.

(29.) Kaye CI, et al. Newborn screening fact sheets. Pediatrics. 2006:118(3): e934-e963.

(30.) Consensus conference. Newborn screening for sickle-cell disease and other hemoglobinopathies. JAMA. 1987;258(9):1205-1209.

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(34.) Pass KA, et al. US newborn screening system guidelines II: follow-up of children, diagnosis, management, and evaluation. Statement of the Council of Regional Networks for Genetic Services (CORN). J Pediatr. 2000:137(4 Suppl):Sl-46.

(35.) Krishnamurti L, et al. Coinheritance of alpha-thalassemia-1 and hemoglobin E/beta zero-thalassemia: practical implications for neonatal screening and genetic counseling. J Pediatr. 1998; 132(5):863-865.

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(37.) Premawardhena A, et al. Hemoglobin E-beta-thalassemia: Progress report from the International Study Group. Ann N YAcad Sci. 2005;1054:33-39.

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(39.) Miller ST, et al. A fast hemoglobin variant on newborn screening is associated with alpha-thalassemia trait. Clin Pediatr. (Phila) 1997;36(2):75-78.

(40.) Old JM, Screening and genetic diagnosis of haemoglobin disorders. Blood Rev. 2003;17(1):43-53.

HEMOGLOBINOPATHIES AND CLINICAL LABORATORY TESTING

MLO and Northern Illinois University (NIU), DeKalb, IL, are co-sponsors in offering continuing education units (CEUs) for this issue's article on HEMOGLOBINOPATHIES AND CLINICAL LABORATORY TESTING. CEUs or contact hours are granted by the College of Health and Human Sciences at NIU, which has been approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS PACE." program (Provider No. 0001) and by the American Medical Technologists Institute for Education (Provider No. 121019; Registry No. 0061). Approval as a provider of continuing education programs has been granted by the state of Florida (Provider No. JP0000496), and for licensed clinical laboratory scientists and personnel in the state of California (Provider No. 351). Continuing education credits awarded for successful completion of this test are acceptable for the ASCP Board of Registry Continuing Competence Recognition Program. After reading the article, answer the following test questions and send your completed test form to NIU along with the nominal fee of S20. Readers who pass the test successfully (scoring 70% or higher) will receive a certificate for 1 contact hour of P.A.C.E.[R] credit. Participants should allow four to six weeks for receipt of certificates.

The fee for this continuing education test is $20.

The Cover Story and Clinical Issues published in this month's MLO are peer-reviewed.

Cover story learning objectives and CE questions were prepared by Jeanne M. Isabel, MSEd, CLSpH(NCA), MT(ASCP), CLS Associate Professor, School of Allied Health and Communicative Disorders, Northern Illinois University in Dekalb, IL.

CE QUESTIONS

1. Hemoglobin is a metalloprotein containing--heme groups.

a. two

b. three

c. four

d. five

2. Beta globin chains are found on chromosome

a. 11.

b. 16.

c. 20.

d. 22.

3. In the healthy neonate, the predominant hemoglobin is

a. Gower 1.

b. Hb A.

c. Hb A2.

d. Hb F.

4. The term for hemoglobinopathies arising from decreased synthesis of globin chains is

a. qualitative.

b. quantitative.

c. semi-quantitative.

5. The hemoglobin structure variant for Hb C is

a. [beta]6(A3) Glu>Val.

b. [beta]6(A3) Glu>Lys.

c. [beta]26(B8) Glu>Lys.

d. none of the above.

6. Consequences of hemolysis from sickle-cell disease include

a. chronic anemia.

b. jaundice.

c. cholelithiasis.

d. all of the above.

7. Thalassemia is classified by which adult globin gene is dysfunctional.

a. TRUE

b. FALSE

8. Loss of three [alpha]-globin genes results in

a. no hematologic effect.

b. thalassemia trait.

c. Hb H disease.

d. Bart's hydrops fetalis.

9. The method of choice for quantification and identification of hemoglobin variants is

a. IEF.

b. HPLC.

c. solubility.

d. electrophoresis.

10. Which of the RBC indices is considered a key diagnostic indicator of thalassemia?

a. MCH

b. MCHC

c. MCV

d. MPV

11. Hemoglobin H inclusions may be seen in stained cells and are comprised of

a. DNA.

b. RNA.

c. [beta]-globin tetramers.

d. reticulum.

12. Hemoglobin electrophoresis as a screening tool to separate major hemoglobins results in some co-migration. Separation of Hb C from HbE is accomplished at

a. acid pH.

b. alkaline pH.

13. The method by which hemoglobin migrates in a pH gradient to a position of 0 (zero) negative charge is

a. HPLC.

b. IEF.

c. PCR.

d. MLPA.

14. Prenatal molecular diagnosis of hemoglobinopathies utilizes DNA extracted from

a. amniotic-fluid cells.

b. white blood cells.

c. chorionic villi samples.

d. a and b.

e. a and c.

15. Neonatal screening provides presymptomatic diagnosis of sickle-cell disease and is followed by prophylactic ampicillin therapy.

a. TRUE

b. FALSE

16. Conditions that produce an FS phenotype on screening at birth are

a. sickle-cell disease.

b. sickle/[beta]-zero thalassemia.

c. sickle/[beta]-plus thalassemia.

d. sickle/delta-[beta] thalassemia.

e. all of the above.

17. State screening programs analyze dried blood spots collected from infants by

a. eluate.

b. dilution.

c. culture media.

d. concentration.

18. PCR techniques for identification of globin-chain mutations or deletions include

a. allele-specific probes.

b. allele-specific primers.

c. amplification with flanking primers.

d. all of the above.

19. According to 2009 CAP proficiency surveys, HPLC accounted for 75% of the methods used for detection of hemoglobinopathies.

a. TRUE

b. FALSE

20. The technique that combines electrophoresis with automated sampling is

a. CIEF.

b. PCR.

c. RBC indices.

d. HPFH.

MLO'S Continuing Education Test is also online.

Both the CE test and a convenient payment feature are available through the auspices of Northern Illinois University.

Go to www.mlo-online.com/CE.aspx to print or to send electronically with payment.

By Charbel Abou-Diwan, PhD; Andrew N. Young, MD, PhD; and Ross J. Molinaro, MT(ASCP), PhD, D(ABCC), F(ACB)

All three authors work in Atlanta at Emory University's School of Medicine, Department of Pathology and Lab Medicine. Charbel Abou-Diwan, PhD, is a clinical chemistry post-Doctoral Fellow there. Andrew N. Young, MO, PhD, is an associate professor there and medical director of the clinical laboratory at Grady Hospital, Ross J. Molinaro, MT(ASCP), PhD, D(ABCC), F(ACB), is an assistant professor in the department as well as medical director of the core laboratory at Emory University Hospital Midtown in Atlanta.
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Article Details
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Title Annotation:COVER STORY
Author:Abou-Diwan, Charbel; Young, Andrew N.; Molinaro, Ross J.
Publication:Medical Laboratory Observer
Article Type:Cover story
Geographic Code:1USA
Date:Aug 1, 2009
Words:6845
Previous Article:Conferences.
Next Article:Acute-care testing at the point of care: now and in the future.
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