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1
Dynacare Kasper Medical Laboratories, 14940 123rd Ave., Edmonton, Alberta T5V 1B4, Canada.
a Author for correspondence. Fax 780-452-8488; e-mail higgins{at}dkml.com
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Abstract |
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 Top Abstract IntroductionMethods of AnalysisClinical ManifestationsReferences |
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-chain production are called -thalassemias. Severity of clinical
manifestations in these disorders relates to the amount of globin chain
produced and the stability of residual chains present in excess. The
thalassemia minor syndromes are characterized clinically by mild anemia
with persistent microcytosis. Thalassemia intermedia (i.e., Hb H
disease) is typified by a moderate, variably compensated hemolytic
anemia that may present with clinical symptoms during a period of
physiologic stress such as infection, pregnancy, or surgery. The
thalassemia major syndromes produce severe, life-threatening anemia.
-Thalassemia major usually is incompatible with extrauterine life;
ß-thalassemia major presents in infancy and requires life-long
transfusion therapy and/or bone marrow transplantation for successful
control of the disease. Double heterozygosity for certain structural
variants and/or thalassemia syndromes may also lead to severe clinical
disease. Several guidelines have been published that outline the
required steps for hemoglobinopathy and thalassemia
investigation. The availability of HPLC has streamlined
many of these requirements, allowing an efficient stepwise
diagnostic strategy for these complex disorders. |
Introduction |
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TopAbstractIntroductionMethods of AnalysisClinical ManifestationsReferences |
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plus ß, 
, or 
), which form a shell
around a central cavity containing four oxygen-binding heme groups each
covalently linked to a globin chain. In healthy adults, ~95% of the
Hb is Hb A (2ß2) with
small amounts (<3.5%) of Hb A2
(22) and Hb F
(22) present. During
embryonic development, "pre alpha" 
globin chains contribute to
embryonic Hb. During fetal development, ß-like globin chains 
and
contribute to the Hb (1).
The -globin chain is encoded in duplicate on chromosome 16, and the
non- chains (ß, , ) are encoded in a cluster on chromosome
11. A diploid cell therefore has four -globin genes and two ß-like
genes. The and ß chains consist of 141 and 146 amino acid
residues, respectively. There is some sequence homology between the two
chains (64 individual amino acid residues in identical positions), and
the ß chain differs from the and chains by 39 and 10
residues, respectively.
Although hemoglobinopathies and thalassemias are two genetically
distinct disease groups, the clinical manifestations of both include
anemia of variable severity and variable pathophysiology. The
thalassemias are characterized by a reduction in the amount of the
normal globin chain produced. This diminution in globin chain
production may result from gene deletion or from mutations that
adversely affect the transcription or stability of mRNA products. The
manifestations range from mild anemia with microcytosis (thalassemia
trait) to fatal severe anemia (Hb Barts hydrops fetalis or
ß-thalassemia major). The hemoglobinopathies, or structural Hb
variants, are attributable to amino acid substitution in either the
or non- chain. More than 700 hemoglobinopathies have been described
to date (2), the majority of which are clinically benign and
fortuitously discovered. The clinically significant hemoglobinopathies
are attributable to amino acid substitutions, primarily in the non-
chain, that bring about changes in the secondary and tertiary structure
of the Hb tetramer. These substitutions are most common at positions in
close proximity to either heme group or globin chain attachment sites.
Hemoglobinopathy nomenclature is an assortment of letters (i.e., Hb S,
C, and D), place names denoting the site of first discovery or
residence of the propositus (e.g., Hb Edmonton), and family names of
the index case (e.g., Hb Lepore). A systematic nomenclature that is
both logical and informative identifies the chain, the location, and
the amino acid substitution on the involved globin chain. Thus Hb
Alberta (ß101 Ala
Pro) is a substitution of proline for alanine
(the normal amino acid) in the 101st position of the ß chain. In the
heterozygous state, the normal Hb is placed first, followed by the
variant, e.g., AS trait.
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Methods of Analysis |
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TopAbstractIntroductionMethods of AnalysisClinical ManifestationsReferences |
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Although electrophoresis at alkaline and acid pH has been used for many years, cation-exchange HPLC (3)(4)(5)(6)(7)(8)(9)(10) is emerging as the method of choice for quantification of Hb A2 and Hb F and identification of Hb variants (11)(12). The use of HPLC streamlines the recommended preliminary and follow-up tests for the identification of hemoglobinopathies and thalassemias and provides for rapid and complete diagnostic work up in a majority of cases. The elements of one approach 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. The relevance of each of these analytic components is discussed below.
cbc
Structural hemoglobinopathies may have an impact on the red cell
indices, and red cell indices are critical to the diagnosis of
thalassemias. The key components of the CBC include: Hb, red
blood cell (RBC) number, mean corpuscular volume (MCV), and red cell
distribution width (RDW).
The thalassemias generally are classified as hypochromic and microcytic anemias. Hence the MCV is a key diagnostic indicator. Virtually all automated hematology analyzers now provide a measurement of MCV that is both precise and accurate. This cell volume, reported in femtoliters, in most adult populations ranges from ~80 to 100 fL. Thalassemic individuals have a reduced MCV, and one study has suggested that an MCV of 72 fL is maximally sensitive and specific for presumptive diagnosis of thalassemia syndromes (13).
The RDW is a measure of the degree of variation in red cell size. Some
causes of microcytic anemia, most notably iron deficiency, are
characterized by an increase in RDW. The thalassemias, in contrast,
tend to produce a uniform microcytic red cell population without a
concomitant increase in RDW. This observation is variable among the
thalassemia syndromes, however, with notable increases in RDW in the
setting of Hb H disease and ß-thalassemia minor (1).
Therefore, the RDW may provide information useful as an adjunct to
diagnosis but is not useful as a lone indicator.
The RBC count is also useful as a diagnostic adjunct because the thalassemias produce a microcytic anemia with an associated increase in the RBC number. 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 typically is 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.
Various indices utilizing these CBC components have been developed with a view to providing a mathematical derivation to reliably differentiate iron deficiency from thalassemia minor. None are useful in all clinical settings, and probably none exceed the value of the MCV alone in selecting cases for subsequent investigations (13).
Hb H INCLUSIONS
Hb H refers to an insoluble Hb tetramer comprising four ß-globin
chains. Hb H arises in the setting of -thalassemia where the
decreased production of -globin chains leads to ß-globin excess.
Oxidation of these tetramers provokes precipitation, which can be
visualized microscopically. In vitro generation of Hb H inclusions is
accomplished by staining unfixed cells with an oxidative dye such as
New Methylene Blue or Brilliant Cresyl Blue. Because batch-to-batch
variability in the dye occurs, positive and negative control slides are
critical (1)(14)(15).
Various authorities differ with respect to the necessity for freshly obtained blood samples (14)(15). Our own unpublished data suggest that freshly collected blood is not critical. Blood film examination is undertaken with a search for cells with typical "golf ball" inclusions.
In the setting of Hb H disease, a disorder in which three of four
-globin chain genes are nonexpressed, 30–100% of red cells contain
typical inclusions. In contrast, -thalassemia minor may be
associated with as few as 1 inclusion-containing cell in 1000–10 000
cells (14). 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.
The Hb H stain is nonspecific in that other nucleic acid or protein precipitates also stain. Reticulin and Howell Jolly bodies do not have the distinctive pattern of Hb H inclusions; however, a brisk reticulocytosis can make identification of a rare H-containing cell difficult.
electrophoresis
Traditionally, electrophoresis has been the method of choice for
identification and quantification of variant Hbs. Commercial, rapid
electrophoretic methods have been developed that allow for separation
at pH 8.4 (alkaline) and pH 6.2 (acid) on agarose gels. These provide a
clear background, allowing for quantification of the Hb present by
densitometric scanning. Visualization of the Hb bands is made possible
by staining with Amino Black and Acid Violet (or similar stains). At
alkaline pH, electrophoretic migration of Hb C, Hb E, Hb
A2, and Hb O is similar. Hb S, Hb D, and Hb G
also comigrate. At acid pH, electrophoretic separation of Hb C from Hb
E, and Hb O and Hb S from Hb D and Hb G is accomplished. It is not
possible to differentiate between Hb E and Hb O, and Hb D and Hb G
using electrophoretic methods.
Electrophoresis, is slow, labor-intensive, and inaccurate in the quantification of low-concentration Hb variants (e.g., Hb A2) or in the detection of fast Hb variants (Hb H, Hb Barts).
The precision and accuracy of Hb A2 measurements using densitometric scanning of electrophoretic gels is poor, especially when compared with HPLC techniques (8). A recent College of American Pathologists (16) hemoglobinopathy survey showed a CV of 33.6% for densitometric scanning of electrophoretic gels at a Hb A2 concentration of 2.41%. For column chromatography, the CV was 14.6% at a mean Hb A2 concentration of 3.21%, and for HPLC, the CV was 4.3% at a mean Hb A2 of 3.47% (16).
Despite the imprecision, 296 of 387 laboratories participating in the College of American Pathologists Hemoglobinopathy Survey program reported results for Hb F, Hb A2, and Hb identification using electrophoretic methods. Some laboratories, including ours, use a combination of electrophoresis and HPLC to identify and quantify Hb.
ief
IEF is an electrophoretic technique with excellent resolution.
Although labor-intensive and time-consuming, it has been used to
identify and quantify Hbs (9)(17). 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. The narrow bands obtained on IEF allow for more precise and
accurate quantification than standard electrophoresis.
capillary ief
Capillary IEF (CIEF)
(5)(18)(19) is a hybrid
technique combining the capillary electrophoresis sensitivity of
detection 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. Separation of the Hb in
this method is related to the isoelectric point of the Hb, and this may
enhance interlaboratory reproducibility.
CIEF has been used to quantify Hb variants Hb A2 and Hb F (18). One comparison (5) of CIEF with cation-exchange chromatography for the qualitative and quantitative analysis of Hb variants found that quantitative data between the two methods were highly correlated and that CIEF gave slightly better resolution of the unusual variants Hb C Harlem and Hb D Punjab.
hplc
Cation-exchange HPLC is emerging as the method of choice
for the initial screening of Hb variants (4) (including
neonatal screening where this is mandated) and for quantification of Hb
A2 and Hb F concentrations. The Bio-Rad Variant
(Bio-Rad Laboratories) is an automated cation-exchange HPLC
instrument that has been used to quantify Hb A2,
Hb F, Hb A, Hb S, and Hb C. College of American Pathologists studies
have shown equivalence or superiority over electrophoretic methods.
HPLC is not without intrinsic interpretive problems. Hbs may co-elute or may elute before instrument peak integration. Using the Bio-Rad Variant ß Thal short program, we have found that Hb E, Hb Osu Christianbourg, and Hb G Copenhagen co-elute with Hb A2, making Hb A2 quantification impossible on samples with these Hb variants, a problem noted by others (6)(18).
The measurement of Hb A2 using cation-exchange HPLC is complicated in individuals with Hb S (20)(21) because the Hb A2 is falsely increased by the presence of Hb S adducts. A capillary zone electrophoretic method has been discussed for quantification of Hb A2 in the presence of Hb S that eliminates interference from these adducts (18). A micro anion-exchange column methodology that eliminates this interference has also been described (21).
In individuals with Hb D trait (22)(23), the Hb A2 value obtained with the Bio-Rad Variant is falsely decreased because of an integration error. Correction for the rising baseline produces Hb A2 values similar to those obtained by anion chromatography.
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. Published Hb F quantities from standard texts often are the result of alkali denaturation/spectrophotometry methods and are higher than those obtained by HPLC techniques. Immunodiffusion techniques are labor-intensive and relatively insensitive. Densitometric scans of an alkaline electrophoretic gel cannot detect Hb F in healthy adults or in those with marginally increased Hb F.
Reversed-phase HPLC has been used as an enhancement for electrospray
ionization spectroscopy in the characterization of Hb Rambam
(ß69 GlyAsp) (24). This represents an additional novel
use of HPLC in Hb variant diagnosis. Liquid chromatography/mass
spectroscopy has been used to characterize other variant Hbs
(25).
HPLC has been used to diagnose thalassemia and hemoglobinopathies,
including detection of -thalassemic genotypes in cord blood
(6).
The presence of a structural Hb variant may adversely affect the measurement of Hb A1C (24)(26)(27)(28)(29). Hb Niigata (28) gives inappropriately high Hb A1C concentrations using HPLC and low results using a latex immunoagglutination method. Hb Sherwood Forest (29), Rambam (24), and Raleigh (27) produce falsely increased Hb A1C results when measured by cation HPLC.
dna analysis
After presumptive identification of hemoglobinopathies and
thalassemia syndromes, and particularly for purposes of genetic
counseling, defining the mutation or deletion present may be required.
Several molecular techniques are available.
DNA from white blood cells, amniocytes, or chorionic tissue may be
utilized for diagnosis of various - and ß-globin chain
abnormalities. Typically, deletional mutations causing -thalassemia
syndromes and some rare ß-thalassemias are diagnosed using Southern
blot hybridization of particular restriction enzyme digests to labeled
complementary gene probes.
PCR techniques using allele-specific probes after globin gene amplification, allele-specific primers, or deletion-dependent amplification with flanking primers are used in definition of known globin chain mutations/deletions, including those for Hb S, E, D, and O, and several ß-thalassemias (14)(30)(31)(32)(33)(34)(35)(36)(37)(38).
For unknown mutations, 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 (37).
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Clinical Manifestations |
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TopAbstractIntroductionMethods of AnalysisClinical ManifestationsReferences |
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sickling disorders
The prototype Hb variant associated with a sickling disorder is Hb
S. The point mutation in the ß-globin gene that produces Hb S exerts
its effect by causing precipitation and polymerization of the
deoxygenated Hb S with resulting sickling of the red cells. These
sickled cells lack deformability, occlude the microvasculature, and
lead to tissue infarctions, which manifest as painful sickle cell
crises. The permanently deformed cells are subsequently removed from
the circulation well before the usual 120-day life span of a healthy
red cell, contributing to a chronic hemolytic anemia. The clinical
manifestations occur only in individuals with homozygous sickle cell
disease. Heterozygotes are said to have sickle cell trait and are
symptom free. Sickle cell trait is common (8% of the African-American
population) and important from a genetic counseling perspective. Double
heterozygosity for certain variants (e.g., Hb S/Hb D Los Angeles, Hb
Montgomery/Hb S) that occur with appreciable frequency in the same
ethnic populations as Hb S may also produce significant sickling
disease (25)(36)(39)(40).
Diagnosis of sickle cell trait and disease depends on a typical HPLC
(Fig. 1
) and Hb electrophoretic pattern. In the case of sickle cell
disease, sickled cells may be apparent on peripheral blood film
examination, and sickling screens (in which a hemolysate is exposed to
a reducing agent) are positive. Sickle cell trait also produces a
positive sickle cell screen. The Hb F concentration is variable in
sickle cell anemia, and increased proportions of Hb F may mitigate the
clinical effects. The focus of several treatment regimens is an
increase in Hb F. Ongoing quantitative assessment of Hb F and Hb S
concentrations is therefore frequently required. For transfusion
therapy, the quantification of Hb A is also monitored.
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Hb C, resulting from a separate single base pair mutation in the ß-globin gene, causes a hemolytic anemia of mild to moderate severity in the homozygous form. Heterozygotes are not symptomatically affected. Peripheral blood film analysis may reveal frequent target cells (Target cells are discoid RBCs that have a central hemoglobinized area in the clear center, producing a target appearance. They result from an increase in the ratio of cell surface area to cell volume.) and an increase in polychromasia. HPLC and electrophoretic patterns are diagnostic. Double heterozygosity for ß C and ß S Hb leads to a severe sickling disorder.
Similarly, Hb O Arab and Hb D Punjab result from point mutations of the ß-globin gene. Although both heterozygous and rare homozygous individuals may be diagnosed on the basis of HPLC and electrophoretic analysis, clinical manifestations are minimal. The major clinical impact of these hemoglobinopathies results from co-inheritance with Hb S, which occurs in a similar population and which if inherited with either Hb O Arab or Hb D Punjab may produce a severe sickling disorder.
thalassemic disorders
Hemoglobinopathies that lead to decreased production of globin
chains ( or ß) produce a clinical syndrome characterized by anemia
of variable severity with hypochromic and microcytic red cells
(41)(42).
The - and ß-thalassemias result, respectively, from deletions or
mutations of the - and ß-globin genes that lead to decreased
transcription or translation of the gene product. In addition to
decreased Hb production, however, manifestations of the thalassemias
are complicated by the resulting chain imbalance: In the
-thalassemias, ß- and -globin chains are produced in excess.
These proteins associate to form unstable tetramers (ß4 = Hb H;
4 = Hb Barts), which precipitate in red cell precursors and
lead to ineffective erythropoiesis with bone marrow expansion and
splenomegaly. In the ß-thalassemias, similarly, -chain excess
leads to ineffective erythropoiesis and interference with erythroid
maturation. In general, the more genes affected, the more significant
the complications of the disorder. In the case of -thalassemia, a
four-gene deletion produces Hb Barts hydrops fetalis, a disorder almost
always incompatible with postnatal life. Pregnant women with an
affected fetus may present in the late second or third trimester of
pregnancy with complications of pregnancy and/or fetal distress. The
fetus typically is hydropic and severely anemic, and frequently in
these cases, prenatal diagnosis is required. The HPLC chromatogram is
diagnostic, showing a typical early elution peak representing Hb Barts
(Fig. 1a
). Review of parental blood films shows features
typical of -thalassemia trait with hypochromia, microcytosis, and an
absence of Hb variants or any increases in Hb A2
or F. The Hb H preparation typically is strongly positive in the
afflicted fetus, with inclusions representing precipitated Hb Barts,
whereas parental blood films stained with Brilliant Cresyl Blue would
be expected to show only rare H-containing cells.
A three-gene deletion of -globin produces a moderately severe
chronic hemolytic anemia characterized by multiple Hb H-containing
cells, hypochromia, microcytosis, and a typical HPLC pattern (Fig. 1d
). Once again, family studies are helpful in diagnosis with
parental features typical of -thalassemia trait expected.
A two -globin gene deletion produces hypochromia and microcytosis
with a normal or mildly decreased Hb concentration and, frequently, an
erythrocytosis. A single -globin gene deletion is clinically and
hematologically silent.
ß-Thalassemias usually result from mutations that affect transcription, translation, or RNA stability (41). When a single ß-globin gene is affected, ß-thalassemia minor results. Erythrocytosis and a mild hypochromic microcytic anemia characterize this heterozygous condition. HPLC analysis shows an increase in Hb A2 and, in some cases, Hb F. Homozygous ß-thalassemia may lead to a marked reduction (ß+-thalassemia) or absence (ß0-thalassemia) of normal ß-chain production. Affected individuals typically present midway through the first year of life with a severe hypochromic microcytic anemia. HPLC and electrophoretic findings include absence or marked reduction in Hb A with a variable Hb F concentration. Interestingly and unlike the pattern in the heterozygotes, Hb A2 is not increased in homozygous ß-thalassemia (42).
Certain structural Hb variants also have thalassemic manifestations.
The most common of these, seen almost exclusively in South East Asians,
is Hb E. Hb E is synthesized at a slower rate than is normal
ß-globin; hence an individual with Hb E trait has hypochromic and
microcytic red cell indices. HPLC and electrophoretic patterns are
diagnostic. The primary clinical importance of Hb E trait is the risk
of co-inheritance with the thalassemia syndromes, which occur in the
same population groups. An Hb E ß-thalassemic double heterozygote
exhibits the clinical syndrome of thalassemia major (42)
(Fig. 1c
).
Hb Lepore is another structural Hb variant that leads to decreased globin chain production with a hypochromic microcytic anemia in the heterozygote and a typical electrophoretic and HPLC pattern.
Additional clinical subgroups of the hemoglobinopathies include those producing cyanosis, erythrocytosis, and a small number of variants that produce unstable Hb tetramers. These variants may be apparent on HPLC but typically produce clinical manifestations that would lead to alternative diagnostic testing, including co-oximetry, spectrophotometric assessment for methemoglobin and unstable Hb testing.
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Footnotes |
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References |
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TopAbstractIntroductionMethods of AnalysisClinical ManifestationsReferences |
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and ß
thalassemia traits. The Thalassemia Working Party of the BCSH General
Haematology Task Force. J Clin Pathol 1994;47:289–95..
-chain variants, Hb Brousscis and Hb Cemenelum characterized by cation-exchange HPLC, iso-electric focusing and peptide sequencing. Clin Chem 1995;41:532-536.
Asp), a pitfall in the assessment of diabetic control: characterization by electrospray mass spectrometry and HPLC. Clin Chem 1998;44:2172-2177.
-thalassemia-2 determinants by PCR. Am J Hematol 1994;46:208-213.
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-thalassemia. Br J Hematol 1992;81:104-108.
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-thalassemias by multiplex polymerase chain reaction. Clin Chem 1994;40:2260-2266.
-thalassemia by polymerase chain reaction and oval restriction enzyme analysis. Hum Genet 1990;85:293-299.
[ISI][Medline]
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Montgomery2ßS2 hybrid tetramers by the Variant apparatus. Clin Chem 2000;46:139-140.
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