Clinica Chimica Acta 439 (2015) 50–57

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Advances in detection of hemoglobinopathies

Dina N. Greene a,⁎,1, Cecily P. Vaugn b,1, Bridgit O. Crews a, Archana M. Agarwal b,c
a
TPMG, Northern California Kaiser Permanente Regional Laboratories, Berkeley, CA, United States
b
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, United States
c
Department of Pathology, University of Utah Health Sciences, Salt Lake City, UT, United States

a r t i c l e i n f o a b s t r a c t

Article history: Hemoglobin disorders are recognized as one of the most common inherited diseases worldwide. Detecting and
Received 18 July 2014 characterizing variant hemoglobins and thalassemias depends primarily on clinical laboratory methods. Multiple
Received in revised form 4 October 2014 biophysical, biochemical, and genetic assays are available to provide phenotypic or genotypic evidence of pathol-
Accepted 4 October 2014
ogy. For many years conventional slab-gel electrophoresis and HPLC were the most commonly utilized laboratory
Available online 12 October 2014
methods. However, the field has rapidly expanded to regularly include capillary zone electrophoresis, molecular
Keywords:
assays, and, more recently, mass spectrometric assays. Interpretation of these techniques is, in general, compli-
Hemoglobinopathy cated because of the involvement of multiple polymorphic genes. Proper characterization of hemoglobin variants
Thalassemia is necessary for diagnosis, primary prevention and genetic counseling for underlying disorders. This review pro-
Molecular vides an overview of the current hemoglobin analysis techniques, and also discusses technologies that have po-
HPLC tential to translate into widespread clinical settings.
Capillary electrophoresis © 2014 Published by Elsevier B.V.
Mass spectrometry

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2. Complete blood count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3. High throughput screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4. Screening tests for the detection of hemoglobin variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
5. Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
6. Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
7. Molecular diagnostics of the hemoglobin disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

1. Introduction responsible for thalassemias and N1500 structurally abnormal hemo-

Hemoglobin, a tetramer with four prosthetic heme moieties, is the
protein responsible for physiological oxygen transport [1]. Defects in
this protein can lead to various pathological complications broadly char-
acterized as thalassemias (decrease in globin expression) or hemoglo-
binopathies (altered structural globin gene product). Diagnosis of
hemoglobin disorders relies heavily on the clinical laboratory, which
uses a combination of biophysical, biochemical, and molecular assays
to support and confirm the diagnosis. With hundreds of gene defects
⁎ Corresponding author. Tel.: +1 510 559 5414.
E-mail address: dina.n.greene@kp.org (D.N. Greene).
1
These authors contributed equally to this work.
globins identified [2], interpretation of results requires a comprehensive
understanding of both the genetics and biochemistry of hemoglobin.
The hemoglobin tetramer is fully assembled when alpha or alpha-
like globins pair with beta or beta-like globins (Table 1). The most abun-
dant hemoglobin in adults, Hb A, consists of two alpha and two beta
subunits. However, hemoglobin composition varies throughout devel-
opment and continues to evolve in the first years of life, a phenomenon
referred to as developmental switching [3]. Gene expression defines the
type of circulating hemoglobins and is key to understanding the hetero-
geneity of hemoglobin tetramers.
The alpha and beta globin gene clusters are located on different
chromosomes [2]. The alpha globin gene cluster is found on chromo-
some 16, with two copies of the alpha gene per chromosome. There is
only one alpha-like protein, zeta, which is expressed in the embryo,

http://dx.doi.org/10.1016/j.cca.2014.10.006
0009-8981/© 2014 Published by Elsevier B.V.
 D.N. Greene et al. / Clinica Chimica Acta 439 (2015) 50–57
Table 1
Subunit composition of the normal and most common variant hemoglobin species
discussed in this article.
Hb name Subunit composition Mutation &/or clinical implication
Hb A Alpha2beta2 n/a
Hb F Alpha2gamma2 n/a
Hb A2 Alpha2delta2 Elevated in beta thalassemia
Hb Bart's Gamma4 Detected in pediatric patients with alpha
thalassemia
Hb H Beta4 Detected in patients with alpha thalassemia
Hb S Alpha2beta2 Beta mutation Glu6Val
Hb C Alpha2beta2 Beta mutation Glu6Lys
Hb E Alpha2beta2 Beta mutation Glu26Lys
but is virtually absent after the first trimester. The beta globin gene clus-
ter is found on chromosome 11, with a single copy of the beta gene per
chromosome and multiple flanking beta-like genes. The embryonic
beta-like globin, epsilon, is expressed in the first trimester only, and is
followed by the production of the fetal beta-like globin, gamma [4].
Hb F forms when gamma-globin is paired with alpha-globin. Expression
of gamma peaks mid-gestation and slowly declines, stabilizing approx-
imately 6 months after birth. Residual expression of gamma remains
throughout life. The final beta-like subunit is delta, expression of
which begins shortly before birth and continues throughout life. Hb
A2, which is critical in the diagnosis of beta thalassemia, occurs when
delta pairs with alpha [5,6]. Hemoglobin disorders are identified by dis-
ruption of the ratios in the normal hemoglobin tetramers described
above or by the appearance of structurally abnormal hemoglobins.
Laboratory diagnosis of hemoglobin disorders is accomplished by in-
tegrating clinical and hematological parameters along with laboratory
hemoglobin analysis, ultimately communicating the clinical significance
of the hemoglobin results to the physicians. Combining these elements
to properly diagnose hemoglobin disorders is essential for treatment of
anemia, primary prevention/genetic counseling, and prevention or
awareness of crisis during physical challenges (for sickling disorders).
This review will highlight the primary biochemical and molecular tech-
niques commonly utilized in the clinical lab today, as well as introduce
the newer technology that is yet to be fully adopted.
2. Complete blood count
Accurate interpretation of hemoglobin analysis is virtually impossi-
ble, especially for thalassemias, without the accompanying hematolog-
ical information obtained from a complete blood count (CBC). Structural
hemoglobin mutations will have variable hematologic changes depend-
ing on the specific mutation. The key hematologic feature in diagnosing
thalassemias is microcytic hypochromic anemia. A mean corpuscular
volume (MCV) b80 fl is often applied as the cut-off for thalassemia sus-
picion in order to maximize sensitivity [7,8]. Although the hypochromic
microcytic anemia observed in thalassemias also occurs in iron deficien-
cy anemia, iron deficiency anemia is commonly accompanied by a de-
creased RBC count and increased red cell distribution width (RDW),
while hemoglobin abnormalities tend towards an increased RBC count
and normal RDW [9]. These parameters are integrated into the Mentzer
index (MCV/RBC count). Diagnoses of iron deficiency anemia may be ac-
companied by a higher Mentzer index (generally greater than ~ 13)
whereas beta thalassemias will generally exhibit a lower index [10]. Ad-
ditional hematologic phenotypes observed in individuals with hemo-
globin abnormalities are: decreased hemoglobin concentration,
decreased MCV, anisopoikilocytosis (altered shape and size of the
RBCs), and presence of target cells.
3. High throughput screens
Quantification of Hb A, Hb A2, Hb F, and any additional hemoglobin
products are integral to hemoglobinopathy diagnosis because various
51
genotypes will result in deviation of normal hemoglobin ratios. High
throughput screens provide a two-fold identification of abnormalities
by first allowing for the visual detection of any variant peaks and second
by quantifying all detected hemoglobin species. These properties
also make the methods amenable to HbA1c quantification for assess-
ment of glycemic control in diagnosis and monitoring of diabetes [11,
12]. In fact, by using these assays to screen for diabetes a number of
additional hemoglobin variants have been identified. The two most
commonly utilized high-throughput screens are high pressure liquid
chromatography (HPLC) [13] and capillary zone electrophoresis (CZE)
[14] (Fig. 1).
Both HPLC and CZE detect hemoglobin species based on their differ-
ential elution or migration using spectrophotometric detection at
415 nm. The primary analytical difference between the two methods
is their mode of separation. HPLC uses cation exchange chromatography
to retain the major hemoglobin species on the column. Separation is ac-
complished using an elution salt gradient and the retention times of the
various hemoglobin species are visualized with a chromatogram. CZE
separates proteins based on buffer pH, pI, and endosmotic flow. CZE
does not have a conventional “solid-phase” and instead applies a charge
to induce migration, classifying it an electrophoretic, rather than a chro-
matographic separation technique. The data output for CZE is called an
electropherogram.
A non-pathological hemoglobin profile has approximately 97% Hb A,
2.5% Hb A2, and b1% Hb F. Beta thalassemias, in particular traits (minor,
intermediate), are characterized by an increase in the relative concen-
tration of Hb A2. An Hb A2 concentration N~4% has close to 100% sensi-
tivity and 90% specificity for beta thalassemia diagnosis [5,6]. Certain
HIV drugs [15], Hb S1C [16], hyperthyroidism, megaloblastic anemia, un-
stable hemoglobins, and congenital dyserythropoietic anemia, type I
can cause false positives [17]. False negatives can be caused by concur-
rent deletions in alpha or delta genes [18,19] or occasionally in
myelodysplastic syndrome, sideroblastic anemia, and acute myelocytic
leukemia [17]. Beta thalassemia major is characterized by the significant
decrease in or absence of Hb A, which is replaced by Hb F (~70–100%)
and Hb A2 (0–4%) [20,21].
Alpha thalassemias pose additional challenges for diagnosis due, in
part, to the presence of 4 alpha globin genes [22]. Deletion of all four
alpha genes (alpha thalassemia major) is not compatible with life. Anal-
ysis of cord blood from affected fetuses reveal virtually 100% Hb Bart, a
gamma-globin tetramer. Newborns with a 3 alpha gene deletion, also
known as Hb H disease, have detectable concentrations of Hb Bart's at
birth, which is replaced with Hb H (beta-globin tetramers) as an adult.
The Hb H concentration in these patients is N 5% and is usually detect-
able using high throughput screens. To our knowledge, no studies
have evaluated the diagnostic sensitivity of HPLC or CZE for diagnosis
of Hb H disease. However, in our laboratory we have seen multiple
cases where Hb H N5% was detected using isoelectric focusing electro-
phoresis, but was undetectable using HPLC or CZE. Similarly, one or
two alpha gene deletions, silent carriers and carriers respectively, can-
not be distinguished from normal individuals using high throughput
screening techniques [23].
In the most obvious cases of a hemoglobin abnormality, the screen-
ing results will detect an unusual peak. This peak may or may not
correspond to pathology, but does signify a mutation in one of the glo-
bin genes leading to the presence of variant hemoglobin. Further,
interpreting the detection/quantification of even common hemoglobin
variants, such as Hb S, can be quite complicated. The quantitative ratio
between Hb A, Hb S, Hb A2, and Hb F is important for detecting under-
lying thalassemias in the S patient, and can also help to distinguish Hb S
carriers from patients with a hemoglobin variant that co-elutes or co-
migrates with Hb S.
Multiple comparisons between HPLC and CZE are available in the lit-
erature [16,24–26]. These studies show that both methods are suitable
for hemoglobinopathy screening, both are quantitative with amenities
like cap-piercing and on-board sample mixing capabilities. The main
52 D.N. Greene et al. / Clinica Chimica Acta 439 (2015) 50–57

Fig. 1. CZE electropherograms corresponding to a wild type (A) and Hb GPhilidelphia carrier (B). The percentage corresponding to each hemoglobin species is quantified using the relative
area under the curve. HPLC quantifies hemoglobin species in a similar fashion, but uses retention time (instead of zones) and has migration patterns specific to the methodology.

analytical differences are summarized in Table 2 and described in detail Hb E and beta thalassemia the HPLC chromatogram will show a much
below. higher level of Hb E with elevated Hb F and an absent or a very small
HPLC is unable to separate Hb E from Hb A2 and therefore neither Hb A peak. For Hb E carriers there will be a prominent Hb A peak and
species can be appropriately quantified. This may result in an inability the Hb E will be b~ 30%. However, if the underlying beta thalassemia
to differentiate an Hb E carrier phenotype from an Hb E carrier with mutation only induces a mild thalassemia, the aforementioned general-
an underlying beta thalassemia. In general, if there is co-inheritance of izations may not apply and proper quantitation of Hb A2 may prove
useful.
Table 2
Falsely elevated Hb A2 by HPLC can also occur in Hb S patients due to
Primary differences between HPLC and CZE. HbS1c coelution, which can similarly complicate interpretation.
CZE has the potential for falsely elevated Hb A2 in Hb C patients due
HPLC CZE
to similar migration times. Our laboratory has also observed that Hb F
Cannot resolve Hb E from Hb A2 Resolves all major Hb variants quantified by CZE will have a positive bias in Hb S patients when com-
Falsely elevated Hb A2 in Hb S Falsely elevated Hb A2 in Hb C patients
pared to quantification by HPLC. This is likely due to the co-migration
patients
Recalibration required No recalibration needed of degraded Hb S with Hb F or the co-migration of post translationally
No additional steps for patients Samples without Hb A require 1:1 dilution modified Hb F with Hb F in CZE. In HPLC most post translationally mod-
without Hb A for interpretation ified species are resolved from the parent compound.
Historical technique; abundance Relatively less literature available An advantage of CZE is that Hb H and Hb Bart's are resolved and can
of literature available
usually be distinguished and quantified at concentrations N~ 3%.
 D.N. Greene et al. / Clinica Chimica Acta 439 (2015) 50–57
However, we feel that it is important to note that we have observed
cases in which the Hb H concentration was N5%, but was undetected
by CZE (DN Greene, unpublished data). These pathological hemoglobin
species are often detected with HPLC, but since they elute in the void
volume they are generally only detected at high concentrations (N5%),
are difficult to accurately quantify, and are prone to false positives
from other early eluting species [27]. A second advantage of CZE is
that it doesn't require calibration. The consistency in the charged capil-
laries allows the migration assignments to be based on the Hb A and Hb
A2 present in each individual sample. However, the negative side of this
is that all patients with absent Hb A (i.e. — homozygous or compound
heterozygous hemoglobinopathies) must be mixed at a 1:1 dilution
with normal whole blood to confirm the identity of the variants relative
to Hb A migration. Visually, CZE electropherograms for hemoglobin
analysis are much cleaner. HPLC chromatograms tend to reveal multiple
post-translational modification and degradation peaks that complicate
interpretation. A practical advantage of CZE is that the commercially
available CZE instruments have unparalleled software that allows for
enhanced interface design and implementation. Finally, one advantage
of HPLC over CZE is the volume of published literature available charac-
terizing HPLC chromatographic patterns of rare variants. This is because
HPLC has been utilized in production for decades longer than CZE. How-
ever, the advantage will eventually be obsolete as CZE based literature
accumulates.
CZE and HPLC have both been successfully used for newborn screen-
ing around the world [28–32]. The premise for hemoglobin separation,
detection, and quantitation are the same as described above, but rather
than venous whole blood as the sample type, cord blood or capillary
heel stick blood applied and dried onto filter paper (dried blood spots,
DBS) are the matrix of choice. High throughput analysis is accomplished
by using on-board automated DBS punching. HPLC and CZE have com-
parable diagnostic sensitivity and specificity for detecting Hb variants
[33]. The sensitivity is basically 100% for detecting the common Hb var-
iants. Additionally, thalassemias can be detected by evaluating the rela-
tive Hb A concentration (decreased in beta thalassemia) or presence of
Hb Bart's (indicative of alpha thalassemia) [33,34].
Migration patterns of variant hemoglobins using biochemical
methods, including HPLC and CZE, are not completely specific. This is
due to the nature of protein mutations. The actual location of the
amino acid substitutions may vary, but the changes to overall charge
that the mutation imposes might be identical to other variant hemoglo-
bins. As such, the College of American Pathology (CAP) requires all labs
performing hemoglobin analysis to support any abnormal interpreta-
tions using the primary screening technique in conjunction to a second-
ary technique. In general, most labs will choose for this to be a slab gel-
based electrophoresis or biophysical screen (both described below).
Further, not all hemoglobin abnormalities are detected by high through-
out screens, and therefore if secondary biochemical techniques are not
performed on all specimens some thalassemias will be undetected. Sec-
ondary confirmation is especially important for alpha thalassemias, and
if clinical suspicion persists and is supported by ethnicity, CBC results,
and iron studies more extensive laboratory tests are advised.
4. Screening tests for the detection of hemoglobin variants
Hemoglobin abnormalities can lead to changes in the biophysical
properties of red cells or the hemoglobin molecule. There are three com-
mon laboratory tests that exploit these changes: sickle cell solubility
[35], Heinz body stain [36], and unstable hemoglobin [37]. In general,
these tests are fairly sensitive, but not specific; a positive result might
point to a hemoglobin abnormality, but does not define the abnormali-
ty. Biophysical screens are powerful tools for resource-poor countries,
where screening for primary prevention may be desirable, but availabil-
ity of automated analyzers (even for red cell indices) may be cost limit-
ing. Additionally, they have the unique ability to provide structural
information related to the underlying hemoglobin variant by exposing
53
phenotypic characteristics that are unable to be detected using HPLC
or CZE.
Sickle cell anemia is caused by the homozygous or compound het-
erozygous inheritance of the Hb S mutation. The point mutation in the
beta globin gene that defines Hb S causes the variant protein to poly-
merize within the red cell. Development of such intracellular rigid poly-
mers leads to changes in red cell morphology and decreased membrane
malleability, ultimately leading to hemolytic anemia. The sickle cell sol-
ubility test exploits the propensity for deoxy-Hb S to polymerize by ex-
posing the hemoglobin molecules to a reducing environment. The assay
is simple: red cells are lysed into a reducing environment and the tur-
bidity of the solution is monitored [38]. Pre-analytical factors that can
lead to false positive results are lipemia or hyperglobulinemia. Red
cells can be washed in saline before lysis to mitigate the effects of
these variables. While quite specific for Hb S, the sickle solubility test
is a qualitative test and will be positive for both Hb S carriers and homo-
zygotes necessitating follow-up for all positive results.
The unstable hemoglobin assay and the Heinz body stain are two
methods used to detect the class of hemoglobin mutations that have
the propensity to cause hemolytic anemia, referred to as unstable he-
moglobins [36,37]. These hemoglobin variants are more prone to oxida-
tive damage ultimately resulting in precipitation of the hemoglobin
molecule, the formation of Heinz body aggregates, and hemolysis. In
vitro assays recapitulate the oxidative state by stressing the already-
weak hemoglobin bonds, either with isopropanol or heat. Under such
conditions Hb A is stable and will remain soluble and unstable hemoglo-
bins will precipitate. A positive unstable hemoglobin result does
not necessarily dictate pathology. High concentrations of Hb F (N5%)
can cause a false positive result. Additionally, approximately 200
hemoglobin variants will form a precipitate, but only about 50% of
these are associated with hemolytic anemia. To detect Heinz bodies
in vivo, a whole blood smear can be incubated with a supravital stain
(ex. — methyl violet, brilliant green, new methylene blue). While the
detection of Heinz bodies using this stain is specific for detecting oxida-
tive damaged red cells, the differential diagnosis for a positive result
must also include G6PD deficiency and drug induced oxidative damage.
Inclusions will also be detected using supravital stains in Hb H disease.
For these variants HPLC or CZE will usually identify an abnormality,
but neither technique will provide a stability assessment.
5. Gel electrophoresis
Similar to the differences observed in elution characteristics
using HPLC and CZE, hemoglobin variants will have variable migration
rates when separated by gel electrophoresis [38]. Acid and alkaline elec-
trophoreses are complimentary methods that separate hemoglobin spe-
cies using a polymer matrix buffered at an acidic or basic pH,
respectively. Isoelectric focusing electrophoresis (IEF) separates hemo-
globin species in the same relative migration pattern as alkaline electro-
phoresis, but with more exquisite resolution of variants. IEF is often
considered the biochemical gold standard for hemoglobin abnormali-
ties. There is a plethora of literature available that defines the migration
patterns expected using any of these gel electrophoresis-based
techniques.
Many laboratories will use both alkaline and acid electrophoresis to
confirm or further characterize any abnormalities detected using HPLC
or CZE. Other laboratories may choose to employ either acid or alkaline
electrophoresis. Neither provides a distinct advantage over the other
and both are available in manufacturer-produced kits. Visualization of
the hemoglobin migration pattern is generally achieved with a non-
specific protein stain.
Although less commonly used, IEF is far from obsolete. A recent pub-
lication endorsed the use of IEF for identification of 1–2 alpha-globin de-
letions [23]. Such genotypes are undetected by HPLC, CZE, and acid/
alkaline electrophoresis because the analytical sensitivity for Hb H is in-
sufficient. The authors hypothesize that because IEF uses a heme specific
54 D.N. Greene et al. / Clinica Chimica Acta 439 (2015) 50–57
stain, the analytical sensitivity is low enough to detect Hb H concentra-
tions well below 2%. IEF therefore offers a cost efficient way to screen
high-risk populations for alpha-thalassemia.
Although valuable, the biggest drawback of gel-electrophoresis tech-
niques is their inability to quantitate hemoglobin species. Semi-
quantitation is available using densitometry, but such methods are not
sensitive enough to diagnose beta thalassemia or to accurately assess
Hb S:A ratios.
6. Mass spectrometry
The application of mass spectrometry for the analysis of hemoglobin
was first reported in 1981 [39]. Since then substantial advances in mass
spectrometry instrumentation and ionization techniques have created
an opportunity for clinical applications of mass spectrometry to detect
both hemoglobinopathies and thalassemias [27]. However, since many
common beta globin chain variants differ from wild-type beta globin
by only 1 Da even high resolution mass spectrometers cannot resolve
intact wild-type from Hb C, D, or E. Mass spectrometry approaches
that detect intact globin chains will have difficulty differentiating be-
tween globin chains in the same sample which differ in mass by less
than 6 Da, and cannot confer location of a given amino acid mutation
[40]. For example, the substitution of 6valine for 6glutamic acid
(Hb S) will result in a mass difference of 30 Da, but the same nominal
mass difference can occur for a substitution of arginine to tryptophan,
threonine to methionine, glycine to serine, or alanine to threonine, in
any position of the globin chain [41]. Further structural elucidation is
possible by fragmenting the intact globin chain via tandem mass spec-
trometry, or by cleaving the globin chain with a proteolytic enzyme
such as trypsin [42–45].
Currently, one of the most promising applications of mass spectrom-
etry for hemoglobin analysis is for large population screening. A
30 minute trypsin digestion of whole blood followed by electrospray
ionization mass spectrometry results in detection of a series of 15
well-characterized peptides (T1–T15) for wild type beta globin [43].
Daniel et al. demonstrated a “pseudo” tandem mass spectrometry ap-
proach targeting three of the 15 peptides and demonstrated 100% sen-
sitivity and specificity (compared to phenotypic and molecular
methods) to detect Hb S, C, DPunjab, OArab, and E in over 200 blood
EDTA samples, with an injection-to-injection cycle time of approxi-
mately 1 min [41]. In a separate study they demonstrated the potential
of the same approach using δ:β globin peptide ratios as a surrogate
marker to quantify Hb A2, and hence diagnose beta thalassemia [46].
However, it is worth noting that amino acid substitutions in a globin
chain can change ionization response, thus quantitation methods
based on peak intensities should be carefully evaluated for this effect.
Boemer et al. validated a similar approach specifically for newborn
screening, which included fragmentation of two peptides from the β
globin chain and one peptide from the gamma globin chain. The method
was designed to screen for Hb S, C, and E, and beta-thalassemia [47].
Validation of the method in over 2000 dried blood spots showed robust
performance (compared to IEF) to identify the main hemoglobinopa-
thies; however, the authors concluded that the methodology could
lead to misinterpretations for rare beta globin variants, and that a clini-
cally insignificant homozygote mutations could be interpreted as major
beta thalassemia, or that a heterozygote variant/Hb S could be confused
with homozygous Hb S. The authors also noted a relatively large CV
(as high as 30.7%) for calculating the Hb S:A ratio.
Most recently, Moat et al. developed newborn screening cutoffs and
a post analytical method based on peptide ratios measured using a
2 minute tandem mass spectrometry method for blood spot tryptic di-
gests [48]. The cutoffs were designed to detect only disease states. The
method targeted Hb S, C, DPunjab, OArab, Leopore and E. The mass spec-
trometry screening cutoffs were developed using a training set, and val-
idated in over 13,000 newborn blood spots concurrently with HPLC. No
disease states were missed, but 8 false positive Hb S carriers were
incorrectly classified as Hb S/C or Hb SS. The authors noted that rare var-
iants on any of the three monitored beta chain peptides could potential-
ly result in a false positive. Inter-assay coefficients of variation for the
assay ranged from 8.9–21.2%.
Mass spectrometry may have the potential to improve the sensitivi-
ty of Hb analysis as a complimentary method to HPLC and IEF. It is also
possible that tandem mass spectrometry methods for newborn screen-
ing will offer several benefits over currently used methods, including
cost-efficient use of expertise and instrumentation already used in new-
born screening laboratories and more automated analysis. Aged blood
spots can also be analyzed by mass spectrometry, which may offer logis-
tical benefits for testing. However, as these applications are relatively
new, increased inter-laboratory comparisons should be performed and
improvements to assay imprecision should be investigated.
7. Molecular diagnostics of the hemoglobin disorders
After presumptive diagnosis of hemoglobin disorders using bio-
chemical methods is made, characterization of deletions or mutations
may be necessary to confirm the clinical phenotype and/or guide genet-
ic counseling. There are a variety of molecular techniques, most PCR-
based, available for the identification of different hemoglobinopathy
mutations and deletions. These methods are summarized in Table 3. Ad-
aptation of a particular method into a laboratory requires assessment of
the available infrastructure and knowledge of the population served to
allow targeting of the specific genetic abnormalities present in the rele-
vant population.
A majority of the molecular assays (many commercially available)
are targeted for specific, characterized mutations. In general these are
robust and cost-effective methodologies. Targeted techniques are capa-
ble of such amenities because they identify specific mutations that
have been previously described and extensively studied. Conversely,
targeting specific mutations is simultaneously a limitation, as mutations
outside of these pre-defined boundaries will be undetected. Targeted
methodologies include: gap-PCR, multiplex ligation-dependent probe
amplification (MLPA), dot blots with allele specific oligonucleotide hy-
bridization (ASO), and allele-specific PCR.
The majority of alpha thalassemias (95%) and a subset of beta thalas-
semias (~5%) are due to large chromosomal deletions. The conventional
method to detect these deletions is Southern blot hybridization. Howev-
er, Southern blot hybridization is a labor-intensive, lengthy technique
and has therefore been replaced with amplification-based methods.
Gap-PCR offers an excellent alternative method for detecting deletions
where the breakpoints have been previously described. PCR primers
are designed to flank the deletion boundaries of the common, character-
ized deletions. In normal, non-deleted alleles, these primers are located
several kilobases apart, PCR elongation time is limited to restrict
amplicon size, and thus, the PCR products for the targeted deletions
will be absent. In contrast, in an allele harboring a specific deletion,
the forward and reverse primers flanking that deletion will be in close
proximity to each other and will therefore amplify a fragment of DNA
spanning the breakpoint. A gap-PCR method to detect the seven
common alpha thalassemia deletions (−α3.7, −α4.2, −(α)20.5, −−SEA,
−−MED, −−FIL, and −−THAI) has been adopted by many laboratories
[49]. The assay is multiplexed, allowing for the amplification of all
seven mutations in a single reaction vessel. Results are visualized and
interpreted by separating the PCR products using agarose or capillary
gel electrophoresis (Fig. 2). The assay will only detect the seven common
alpha thalassemia deletions listed above; rare mutations or deletions
will not be detected. Gap-PCR can also be utilized for detection of dele-
tions leading to beta-thalassemias [15], but the same limitations apply.
MLPA is a relatively new technique employed for the multiplex de-
tection of gene-specific deletions and/or duplications [50,51]. A probe
set comprised of two adjacent oligonucleotides is designed for each re-
gion of interest. The probe sets are hybridized to the patient DNA and li-
gated. Ligated probe sets are then amplified using universal primer
 D.N. Greene et al. / Clinica Chimica Acta 439 (2015) 50–57 55

Table 3
Summary of the primary molecular methods used for hemoglobin analysis.

Methodology Application Limitations

Gap-PCR Detection of common deletions in alpha and/or
beta globin genes
MLPA Detection of both common and uncommon
deletions and duplications in both alpha and beta
globin genes
Dot blot/allele-specific oligonucleotide (ASO) Detection of targeted point mutations
Allele-specific PCR/ARMS Detection of targeted point mutations
High-resolution melting (HRM) Detection of point mutations and small insertions
and deletions
Denaturing gradient gel electrophoresis (DGGE) Detection of point mutations and small insertions
and deletions
Denaturing high performance liquid Detection of point mutations and small insertions
chromatography (dHPLC) and deletions
Single-stand confirmation polymorphism (SSCP) Detection of point mutations and small insertions
and deletions
Sequencing Detection of point mutations and small insertions
and deletions, including novel variants; useful for
exact identification of variants detected by
screening methods
Only detects the deletions specifically targeted by the
assay
Does not detect exact breakpoints; similar variants may
be indistinguishable
Only detects variants specifically targeted by the assay
Only detects variants specifically targeted by the assay
Novel variants may confound results; follow-up
sequencing may be required
May be better suited for screening rather than
identification of exact variants
May be better suited for screening rather than
identification of exact variants
May be better suited for screening rather than
identification of exact variants
Not suitable for detecting large deletions

sequences present on the ends of the probes. Only probes that hybrid-
ized to target DNA, allowing subsequent ligation, will be amplified.
The amplified probes are separated by capillary electrophoresis and
the peak height of each amplified probe is then compared to the corre-
sponding peak height of a reference sample to generate a ratio.
Wildtype alleles give a ratio of 1.0, corresponding to two alleles in the
patient sample and two alleles in the reference samples (Fig. 3). Gener-
ally, a deletion in a patient sample produces a ratio of 0.5 (representing
one allele compared to the two alleles in the reference sample), while a
duplication produces a ratio of 1.5 (representing three alleles in the pa-
tient compared to two alleles in the reference sample). This pattern
holds for beta globin results, but is complicated in alpha globin analysis
because there are two genes per allele. Commercial kits for both the
alpha and beta globin gene clusters are available from MRC Holland. Be-
cause multiple probe sets can be multiplexed in a single reaction, MLPA
is adept at detecting deletions or duplication in several regions in a sin-
gle tube. Additionally, the MLPA kit for alpha globin includes detection
of the Constant Spring mutation and the kit for the beta globin cluster
includes detection of the Hb S mutation. In comparison to gap-PCR,
which requires knowledge of exact breakpoints, MLPA can be used
to identify novel deletions or duplications [52,53]. At the same
time, since exact breakpoints are not identified with MPLA, the same
pattern of probe ratios can apply to multiple hemoglobin variants, re-
quiring interpretation in conjunction with biochemical analysis. More
precise characterization of the breakpoints of novel variants can be
achieved with high-resolution array comparative genomic hybridiza-
tion [54,55]; however, this method is not well suited to routine clinical
work due to high cost and complexity.
Detection of characterized point mutations can be accomplished
using dot blot or allele specific oligonucleotide (ASO) hybridization. In
this method, amplified patient DNA that includes the region of interest
is spot onto nylon membranes. Each patient sample is spotted as a sep-
arate dot onto two membranes, one membrane for each subsequent
probe. Two ASO probes are used for each mutation, one complementary
to the mutant DNA sequence and the other to the wildtype sequence.
These labeled ASOs are hybridized to the amplified, bound DNA. A de-
tected signal for a specific dot on the membrane indicates hybridization
of the probe and the amplicon, signaling that the specified allele
(wildtype or mutant) is present; heterozygous patients would exhibit
signal for dots corresponding to both the wildtype and the mutant
probes. In the reverse dot blot, it is the allele-specific probes (unlabeled
in this case) that are fixed to a nylon membrane strip in the form of dots.
Patient genomic DNA is amplified, labeled and hybridized to the filter.

Fig. 2. Gap-PCR agarose gel electrophoresis results for detection of alpha thalassemia de-
letions. The multiplex PCR reaction contains primers for a control gene, LIS1 (visible in
lanes 1, 2, 3, and 5 at 2350 bp), a region of the α2 gene (1800 bp; will only be amplified
in wild type alleles), and seven common alpha globin deletions. Lane 1: bands correspond-
ing to amplification of LIS1 and α2 in a normal patient; lane 2: bands corresponding to LIS1,
−a3.7 (2022 bp), and −α 20.5 (1007 bp); lane 3: bands corresponding to LIS1, α2, and
−α4.2 (1628 bp); lane 4: bands corresponding to α2 and −−FIL (546 bp); lane 5: bands
corresponding to LIS1, α2, and −−SEA (1349 bp); lane 6: bands corresponding to −α3.7
(2022 bp) and −−SEA (1349 bp).
Fig. 3. MLPA ratio results for probes in a single sample harboring a 619-bp deletion causing
a β0 phenotype. Probes hybridizing to two alleles produce ratios of ~1 (green squares for
control regions and blue squares for beta globin gene regions of interest). Probes hybrid-
izing to a single allele produce a ratio of ~0.5 (red squares). In this sample the two red
squares correspond to regions in beta globin exon 3, indicating a single allele deletion in
this region. The MLPA kit for beta globin also includes a probe set for the Hb S mutation;
this probe set did not hybridize in this sample, due to a lack of the Hb S mutation, and
thus shows a ratio of 0 (represented by the white square).
56 D.N. Greene et al. / Clinica Chimica Acta 439 (2015) 50–57
Here, multiple mutations, rather than multiple patients, can be tested in
one hybridization reaction. These techniques are inexpensive and useful
for a limited number of known mutations; consequently they have been
used in many laboratories worldwide for diagnostic as well as prenatal
screening [56–58].
Similarly, allele-specific PCR (AS-PCR), also known as amplification
refractory mutation system (ARMS), can be used to detect pre-defined
point mutations in beta globin genes and is a cost effective way to detect
population-specific or common mutations [59,60]. Two different PCR
reactions are performed for each variant position using forward primers
specific for either the wildtype allele or the mutant allele and a common
reverse primer. Specificity is obtained by using stringent binding
criteria. Reactions may also be multiplexed, with mutant-specific
primers for all targets in one tube and wildtype primers in the other
tube. The PCR products are analyzed by electrophoresis; detected
bands correspond to patient genotype. More specifically, PCR product
obtained with the wildtype primers only denotes a patient that does
not have mutations in the amplified region; PCR product obtained
with the mutation-specific primer only, denotes that the patient is ho-
mozygous for the mutation being evaluated; PCR products obtained
with both mutant and wildtype specific primers denotes that the pa-
tient is heterozygous for the mutation of interest.
Methodologies such as high resolution melting (HRM), denaturing
gradient gel electrophoresis (DGGE), denaturing high performance liq-
uid chromatography (dHPLC), and single-strand conformation poly-
morphism (SSCP) can also be employed to detect hemoglobin point
mutations; these methods are also suitable for the detection of small in-
sertions and deletions. These techniques target a specific area of the
gene, rather than a specific mutation and therefore have broader speci-
ficity, with less precision for identifying exact mutations. Each of these
methods includes an initial amplification of the region of interest and
relies on differences in the melting properties or secondary structure
of an amplicon harboring a mutation — as compared to a non-mutated
amplicon — during the detection step. In HRM, the reaction vessel is
cooled following amplification and then slowly heated in a step-wise
fashion. Fluorescence of a double-stranded DNA-binding dye is moni-
tored to determine the melting temperature of the amplicon in each
tube. Since the melting temperature is a function of the exact sequence
of the amplicon, any variation in sequence will produce a slightly al-
tered melting profile as compared to the wildtype sequence. Data is vi-
sualized as a melting cure, derivative melting peak, or normalized
difference plot. A significant advantage of HRM is that it does not require
post-PCR manipulation; reactions are completed in a closed-tube for-
mat making it an efficient and cost-effective way to detect mutations.
However, careful assay design is crucial for HRM analysis to maximize
differences between melting temperatures of mutated and wildtype al-
leles; small amplicons are generally preferred. Additionally, it may be
necessary to do an initial long-range PCR to isolate the alpha-1 and
alpha-2 genes, followed by amplification of smaller amplicons [61]. Fur-
ther, previously unidentified polymorphisms or mutations can con-
found the results; it may be necessary to sequence confirm all samples
with non-wildtype alleles to verify the presence of specific mutations.
If in either of these techniques an initial long-range PCR or post-HRM se-
quencing confirmation is used, the methodology loses appeal, as it is no
longer a closed-tube format.
Alternatively, DGGE or dHPLC, although less commonly used in
United States, may be utilized for rapid detection of both point muta-
tions and small insertions/deletions [62,63]. In DGGE, after PCR amplifi-
cation, double stranded DNA is electrophoresed through a linearly
increasing denaturing gradient gel. The electrophoresis pattern is
based on the melting properties of double stranded DNA that can be dif-
ferent even if there is a single base-pair change. Although cost effective,
it is technically demanding and may need definitive diagnosis either by
a targeted approach or direct sequencing. In dHPLC, the region of
interest is amplified using PCR, followed by denaturation and re-
hybridization to form heteroduplexes. The resulting products are then
applied to a heated reverse-phase liquid chromatography column. The
heteroduplexes have different melting temperatures and thus give dis-
tinct peaks in the resulting chromatogram. This method can be auto-
mated and is cost effective for population screening. Optimization of
the column temperature is required when utilizing this methodology;
while it is easy to distinguish between homozygous and heterozygous
samples, it may be difficult to differentiate homozygous variants from
one another or determine the exact identity of heterozygous variants.
Detection of variants by SSCP has also been described [64–66]. This
method relies on difference in secondary structure of single-stranded
amplicons; mutations are detected by observing differences in the mi-
gration of the products in a gel. Similar to the above methods, this meth-
od may require additional testing to determine the exact identity of a
detected variant.
Lastly, Sanger sequencing can be utilized to detect point mutations
for the characterization of hemoglobinopathies and beta thalassemias
[28,67]. The beta and alpha globin genes are relatively small, 1.2 kb
and 1.8 kb respectively, and easily amenable to sequencing. PCR ampli-
fication followed by bidirectional sequencing is performed. The coding
region, deep intronic regions, exon-intron boundaries, proximal pro-
moter and 5′ and 3′ untranslated regions are sequenced for beta globin
and alpha globin (A1 and A2) genes because mutations have described
in these areas that can lead to beta thalassemia. Sequencing can be ad-
vantageous because it does not require targeting each mutation sepa-
rately (in contrast to the targeted methods) and it also identifies the
exact mutation (in contrast to the scanning methods). Sequencing is
not well suited to the detection of large deletions/duplications, and
thus is not appropriate for alpha thalassemia testing.
8. Conclusion
The diagnosis of hemoglobinopathies and thalassemias has been a
component of the clinical lab for almost a century. Even still, the physi-
ology and biochemistry associated with these common genetic disor-
ders encourages advances in their detection. As the field continues to
develop more sophisticated reflex strategies will likely be designed to
integrate mass spectrometry, molecular techniques, and classical bio-
chemical methods. Regardless of the techniques implemented, all re-
sults must align with the clinical picture.
Acknowledgments
The authors would like to thank Genevieve Pont-Kingdon and
N. Scott Reading for consultations regarding molecular assays.
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