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Hemoglobinopathies and Thalassemias
The ABCs of Lab Evaluation
By Shirley L. Welch, PhD
http://www.aacc.org/publications/cln/2009/october/Pages/Series1009.aspx#
Idividuals with one copy of HbS, called HbS trait, usually are asymptomatic and have normal red blood
cell indices and morphology. Typical laboratory findings for these patients are 35 to 45% HbS. In simple
cases of HbS trait, the percentage of HbS is always greater than the percentage of HbA. For individuals
with HbS levels <30%, labs should consider concurrent iron deficiency or α-thalassemia trait.
Individuals with two copies of HbS develop sickle cell disease. Symptoms include hemolytic anemia, with
Hb levels of 6 to 10 g/dL. In addition, peripheral-blood smears show sickle cells, target cells, and HowellJolly bodies. Their electrophoretic patterns and HPLC results typically show 90 to 95% HbS, no HbA, and
often slightly elevated HbF in the 5 to 10% range. It is important to know a patient’s transfusion history
in cases of sickle cell disease, as small amounts of HbA post-transfusion can produce misleading test
results. In neonates, blood counts are usually normal, but as HbS levels increase and HbF levels
decrease, hematologic abnormalities appear.
Individuals can also have HbS in combination with other variants and/or mutations. For example, a
person may co-inherit a β−chain variant such as HbC, an α-chain variant such as HbG-Philadelphia, or
have concurrent β-thalassemia and/or α-thalassemia. (See HbC section below.) A diagnosis of concurrent
β+-thalassemia is likely when test results show HbS >50% and HbA <50% with increased HbA 2 and HbF.
If HbA is not present and HbA2 and HbF are elevated, concurrent β0 thalassemia should be considered.
Patients with S/β0-thalassemia have a clinical course similar to sickle cell disease, but patients with S/β+thalassemia generally have milder disease due to the presence of some HbA.
α-Thalassemias
Thalassemias are caused either by mutations that reduce the rate of synthesis of a globin chain or by
deletion of one or more of the globin genes. α-Thalassemias usually are caused by deletions of one or
more of the four α-globin genes. These deletions decrease the synthesis of the protein, thereby creating
an overabundance of γ-chains in a fetus or β-chains after HbF disappears. These γ- or β- chains can
aggregate and form HbBart’s or HbH, respectively.
For patients whose Hb electrophoresis or HPLC analyses and iron studies are normal and the MCV is low,
α-thalassemia trait should be considered. However, DNA studies are required for a definitive diagnosis.
Table 3 summarizes the effects of α-gene deletions.
Table 3
Common α-Thalassemias
1
Genotype
Genes
Deleted
(-α/αα)
1
Silent Carrier
None
(-α/-α)
2
Homozygous α2Thalassemia
Microcytosis
(--/αα)
2
α-thalassemia 1
trait
+/- anemia
(--/-α)
3
HbH Disease
Chronic hemolytic
anemia
(--/--)
4
Hydrops fetalis
Lethal
Phenotype
Clinical
Findings
β-Thalassemias
At least 150 mutations are known to cause β-thalassemia. The condition occurs mainly in people from the
Mediterranean region, the Middle East, India, and Southeast Asia. These mutations have been divided
into two categories: β0-thalassemias, which involve complete absence of β-chain production; and β+
thalassemias, which result in reduced synthesis of the β-chain. The severity of the disorder varies widely
depending on the amount of β-globin produced.
Laboratory findings for individuals with β-thalassemia trait include microcytosis, hypochromia, no or mild
anemia, and normal or slightly increased RBCs. HbA 2 levels are elevated in these individuals, and HbF
may be normal or increased. Inheritance of two β-thalassemia genes causes more severe disease ranging
from β-thalassemia intermedia to Cooley’s anemia or β-thalassemia major.
Quantitation of HbA2 and HbF
Diagnosis of β-thalassemia requires quantitation of HbA2. In general, labs use anion-exchange column
chromatography, HPLC or CE for this analysis. Both HPLC and CE provide acceptable quantitation of HbA 2
and HbF in the presence of many Hb variants; however, anion exchange chromatography only measures
HbA2 quantitatively. Therefore, labs should use another method in conjunction with anion exchange
chromatography to identify Hb variants.
Labs can also measure HbF by alkali denaturation if the levels are >50%. Labs should confirm by a
second method levels of HbF >10% detected by HPLC.
Sickle Solubility Test
Another test used by labs specifically for screening or confirming the presence of HbS is the sickling
solubility test. Several commercial kits are available that can detect HbS down to a level of 20%.
Solubility testing as a screen is not indicated in infants under 6 months of age, as there is a high potential
for false-negative results.
Significant haemoglobinopathies: guidelines for screening and
2
diagnosis
Kate Ryan,1 Barbara J. Bain,2 David Worthington,3 Jacky James,4 Dianne Plews,5 Anthony Mason,6 David Roper,7 David
C. Rees,8 Barbara de la Salle9 and Allison Streetly10 Writing group: On behalf of the British Committee for Standards in
Haematology
http://www.bcshguidelines.com/documents/sign_haemoglo_bjh_042010.pdf
Cellulose acetate electrophoresis
Haemoglobin electrophoresis at pH 8Æ4–8Æ6 using a cellulose
acetate membrane is simple, reliable and rapid. It enables the
provisional identification of haemoglobins A, F, S/G/D, C/E/
O-Arab, H and a number of less common variant haemoglobins.
Differentiation between haemoglobins migrating to a
similar position can be obtained by using electrophoresis on acid (agarose) gels, HPLC or IEF. The provisional
identification of any variant haemoglobin should be supported by at least one further unrelated method.
Application of an alternative technique will exclude the possibility that a single band in either the S or C
position represents a compound heterozygous state such as SD or SG and CE or CO-Arab respectively. If a
patient has microcytosis the possibility that a single band represents compound heterozygosity for a variant
haemoglobin and b0 thalassaemia must also be considered.
Sickle solubility test
The kits for sickle cell solubility tests that are predominantly used in the UK will detect
haemoglobin S down to a concentration of 20% (and sometimes below; in some cases as low as
8%) (BCSH 1998). The method of Lewis et al (2006), although less sensitive than some commercial
kits, can detect Hb S down to a concentration of 20%. The methods are therefore capable of
detecting all cases of sickle
cell trait beyond the period of infancy, even when there is coexisting a thalassaemia trait (but
possibly not when there is coexisting Hb H disease). False positives have been described in patients
with high plasma protein levels (Canning & Huntsman, 1970) and in anaemic patients when double
the volume of blood is used in the test (Arras & Perry, 1972; Lilleyman et al, 1972). The latter
problem can be avoided, however, by using a more concentrated sample of blood or washing the
red cells.
All positive and equivocal sickle solubility tests should be confirmed by HPLC or an alternative
technique both for confirmation of the presence of Hb S and to distinguish sickle cell trait from
sickle cell anaemia and from compound heterozygous states. In an emergency, e.g. preanaesthesia, this distinction can be made with reasonable accuracy with a sickle solubility test
combined with a blood film and a blood count. It is also recommended that all negative sickle
solubility tests be confirmed by HPLC or an alternative technique.
3
Conversely, sickle solubility testing should be employed whenever an unknown haemoglobin is
encountered that elutes in the position of the ‘Hb S Window’ by HPLC or is in the position of Hb S
on CAE or IEF.
In general, a sickle solubility test is not indicated in an infant before the age of 6 months because a
negative result may be misleading. However, a sickle solubility test can sensibly be performed in
an emergency, prior to anaesthesia, as if it is negative it is unlikely that anaesthesia will cause any
clinical problems because the Hb S % will be too low. The wording of the report on such a test
must state that a negative test does not exclude the presence of a low percentage of haemoglobin
S and that further testing is necessary and will follow.
If the patient is anemic, it is necessary to correct the hematocrit to 0.5 in order to avoid false
negative results.
All negative or equivocal sickle solubility tests be centrifuged before reading in order to increase
sensitivity and reliability. It is important not to omit this step if samples have low percentage of Hb
S.
All sickle solubility tests, whether, positive, negative or equivocal, should be confirmed by Hb
electrophoresis or HPLC
If rapid results are required, such as before emergency anaesthesia, the distinction between sickle
cell trait and sickle cell anaemia or compound heterozugous states can be made with reasonable
accuracy by combining a sickle solubility test with a blood film and a blood count.
Significant haemoglobinopathies: guidelines for screening and
diagnosis
Kate Ryan,1 Barbara J. Bain,2 David Worthington,3 Jacky James,4 Dianne Plews,5 Anthony Mason,6 David
Roper,7 David
C. Rees,8 Barbara de la Salle9 and Allison Streetly10 Writing group: On behalf of the British Committee for
Standards in Haematology
http://www.bcshguidelines.com/documents/sign_haemoglo_bjh_042010.pdf
Pre-operative/pre-anaesthesia
It is important to detect SCD prior to anaesthesia because its presence will influence clinical
management. Testing should be initiated by clinical staff on the basis of a clinical history and
assessment of family origin. All patients from groups with a high prevalence of Hb S (Table I)
should be offered testing as some cases of milder disease may be unrecognized and the presence
of Hb S heterozygosity may also influence perioperative
techniques.
Haemoglobin S
African including North Africans, African-Caribbeans, African-Americans, Black
British and any other African ethnicity (e.g. Central and South Americans of
partly African ethnicity), Greeks, Southern Italians including Sicilians, Turks,
4
Arabs, Indians
α thalassaemia
Chinese, Taiwanese, South-East Asian (Thai, Laotian, Cambodian, Vietnamese,
Burmese, Malaysian, Singaporean, Indonesian, Philippino), Cypriot, Greek,
Turkish and Sardinian
β thalassaemia
All ethnic groups other than Northern Europeans
Table I. Ethnic groups with a clinically significant prevalence of haemoglobin S and α0 and β thalassaemia.
0
Table 2. Geographic distribution of ethnic populations at increased risk for
thalassemia or sickle cell disorders
Regions of Origin
Africa
Mediterranean region
e.g., Sardinia, Corsica, Sicily, Italy, Spain,
Portugal, Greece, Cyprus,
Turkey, Egypt, Algeria, Libya, Tunisia,
Morocco, Malta
Middle East
e.g., Iran, Iraq, Syria, Jordan, Saudi
Arabia and other Arabian peninsula
countries, Qatar, Lebanon, Palestine,
Israel (both Arabs and Sephardic
Jews affected), Kuwait
South East Asia
e.g., India, Afghanistan, Pakistan,
Indonesia, Bangladesh, Thailand,
Myanmar
Western Pacific region
e.g., China, Vietnam, Philippines,
Malaysia, Cambodia, Laos
Caribbean countries
South American countries
Thalassemia
Sickle Cell Disease
↑
↑
↑
↑
↑
↑
↑
↑in parts of India
↑
_
↑
↑
↑
↑

For routine operations, FBC and haemoglobin analysis using HPLC or a suitable alternative diagnostic
method should be performed at the pre-assessment visit. In an emergency, an FBC and a sickle solubility
test should be performed. Results in this situation should be evaluated clinically and must be followed by
definitive testing (see below).
Investigation of microcytosis outside the antenatal situation
Recommendation
 Preoperative testing should be carried out in patients from ethnic groups in which there
is a significant prevalence of sickle cell haemoglobin.
 Emergency screening with a sickle solubility test and full blood count must always be
followed by definitive analysis.
 The need to investigate for thalassaemia and haemoglobinopathies should be considered
in patients with unexplained microcytosis.
5
Haemoglobin electrophoresis at pH 8.4 8.6 using a cellulose acetate membrane is simple,
reliable and rapid.
It enables the provisional identification of haemoglobins A, F, S/G/D, C/E/O-Arab, H and a
number of less common variant haemoglobins.
Differentiation between haemoglobins migrating to a similar position can be obtained by using
electrophoresis on acid (agarose) gels, HPLC or IEF.
The provisional identification of any variant haemoglobin should be supported by at
least one further unrelated method.
Application of an alternative technique will exclude the possibility that a single band
in either the S or C position represents a compound heterozygous state such as SD or SG and CE
or CO-Arab respectively.
If a patient has microcytosis the possibility that a single band represents compound
heterozygosity for a variant haemoglobin and β0 thalassaemia must also be considered.
Hydropic fetus
Neonate or infant with anaemia and either
haemoglobin F only or unexpectedly low
percentage of haemoglobin A
Haemoglobin Bart’s hydrops fetalis
β thalassaemia major
Unexplained anaemia and splenomegaly
β thalassaemia major or intermedia,
haemoglobin H disease, unstable
haemoglobin
Thalassaemias including haemoglobin
H disease
Haemoglobin S and interacting
haemoglobins (C, D-Punjab, O-Arab)
or β thalassaemia
Haemoglobin H disease, unstable
haemoglobin
Thalassaemia, variant haemoglobin
Suspected thalassaemia or unexplained
microcytosis
Clinical and haematological features suggestive of
sickle cell disease
Unexplained haemolysis
Unexplained target cells
Unexplained irregularly contracted cells
Unexplained polycythaemia
Unexplained cyanosis with normal oxygen
saturation
Variant haemoglobin, particularly
haemoglobin C or an unstable
haemoglobin
High affinity haemoglobin
Haemoglobin M
Table 2. Conditions for haemoglobinopathy investigations in investigation of clinical disorders.
Variant haemoglobins, such as Hb S can be quantified by scanning densitometry after
electrophoresis/staining; however quantification of haemoglobin A2 by this method is not
recommended as the precision is not good enough for the diagnosis of b thalassaemia trait
(BCSH 1998).
High-performance liquid chromatography (HPLC)
High-performance liquid chromatography can be used for the quantification of haemoglobins S,
A2 and F and for the detection, provisional identification and quantification of many
6
variant haemoglobins. HPLC usually provides accurate quantification of Hb A2 and is therefore
suitable for the diagnosis of b thalassaemia trait.
High-performance liquid chromatography usually separates haemoglobins A, A2, F, S, C, DPunjab and G-Philadelphia from each other. However, both Hb E and Hb Lepore often co-elute
with A2 (as other haemoglobins co-elute with A, S and F) but may be recognized by alternative
techniques. HPLC has the disadvantage that it also separates glycosylated and other derivative
forms of haemoglobin, which can make interpretation more difficult. For example, derivatives of
haemoglobin S co-elute with haemoglobin A2, rendering its quantification inaccurate. Careful
examination of every chromatogram is essential. As with every method of haemoglobin analysis,
controls should be run with every batch. Identification of variants is only provisional, and
unrelated second-line methods should be used for confirmation.
If HPLC is used as the screening technique, it is essential to check and maintain the positions of
the windows, which are used as the first stage identification of any variants found. This is
generally done by adjusting the column temperature or the flow rate so that the Hb A2 peak
appears at a standard time.
This is just as important as the calibration of the Hb A2 and Hb F levels and should be checked
daily. Appropriate controls should be included wherever possible.
Sickle solubility test
The kits for sickle cell solubility tests that are predominantly used in the UK will detect
haemoglobin S down to a concentration of 20% (and sometimes below; in some cases as low as
8%) (BCSH 1998).
The methods are therefore capable of detecting all cases of sickle cell trait beyond the period of
infancy, even when there is coexisting a thalassaemia trait (but possibly not when there is
coexisting Hb H disease). False positives have been described in patients with high plasma
protein levels (Canning & Huntsman, 1970) and in anaemic patients when double the volume of
blood is used in the test (Arras & Perry, 1972; Lilleyman et al, 1972). The latter problem can be
avoided,
however, by using a more concentrated sample of blood or washing the red cells.
All positive and equivocal sickle solubility tests should be confirmed by HPLC or an alternative
technique both for confirmation of the presence of Hb S and to distinguish sickle cell trait from
sickle cell anaemia and from compound heterozygous states. In an emergency, e.g. preanaesthesia,
this distinction can be made with reasonable accuracy with a sickle solubility test combined with
a blood film and a blood count. It is also recommended that all negative sickle solubility
tests be confirmed by HPLC or an alternative technique.
Conversely, sickle solubility testing should be employed whenever an unknown haemoglobin is
encountered that elutes in the position of the ‘Hb S Window’ by HPLC or is in the position of Hb
S on CAE or IEF.
In general, a sickle solubility test is not indicated in an infant before the age of 6 months because
a negative result may be misleading. However, a sickle solubility test can sensibly be performed
7
in an emergency, prior to anaesthesia, as if it is negative it is unlikely that anaesthesia will cause
any
clinical problems because the Hb S % will be too low. The wording of the report on such a test
must state that a negative test does not exclude the presence of a low percentage of
haemoglobin S and that further testing is necessary and will follow.
The following techniques can be used for haemoglobin variants:
• High performance liquid chromatography (HPLC)
• Isoelectric focusing (IEF)
• Cellulose acetate electrophoresis (CAE)
Abnormal results should be confirmed by a different technique that is appropriate for the likely
variant.
Another technique that can be used for confirmation, besides those listed above, is acid agar or
acid agarose electrophoresis although this is not suitable as a screening technique.
Sickle solubility testing can be used as confirmation of an initial screen that suggests the
presence of sickle haemoglobin.
Screening for thalassaemia.
Methods used are red cell indices in conjunction with measurement of Hb A2 levels. Routine
measurement of blood indices includes measurements of MCH and mean cell volume (MCV); it is
recommended that MCH is used to screen for thalassaemia as this parameter is more stable than
MCV. Hb A2 is quantified by HPLC or microcolumn chromatography.
HPLC system
A national recommended cut-off Hb A2 of 3.5% or above has been set as the action point in the
diagnosis of carriers of b thalassaemia. A value of 5.0% for Hb F has been set for the
investigation of a raised fetal haemoglobin in pregnancy.
In a patient with an MCH below the cut-off point (<27 pg), further investigation will be required
if the total Hb A2 is above 3.5%.
The a thalassaemia risk needs to be considered in the light of the family origin of the patient.
The major risk is for b thalassaemia, but the risk of Hb Bart’s hydrops fetalis should not be
overlooked.
Hb A2 values >4.0% with normal indices may indicate b thalassaemia trait with or without coexisting a thalassaemia.
8
In this case:
• Re-analyse FBC
• Repeat Hb A2
• Consider B12/folate deficiency, drugs, liver disease/alcohol or HIV infection
Hb A2 values ≤4.0% with normal red cell indices and a normal Hb F level can usually be regarded
as normal, although some mild b thalassaemia alleles (mainly in subjects of Mediterranean
origin) are associated with an A2 of 3.5–4.0%.
Interpretation of results in the presence of iron deficiency
Severe iron deficiency anaemia(Hb<80 g/l)can reduce theHbA2
level slightly (by up to 0.5%). Outside of pregnancy, anaemia
should be treated and the haemoglobin analysis repeated when
the patient is iron replete. In pregnant women there is no
justification for delaying the investigation for haemoglobinopathies
whilst treating iron deficiency presumptively, as this will
delay the process of identifying at-risk carrier couples, who could
be offered prenatal diagnosis.
Neonatal samples
Neonatal samples are typically composed of
mostly Hb F (approximately 75%) with approximately 25%
Hb A and small quantities of acetylated Hb F and sometimes
Hb Bart’s.
It is also important to realize that occasionally the
presumptive identification of a haemoglobin variant using
screening methods is incorrect, because some variants give
exactly the same results using current screening techniques.
The sensitivity and specificity are approximately 99% for the
methods used. Unequivocal identification of haemoglobin
variants can only be achieved by either protein analysis (e.g.
mass spectrometry) or DNA analysis.
Recommendation
 Abnormal laboratory screening results should be confirmed
by a different technique that is appropriate for the likely
abnormality.
 Quantification of Hb A2 by CAE plus scanning densitometry
9







is not recommended.
 A sickle cell solubility test is not generally indicated in
infants below the age of 6 months and is not recommended
as a primary screening tool except in an emergency
situation.
 All sickle solubility tests should be confirmed by
HPLC or an alternative method.
Assessment of iron status may be useful in the interpretation
of laboratory tests since in severe iron deficiency anaemia(Hb<80 g/l)can reduce the
HbA2 level slightly (by up to 0.5%).
Examination for Hb H bodies cannot reliably distinguish
between a thalassaemia traits and should not be used for
screening.
Laboratories should be aware of the effect of
blood transfusion on the interpretation of results. Haemoglobin interpretation is
misleading after a recent blood transfusion and necessitates
repeat testing after 4 months if a pre-transfusion sample has
not been analysed. DNA testing for
the sickle gene is now recommended for transfused neonates to
avoid the need for a repeat specimen at 4 months posttransfusion,
although a pre-transfusion specimen is still the
preferred specimen.
No technique can identify all abnormalities but the combined sensitivity/specificity of the
HPLC and IEF techniques for haemoglobins present at the time of screening is
approximately 99%. The pattern of haemoglobin variants is not unique however and
whilst some will be clarified by using the second technique, unequivocal identification
can usually only be made by DNA analysis or mass spectrometry.
The diagnosis of a thalassaemia is more complicated because
DNA analysis is the only accurate way to distinguish between
a+ and a0 thalassaemia. However it is not practical to seek to
confirm all potential cases of a thalassaemia by DNA analysis
because the a+ form is too common and not usually clinically
important; it is not cost-effective for DNA laboratories to
perform analysis on all such cases. Furthermore, non-deletional
forms of a+ thalassaemia are more common than was thought and rapid methods for
their detection are not available.
If a blood transfusion has been received within 4 months,
misleading data and conclusions may result.
Analytical fact should be separated from interpretative
opinion. The factual results should be given first and should
10
be followed by a clear conclusion, which may include
recommendations.




If information from the blood count is used in coming to a
conclusion about the significance of the analytical data (as in
probable a thalassaemia) then those aspects of the blood
count used (such as haemoglobin concentration, red cell
count, MCH, MCV) must be included in the haemoglobinopathy
report.
Similarly, if information on ethnicity/family origin is used, it
should be stated in the report.
Results of the sickle solubility test, in the absence of resultsfrom
an unrelated confirmatory method, should only be reported as
an ‘interim’ report. The final report with information from the
blood film, HPLC and/or electrophoresis and any other
appropriate tests should follow as soon as possible.
As it improves clarity, the conclusion should always be given
both in full text and in standard abbreviation form in
parentheses. For example: Sickle Cell Carrier (AS) or Sickle
Cell Anaemia (SS).
Laboratory Investigation of Hemoglobinopathies and
Thalassemias: Review and Update
1.
2.
Gwendolyn M. Clarke1 and
Trefor N. Higginsa,1
+ Author Affiliations
1.
1.
Dynacare Kasper Medical Laboratories, 14940 123rd Ave., Edmonton, Alberta T5V 1B4, Canada.
1
↵aAuthor for correspondence. Fax 780-452-8488; e-mail [email protected].
http://www.clinchem.org/content/46/8/1284.full
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).
11
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 δ β-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).
Carrier Screening for Thalassemia and
Hemoglobinopathies in Canada
2008
http://www.sogc.org/guidelines/documents/gui218CPG0810.pdf
Individuals with two deleted copies of the a-globin gene
have α-thalassemia trait. These individuals are either heterozygotes
for α0-thalassemia (αα/—) or homozygotes for
α+-thalassemia (α-/α-). Both of these types of
α-thalassemia trait (i.e. αα/—or α-/α-) are essentially
identical clinically and on routine hematology testing.
Patients are generally asymptomatic. A CBC will typically
show microcytosis (low MCV, e.g., < 80 fL) and
hypochromia (low MCH, e.g., < 27 pg); the patient may also
be mildly anemic. Despite anemia, the red blood cell count
is often mildly elevated. Hb electrophoresis and Hb HPLC
are normal in -thalassemia trait after the newborn period,
and the HbA2 level is normal, which is not the case in
-thalassemia trait (see below for a discussion of HbA2 in
-thalassemia). -Thalassemia trait is usually diagnosed by
staining a peripheral blood smear with brilliant cresyl blue to detect abnormal red blood
cell inclusions called H bodies.
12
Patients with Hb H disease are usually anemic, microcytic,
and hypochromic on routine hematologic testing. Hb electrophoresis
and HPLC may show an abnormal hemoglobin,
Hb H, although in some cases this may be difficult to detect
or require special techniques. On H-body staining, almost
every red blood cell will show HbHinclusions.
Disease
α+-thalassemia
silent carriers
α-thalassemia trait
Genotype/
Clinical Findings
Ethnic Group at
Risk
(αα/α-)
These individuals
are asymptomatic
usually found
by chance
among
various ethnic
populations,
particularly
African
American
Individuals with
two deleted
copies of the aglobin gene:
either
heterozygotes
for α0thalassemia
(αα/—) or
homozygotes
for
α+-thalassemia
(α-/α-)
Patients are
generally
asymptomatic.
The patient may
be mildly anemic
This condition
is
encountered
mainly in
Southeast Asia,
13
Haematological Findings
These generally
have normal routine hematologic
findings:
normal Hb, MCV, and MCH.
Rarely the MCV and/or MCH can
be low.
Hb electrophoresis, Hb HPLC, and
H
body staining of a peripheral
blood smear are usually negative
outside the newborn period.
In this form, the diagnosis cannot
be confirmed based on
hemoglobin electrophoresis
results, which are usually normal
in all α thalassemia trait.
Genetic, molecular (DNA) testing
testing is necessary to confirm
the diagnosis.
A CBC will typically
show microcytosis (low MCV,
e.g., < 80 fL) and hypochromia
(low MCH, < 27 pg); Occasional
target cells;
the red blood cell count is often
mildly elevated. Hb
electrophoresis and Hb HPLC are
normal, and the HbA2 level is
normal.
It is usually diagnosed by staining
a peripheral blood smear with
brilliant cresyl blue to detect
abnormal red blood cell
inclusions H bodies. the finding
of H bodies
in the right clinical and ethnic
context is typically considered
diagnostic of α-thalassemia trait.
the Indian
subcontinent,
and some parts
of the Middle
East.
Hb H disease
( α thalassemia
intermedia)
( α-/—)
three-gene
deletion.
Patients with
Hb H disease
have one
functioning α
globin gene
they inherit α0thalassemia (—
/) from one
parent and
α+-thalassemia
(α-/) from the
other.
This condition is in
most cases clinically
mild: many patients
require occasional
transfusion of red
blood cells, but
these patients are
usually not
considered
“transfusion
dependent.”
However, marked
phenotypic
variability has been
noted, and some
patients do
require regular
transfusions to
survive. There are
even occasional
cases of Hb H
disease presenting
as hydrops
fetalis.Patients with
Hb H disease are
usually anemic
14
(αα/—) and
patients with single deletions on
both chromosomes
(α-/α-) have essentially the same
results in all these tests,
and can be differentiated only by
molecular methods. Such
molecular distinction is crucial
for the identification of a
couple at risk for having a fetus
with four-gene deletion
α-thalassemia (hemoglobin
Bart’s hydrops fetalis, or Hb
Bart’s disease,—/—). Hb Bart’s
disease can occur only
when the fetus inherits a double
deletion from each parent,
that is, when both parents are
carriers of α0-thalassemia,
(αα/—). In such a situation, the
couple has a 25% risk of
having a fetus with all four α
globin genes deleted.
A CBC will typically
show microcytosis (low MCV,
e.g., < 80 fL) and hypochromia
(low MCH, e.g., < 27 pg). Hb
electrophoresis
and HPLC may show an abnormal
hemoglobin,
When peripheral blood films
stained with supravital stain or
reticulocyte preparations are
examined, unique inclusions in
the RBCs (Hemoglobin H bodies)
are typically observed in almost
every red blood cell.
These inclusions represent β
chain tetramers (Hemoglobin H),
which are unstable and
precipitate in the RBC, giving it
the appearance of a
golf ball.
Hemoglobin Bart’s
hydrops
( α Thalassemia
Major)
(—/—)
four-gene
deletion
Silent Carrier β
Thalassemia
The mutation
that causes the
thalassemia
is a β+
thalassemia
mutation which
diminishes but
does not
eliminate beta
globin chain
production
from one of
two beta globin
genes.
individuals with
one normal
gene and one
affected gene
(genotypically
represented as
β-thalassemia trait or
β-thalassemia minor
Svere intrauterine
anemia resulting in
fetal hydrops and,
in
almost all cases,
intrauterine death.
There are only rare
case reports of
infants surviving
with this condition.
Without any α
globin production,
these fetuses are
unable to make any
fetal (HbF) or adult
(HbA) hemoglobin.
Instead, they can
produce only
embryonic
hemoglobins, which
generally cannot
support life past the
third trimester.
Some of these
fetuses will also
have congenital
abnormalities such
as terminal
limb defects.
these patients have minor changes in RBC indices
no symptoms
often
asymptomatic.
Patients have mild
anemia.
CBC will show mild or no anemia,
and MCV and MCH are usually
low
RBC count is often high.
The routine diagnostic test is Hb
15
B/B+ or B/B0).
The production
of β chains
from the
abnormal allele
varies from
complete
absence to
variable
degrees of
deficiency.
β -thalassemia
intermedia
or β -thalassemia
major
HbS/ β -thalassemia
Two mutated β
globin genes
are inherited,
one from each
Parent.
Genotypically,
intermedia is
usually β+/ β+
or possibly
β+/ β0
Two mutated β
globin genes
are inherited,
one from each
Parent.
there is either
zero or almost
zero β globin
chain synthesis
in β thalassemia
major (β+/ β0 or
β0/ β0).
The most
common
variant
hemoglobins
that may be
electrophoresis or Hb HPLC:
these
will demonstrate increases in
HbA2 (i.e., > 3.5% of total
hemoglobin) and usually HbF
(i.e., >1%). In the right clinical
and ethnic context, an elevated
HbA2 is considered diagnostic of
β-thalassemia trait.
This condition
results in anemia of
intermediate
severity, which
typically does not
require regular
blood transfusions
This condition is
characterized by
transfusiondependent anemia,
massive
splenomegaly,
bone deformities,
growth retardation,
and peculiar facies
in untreated
individuals, 80% of
whom die within
the first 5 years of
life from
complications of
anemia.
can produce
a sickling syndrome
of variable severity
16
Peripheral blood film
examination usually reveals
marked hypochromia and
microcytosis
(without the anisocytosis usually
encountered in iron deficiency
anemia), target cells, and
faint basophilic stippling.
patients will be
anemic,microcytic, and
hypochromic, and Hb
electrophoresis or
HPLC will show elevated HbA2.
The peripheral blood films reveal
hypochromia, microcytosis
And anisocytosis
By 6 to 12 months of age,
patients will be
anemic,microcytic, and
hypochromic, and Hb
electrophoresis or
HPLC will show elevated HbA2.
The peripheral blood films reveal
severe hypochromia and
microcytosis, marked
anisocytosis,
fragmented RBCs, polychromasia,
nucleated RBCs, and, on
occasion, immature leukocytes.
HbE/ β-thalassemia
co-inherited
with βthalassemia
HbE mutation
from one
parent and a βthalassemia
mutation from
another.
Extremely
common in
some
Southeast
Asian
populations,
reaching a gene
frequency of up
to 70% in
northern
Thailand.
Hemoglobin
C/ β-thalassemia
Sickle Cell Anemia
(HbSS)
Homozygous
HbS disease
No normal HbA
is produced:
instead, red
cells contain
May vary in its
clinical severity
from as mild as
thalassemia
intermedia to
as severe as β
thalassemia major
HbE/ β-thalassemia
is one of the most
important causes of
clinically severe
thalassemia
worldwide.
Hemoglobin
C/ β-thalassemia is
clinically and
hematologically
very
heterogeneous,
ranging from very
mild to very severe.
Individuals with Hb
C Beta +
thalassemia have a
mild anemia, low
MCV, and target
cells. Individuals
with Hb C Beta 0
thalassemia have a
moderately severe
anemia,
splenomegaly, and
may have bone
changes.
If they inherit B+ thalassemia
there is 65 - 70% Hb C, 20 - 30%
Hb A, and increased Hb F. If they
inherit Beta 0 thalassemia on
electrophoresis there is no Hb A
and increased Hb F with Hb C.
In HbSS,
sickled RBCs have a
considerably
shorter life span
than normal
RBCs (due to
extravascular
sickled RBCs and target cells.
Howell- Jolly bodies seen when
there is splenic atrophy.
17
primarily
HbS (with small
amounts of
HbA2 <1%
and HbF (5%10%).
HbS heterozygotes
HbAS
SC disease
co-inheritance
of HbS
and HbC
Sickle-β+ thalassemia
co-inheritance
of HbS with Bthalassemia
S/B+ or S/Bo
β+thal/βs
hemolysis) and
cause intermittent
episodes of vascular
occlusion under
conditions of
decreased oxygen
tension. This causes
tissue ischemia, and
acute and chronic
organ dysfunction
involving the
spleen,
brain, lungs, and
kidneys. Pain and
swelling of hands
and
feet (hand-foot
disease) is a
frequent early
presentation of
this disease in
infants and young
children.
The hemolysis leads
to chronic anemia
and predisposes
the patient to
aplastic crises.
heterozygous
have normal red cell indices on
carriers are
CBC, with normal RBC
asymptomatic.
morphology on
peripheral blood smear. Both
HbSS and HbAS are routinely
diagnosed by Hb electrophoresis
or HPLC.
Mild anemia, vasoocclusive problems.
they have a sickle
syndrome which is
very similar to
sickle cell anemia
Mild sickle cell
hematological findings are
disease
comparable to sickle cell anemia
(with some differences, such as
microcytosis in
S/B- thalassemia but not in HbSS)
Differentiating S/Bo -thalassemia
from HbSS requires not only Hb
HPLC but also additional
18
nformation such as CBC results,
physical examination findings,
and occasionally family or
molecular studies.
SD disease
co-inheritance
of HbS with
HbD
βs/βD Punjab
inherit a Hb C
gene from each
parent
As for sickle cell
anemia
Hemoglobin C
carriers
inherit Hb C
from one
parent and Hb
A from the
other.
They have no
Usually have target cells on blood
anemia but Usually smear and may have a slightly
have target cells on low MCV.
blood smear and
may have a slightly
low MCV. There are
no other clinical
problems.
HbSC disease
HbA is not
present.
The RBCs
contain 50%
HbS and 50%
HbC. Anemia is
much milder,
with Hb levels
of 11 g/dL or
higher.
Hb CC disease
They have a mild
hemolytic anemia.
There may be very
occasional episodes
of joint and
abdominal pain
which are
attributed to Hb CC
disease.
Splenomegaly is
common. Aplastic
crises and gall
stones may occur.
microcytosis, and target cell
formation
The peripheral blood smear may
have some sickled cells and a
high proportion of target cells. In
addition, microcytic, dehydrated,
dense RBCs are seen. These may
contain crystal-like
condensations.
HbS variants may
occur as double
heterozygotes with
other Hb variants.
These include HbD,
HbE, and HbO Arab.
Double
19
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
decreased (β+ ) or absent (β0) β globin production
Recommendations for thalassemia investigation

An MCV ≥ 80 fL and an MCH ≥ 27 pg, with a normal electrophoresis or HPLC, requires no
further testing. The finding of a normal MCV (i.e.,≥ 80 fL) in combination with a normal
MCH (i.e., ≥ 27 pg) would rule out most cases of thalassemia and would require no
additional thalassemia testing.

Individuals with an MCV < 80 fL or MCH < 27 pg can have α- and/or β-thalassemia and/or
iron deficiency anemia.

For individuals with MCV < 80 fL or MCH < 27 pg, the next step is hemoglobin
electrophoresis or HPLC, quantitation of HbA2 and HbF, and a blood smear stained for H
bodies.

It is appropriate to recommend that all pregnant women from an ethnic background at
increased risk of hemoglobinopathy and/or thalassemia be screened by both CBC, to assess
the MCV and MCH, and a hemoglobin electrophoresis or HPLC.

In general, β-thalassemia trait can be reliably diagnosed by hemoglobin electrophoresis or
HPLC, with HbA2 and HbF quantitation. Patients with β-thalassemia trait have an elevated
HbA2, i.e., > 3.5%.
20

In patients with a low MCV, but with a normal Hb electrophoresis/HPLC and HbA2 and HbF
quantitation, the differential diagnosis includes iron deficiency anemia and α-thalassemia.
A serum ferritin (to exclude iron deficiency anemia) and a smear to screen for the H bodies
of α-thalassemia are therefore required. For pregnant patients, these tests (ferritin and
H body stain) should be ordered concurrently.

In the right clinical context (e.g., microcytic anemia in an “ethnically appropriate” patient),
the presence of H bodies can identify the patient as a carrier of α-thalassemia. H body
testing is not 100% sensitive (see below), and therefore the absence of H bodies does not
completely exclude α-thalassemia carrier status in an ethnically at-risk patient. If iron
deficiency is ruled out in a pregnant woman with a negative H body test, testing of
the partner remains crucial to determine the risk of having an affected fetus with
Hemoglobin Bart’s hydrops fetalis. Molecular studies may also be done to confirm or
exclude carrier status for α-thalassemia.

Hemoglobin electrophoresis or HPLC will allow identification of Hb variants, such as HbS, C,
D, and E. A phenotypic sickle cell preparation (such as a slide or tube sickling test) is not
helpful in identifying other types of β globin variants besides HbS, so should not be used as
a carrier screen for hemoglobinopathies.

The finding of any abnormality (e.g., low MCV or MCH, or abnormal hemoglobin
electrophoresis or HPLC) requires screening of the partner, which entails CBC, smear for H
bodies, hemoglobin electrophoresis or HPLC, and HbA2 and HbF quantitation. If the
patient is pregnant, testing of the partner should be done promptly.
B-thalassemia
trait or -thalassemia minor. The CBC will show mild or no
anemia, and MCV and MCH are usually low. Like
-thalassemia trait, the RBC count is often high. Because
reduced production of globin means an inability to generate
as much normal HbA (22), they compensate by
increasing production of other -like chains, namely and
, leading to increases in the levels of the minor
hemoglobins HbA2 (22) and HbF (22). The routine
diagnostic test is Hb electrophoresis or Hb HPLC: these
will demonstrate increases in HbA2 (i.e., > 3.5% of total
hemoglobin) and usually HbF (i.e., >1%).12 In the right clinical
21
and ethnic context, an elevated HbA2 is considered
diagnostic of -thalassemia trait.
22