INTRODUCTION

Auxiliary hemoglobin (Hb) variations regularly are based on a point transformation in a

globin quality that produce a single amino corrosive substitution in a globin chain. Albeit
most are of constrained clinical centrality, a couple significant subtypes have been related to
a few recurrence. Homozygous Hb C and Hb S (sickle cell ailment) produce critical clinical
indications,while Hb E and Hb D homozygotes might be gently symptomatic. Despite the
fact that heterozygotes for these variations are regularly asymptomatic, determination might
be significant for hereditary directing. Thalassemia, interestingly, results from quantitative
decreases in globin chain union. Those with reduced beta-globin chains are named
betalphalpha-Thalassemias, while those with diminished alpha-chain creation are known as
alpha-Thalassemias. Seriousness of clinical indications in these catters identifies with the
measure of globin chain delivered and the steadiness of lingering chains present in
abundance. The thalassemia minor disorders are described clinically by mellow frailty with
tireless microcytosis. Thalassemia intermedia (i.e., Hb H ailment) is exemplified by a
moderate, dynamically repaid hemolytic weakness that may give clinical side effects during a
time of physiologic pressure for example, disease, pregnancy, or medical procedure. The
thalassemia real disorders produce serious, hazardous sickliness. alpha-Thalassemia major
generally is contrary with extrauterine life; beta-Thalassemia real exhibits in early stages and
requires long lasting transfusion treatment or potentially bone marrow transplantation for
fruitful control of the illness. Twofold heterozygosity beyond a shadow of a doubt auxiliary
variations as well as thalassemia disorders may likewise lead to extreme clinical illness. A
few rules have been distributed that layout the required strides for hemoglobinopathy and
thalassemia examination. The accessibility of HPLC has streamlined huge numbers of these
prerequisites, permitting an effective stepwise demonstrative methodology for these
unpredictable issue.

Hemoglobin (Hb;1 Mr 68 000) is the oxygen-conveying moiety of erythrocytes. It is a

polypeptide tetramer, globular in structure, and comprising of two sets of not at all like globin
chains (i.e., alpha or more beta, delta, or gamma), which structure a shell around a focal hole
containing four oxygen-restricting heme bunches each covalently connected to a globin
chain. In sound grown-ups, ~95% of the Hb will be Hb An (alpha2-beta2) with modest
quantities(less then3.5%) of Hb A2 (alpha2-delta2) and Hb F (alpha2-gamma2) present.
During embryonic improvement, "pre alpha" z globin chains add to embryonic Hb. During
fetal improvement,b-like globin chains e and g add to the Hb (1).

The alpha-globin chain is encoded in copy on chromosome 16, and the non-alpha chains
(beta, delta, gamma) are encoded in a bunch on chromosome 11. A diploid cell in this manner
has four alpha-globin qualities and two beta-like qualities. The alpha and beta chains
comprise of 141 and 146 amino corrosive deposits, individually. There is some arrangement
homology between the two chains (64 singular amino corrosive deposits in indistinguishable
positions), and the beta chain contrasts from the delta and gamma chains by 39 and 10
deposits, individually.

In spite of the fact that hemoglobinopathies and thalassemias are two hereditarily particular
illness gatherings, the clinical indications of both incorporate pallor of variable seriousness
and variable pathophysiology. The thalassemias are described by a decrease in the measure of
the typical globin chain created. This lessening in globin chain creation may result from
quality erasure or from changes that unfavorably influence the translation or solidness of
mRNA items. The indications extend from gentle pallor with microcytosis (thalassemia
attribute) to deadly serious frailty (Hb Barts hydrops fetalis or beta-thalassemia major). The
hemoglobinopathies, or auxiliary Hb variations, are owing to amino corrosive substitution in
either the an or non-alpha chain. In excess of 700 hemoglobinopathies have been depicted to
date (2), most of which are clinically considerate and serendipitously found. The clinically
critical hemoglobinopathies are inferable from amino corrosive substitutions, essentially in
the non-alpha chain, that achieve changes in the optional and tertiary structure of the Hb
tetramer. These substitutions are most normal at positions in nearness to either heme
gathering or globin chain connection destinations.

Hemoglobinopathy terminology is an arrangement of letters (i.e., Hb S, C, and D), place

names indicating the site of first revelation or habitation of the propositus (e.g., Hb
Edmonton), and family names of the list case (e.g., Hb Lepore). A methodical terminology
that is both consistent also, useful recognizes the chain, the area, and the amino corrosive
substitution on the included globin chain. Along these lines Hb Alberta (beta101 Ala-Pro) is a
substitution of proline for alanine (the ordinary amino corrosive) in the 101st position of the
beta chain. In the heterozygous express, the typical Hb is set first, trailed by the variation,
e.g., AS characteristic.
 AIM & OBJECTIVE

AIM:

1. The aim of the study was to know the incidence and prevalence of
hemoglobinopathies in anand district by High-parformance liquid chromatography.
OBJECTIVES:

 To find the types of haemoglobin abnormalities among all cases of

hemoglobinopathies.
 To explore possibility of screeining &proper counselling &suggest protocol to
prevent & decrease the incidence of disease in affected population.
 To observe the percentage pf people are suffer from heamoglobinopathies in
[Link] how much people known about this disease.
 In heterozygous condition with coinheritance of alpha & beta-thasassemia, patient
show a degree of clinical variability.
 To find out the percentage of peoples are affected with haemoglobinopathies in
Anand district . And how much people are aware about this disease.

REVIEW OF LITRETURE
HIGH PERFORMANCE LIQUID CHROMETOGRAPHY:

The HPLC strategy is fundamentally the same as the conventional column chromatography,
with the exception of that the dissolvable is driven through the column by applying positive
weight. This enabled most detachments to be performed in under 20 minutes, with improved
partitions contrasted with the old strategy (1). Current flash chromatography frameworks are
sold as pre-stuffed plastic cartridges, and the dissolvable is siphoned through the cartridge.
Frameworks may likewise be connected with locators and portion authorities giving
computerization. The presentation of slope siphons brought about snappier divisions and less
solvent usage(2).

In extended bed adsorption, a fluidized bed is utilized, as opposed to a solid phage made by a
stuffed bed. This permits exclusion of beginning clearing steps, for example, centrifugation
and filtration, for culture juices or slurries of broken cells (3).

Phosphocellulose chromatography uses the coupling affiity of numerous DNAbinding

proteins for phosphocellulose. The more grounded a protein's association with DNA, the
higher the salt focus expected to elute that protein (4).
A representative HPLC unit may contains (A) Solvent reservoirs, (B) Solvent
degasser, (C) Gradient valve, (D) Mixing vessel for delivery of the mobile phase, (E) High-
pressure pump, (F) Switching valve in "inject position", (G) Switchng valve in "load
position", (H) Sample injection loop, (I) Pre-column (guard column), (J) Analytical
column, (K) Detector, (L) Data acquisition, (M) Waste or fraction collector (5).

High performance liquid chromatography (HPLC earlier alluded to as high-pressure fluid

chromatography), is a system in investigative science used to isolate, recognize, and measure
every part in a blend. It depends on siphons to pass a pressurized fluid dissolvable containing
the example blend through a segment loaded up with a strong adsorbent material. Every
segment in the example associates somewhat distinctively with the adsorbent material,
causing diverse stream rates for the various parts and prompting the partition of the segments
as they stream out the section (6). HPLC has been utilized for assembling (for example
during the creation procedure of pharmaceutical and natural items), legitimate (for example
distinguishing execution improvement tranquilizes in pee), explore (for example isolating the
segments of a complex natural example, or of comparable manufactured synthetic
compounds from one another), and restorative (for example recognizing nutrient D levels in
blood serum) purposes (7).

Chromatography can be portrayed as a mass exchange procedure including adsorption. HPLC

depends on siphons to pass a pressurized fluid and an example blend through a section loaded
up with adsorbent, prompting the partition of the example segments. The dynamic segment of
the segment, the adsorbent,is ordinarily a granular material made of strong particles (for
example silica, polymers, and so on.), 2–50 micrometers in size. The segments of the
example blend are isolated from one another because of their various degrees of collaboration
with the retentive particles (8). The pressurized fluid is commonly a blend of solvents (for
example water, acetonitrile as well as methanol) and is alluded to as a "versatile stage". Its
sythesis and temperature assume a noteworthy job in the detachment procedure by impacting
the associations occurring between test segments and adsorbent. These connections are
physical in nature, for example, hydrophobic (dispersive), dipole–dipole and ionic, frequently
a blend (9).

HPLC is recognized from conventional ("low weight") fluid chromatography on the grounds
that operational weights are fundamentally higher (50–350 bar), while common fluid
chromatography ordinarily depends on the power of gravity to pass the versatile stage
through the section. Because of the little example sum isolated in logical HPLC, average
segment measurements are 2.1–4.6 mm distance across, and 30–250 mm length. Additionally
HPLC sections are made with littler sorbent particles (2–50 micrometer in normal molecule
size). This gives HPLC prevalent settling power (the capacity to recognize mixes) when
isolating blends, which makes it a prominent chromatographic system (10).

The schematic of a HPLC instrument ordinarily incorporates a sampler, siphons, and a

locator. The sampler brings the example blend into the versatile stage stream which conveys
it into the segment. The siphons convey the ideal stream and organization of the versatile
stage through the section. The indicator creates a sign relative to the measure of test segment
rising up out of the section, thus taking into account quantitative investigation of the example
segments. A computerized chip and client programming control the HPLC instrument and
give information investigation. A few models of mechanical siphons in a HPLC instrument
can combine different solvents in proportions changing in time, producing a sythesis angle in
the portable stage. Different finders are in like manner use, for example, UV/Vis, photodiode
exhibit (PDA) or dependent on mass spectrometry. Most HPLC instruments likewise have a
section broiler that takes into consideration modifying the temperature (11).

High parformance Liquid Chromatography (HPLC) is a type of section chromatography that

siphons an example blend or analyte in a dissolvable (known as the portable stage) at high
weight through a segment with chromatographic pressing material (stationary stage). The
example is conveyed by a moving bearer gas stream of helium or nitrogen. HPLC can
separate, and recognize aggravates that are available in any example that can be broken up in
a fluid in follow focuses as low as parts per trillion. In light of this adaptability, HPLC is
utilized in an assortment of modern and logical applications, for example, pharmaceutical,
ecological, crime scene investigation, and synthetic concoctions (12).
Test maintenance time will fluctuate contingent upon the collaboration between the stationary
stage, the particles being investigated, and the dissolvable, or solvents utilized. As the
example goes through the section it connects between the two stages at various rate, basically
because of various polarities in the analytes. Analytes that have minimal measure of
association with the stationary stage or the most measure of communication with the versatile
stage will leave the segment quicker (13).

Traditional sections utilized for HPLC are commonly made of hardened steel and are
produced so they can withstand weight of up to 5.5x107pa. The segments are 3-50cm long
and around 4 mm inward breadth, with stream paces of 1-3 cm 3 min smaller scale bore or
open cylindrical sections have an inside width of1 to 2 mm and are commonly 25 - 50 cm
long. They can support stream paces of 5-20 mm3 min-1. Miniaturized scale bore segments
have three significant preferences over customary sections: that lessens eluent utilization
attributable to the more slow stream rates. Perfect are interfacing with a mass spectrometer
inferable from the diminished stream rate. Expanded affectability attributable to the higher
convergence of analyte can be utilized (14).

THALASSEMIA SYNDROMES:

This term consists of all thalassemic Hb synthesis issues .These are autosomal recessive
conditions. α- and β-thalassemias have the best clinical importance (15,16,17).

Heterozygous thalassemia vendors aren't completely healthy: they constantly have signs that
require rationalization with moderate, iron-refractory, microcytic hypochromic anemia.
Homozygous fundamental paperwork are followed by severe, hypochromic hemolytic
anemias and complicated diseases.

α-thalassemias:
α-thalassemias are brought about by a α-globin chain blend deformity. At the sub-atomic
level, they result from fractional (α+) or complete (α0) cancellations, or all the more once in a
while transformations, of at least one of the four α-globin qualities (αα/αα)(15).

β-thalassemias:
β-thalassemia disorders are the aftereffect of lacking (β+) or missing (β0) generation of β-
globin chains. Their atomic causes are β-globin quality transformations. Most patients
originate from Mediterranean nations, South-East Europe, Arab countries, and Asia.
Hematological changes become show from between the ages of a quarter of a year and a half
year onwards (15,18,19).

ABNORMAL HEMOGLOBIN:
HbS and sickle-cell disease:
The expression "sickle-cell malady" incorporates all indications of anomalous HbS levels
(extent of HbS >50%). These incorporate homozygous sickle-cell ailment (HbSS) and a
scope of blended heterozygous hemoglobinopathies (HbS/β-thalassemia, HbSC illness, and
different mixes) (20).
As per the International Nomenclature, the beforehand usually utilized term "sickle-cell iron
deficiency" ought not be utilized, as the overwhelming parts of the infection are vascular
decimations and the organ harm they cause, not weakness.

HbS is the most perilous all things considered. The sickle cells brought about by an absence
of oxygen lead to vascular demolitions, so areas of localized necrosis with tissue passing can
happen in practically all organs (skin, liver, spleen, bone, kidneys, retina, CNS). Constant
hemolytic iron deficiency can typically be very much endured (20). Aplastic emergencies are
seen with serious sickliness following viral diseases (21).

HbC abnormality and HbC disease:

HbC homozygosity, or HbC illness, advances along these lines to sickle-cell infection,
however is less genuine (21,22). Variable hemolytic pallor is the most overwhelming
structure. Heterozygous HbC quality transporters appreciate total clinical wellbeing.

HbE abnormality and HbE disease:

HbE is a very basic Hb variation local to South-East Asia. Its ailment example is like that of
β-thalassemias. Hb is likewise precarious, which implies that hemolysis can be brought about
by viral contaminations and drugs. HbE is frequently joined with thalassemias, which may
bring about genuine major-structure hemoglobinopathies (21,22,23).

HbE homozygosity (HbE sickness): Typically, this is moderate, microcytic hypochromic

pallor with conceivable hemolysis because of exogenous causes.

HbE heterozygosity: Patients have variable hypochromic iron deficiency like that found in β-
thalassemia minor.
 MATERIAL&
METHODOLOGY
Present study was conducted as a pilot programme for screening of heamoglobinopathies in a
APMS campus. The total number of subjects included in the study was [Link] target group
comprised of students age of 19 – 25 years. A detailed hematological workup including
complete blood count (CBC) with hemoglobin levels, RBC indices including MCV, MCH,
MCHC, RBC Count, RDW-CV was performed on 5-part differential automated blood cell
counter (BC-5800) of Shenzhen Mindray Bio Medical Electronics based on the principle of
electrical Impedance.

Haematological diagnosis:
The normal reference ranges taken as laboratory control included: Hb: 11-16 gm%, RBC
Count: 3.5-5.5 x106/μl, Hct: 37-54%, MCV: 80-100 fl, MCH: 27-34 pg, MCHC: 32-36
gm/dl, RDW: 11-16%. Typing of beta thalassemia was done after peripheral blood smears
were evaluated for red cell morphology and correlated with RBC indices. Morphologically
beta thalassemia were classified as microcytic hypochromic (MCV<80fl, MCH<27 pg) and
macrocytic anemia (if MCV>100 fl).

Peripheral blood smear examination:

Anemia cases were further screened by high performance liquid chromatography (HPLC)
Biorad (Variant II) system for presence of any hemoglobinopathy. Interpretation of HPLC
results was done using various parameters (as described in company module) like flat
baseline, total peak area, peak profile and shape, relative percentages of the hemoglobin
fractions found after reviewing CBC data from 5-part cell counter to determine whether a
variant was present.
Beta Thalassemia carriers show RBC microcytosis, hypochromia, anisocytosis, poikilocytosis
(spiculated tear-drop and elongated cells).

HPLC Studies:

Patient with β-thalassemia minor/trait (heterozygous) has raised RBC count (>4.5 million/μl)
and HbA2 levels (4-9%) but HbF is normal (<2%).

The Hb pattern in beta-thalassemia varies according to beta-thalassemia type. HbA2 is

enhanced in beta thalassemia minor.10 Figure 2a and 2b shows the HPLC pattern in normal
individual and patient with ß Thalassemia trait. Hb electrophoresis pattern in various
conditions is shown in figure 3.

1. Cao A, Galanello R. Beta-thalassemia. Genet Med. 2010;12:61-76. Crossref

1.
 RESULT

[Link] sample positive sample:

Total [Link] sample Positive number of Male Female

 sample
357 25 11 14

[Link] on the different series on haemoglobinopathies:

Haemoglobinopathies No. of positive sample

Hb c/o thal 10
Typical thal 10
Siackle cell 1
Hb-D 2
Double heterozygous 1
HPFH 1
Toal 25

3. male / female:

Haemoglobinopathies Male Female

Hb c/o 4 6
Typical thal 4 6
Sickle cell 1 0
Hb-D 1 1
Double heterozygous 0 0
HPFH 1 1
Total 11 14

[Link] on district:

District Hbs/co Typical Hb-D Sickle Double HPF Total Per.

thal heterozygou H (%)
s

Anand 2 4 1 - - - 7 28
Kheda 1 2 - - - - 3 12

Bharuch 1 1 - - - - 2 8

Surat 4 1 - - - - 5 20

Vadodara 2 - 1 - - 1 3 12

Panchmahal 1 1 - - - - 2 8

Kutch - 1 - - - - 1 4

Bhavnagar - 1 - - - - 1 4

Amreli - - 1 - - - 1 4

[Link] on community:

community HPFH HbS Typical thal Hbs co HbD DH

Vankar 1 - - - - -
 Rathva - - - 2 - -

Makwana - - - 1 - -

Bhil - 1 - - - -

Rajput - - 2 1 1 -

Sindhi - - 1 - - -

Vasava - - - 2 - 1

Valand - - 1 - - -

Patel - - 3 - 1 -

Thakor - - 1 - - -

Chaudhari - - - 2 - -

Gamit - - - 1 - -

Ramanuj - - 1 - - -

Suthar - - 1 - - -

Prajapati - - 1 - - -

6. on marital and premarital:

Based haemoglobinopathies marital Premarital

Hbs c/o 0 10
Typical thal 0 10
Sickle cell 0 1
 Hb d 0 2
HPFH 0 1
Double heterozygous 0 1
Total 0 25

[Link] on hb value:

Hb value Hbs Hb-D HPFH Typical thal Hbs co DH

Normal 1 2 1 4 3 -
Mild - - - 4 6 -
Moderate - - - 2 1 1
Severe - - - - - -

8. based on CBC:

Hbpa MCV MCH MCHC RDW

thiees Nor Incr Decr Nor Incr Decr Nor Incr Decr Nor Incr Decr
mal ease ease mal ease ease mal ease ease mal ease ease

Typic - - 10 - - 10 10 - - - 10 -
al
thal
 Sickl - - 1 1 - - 1 - - 1 - -
e trait

Hbs - - 10 - - 10 9 - 1 - 10 -
c/o

DH - - 1 - - 1 1 - - - 1 -

HbD - 1 - - 1 - - 1 - - 1 -

HPF 1 - - - 1 - - 1 - - 1 -
H

[Link] on hplc:

[Link] Sample I'd no. HbA HbA2 HbF Hb S HbD HbE

% % % % % %
(Typical thal)
1 3248305 93.80 4.90 1.30 - - -
2 3248395 94.60 5.00 0.40 - - -
3 3248405 93.50 5.30 0.20 - - -
4 3248408 94.90 4.70 0.40 - - -
5 3248451 94.60 5.00 0.40 - - -
6 3248452 95.90 3.60 0.50 - - -
7 3248488 94.50 5.30 0.20 - - -
8 3248496 94.00 5.00 1.00 - - -
9 3248497 92.90 5.60 1.50 - - -
10 3248804 93.30 5.90 0.80 - - -
Hbs c/o

12 3248325 72.00 3.60 0.50 23.90 - -

13 3248334 73.60 3.50 0.50 22.40 - -
14 3248350 71.60 3.90 1.80 22.70 - -
15 3248426 70.00 3.30 0.80 25.90 - -
16 3248454 73.10 3.20 0.80 22.90 - -
17 3248770 73.40 3.30 0.40 22.90 - -
18 3248778 75.90 4.00 0.60 23.10 - -
19 3248783 66.10 3.80 1.50 28.60 - -
20 3248784 72.40 3.30 0.40 23.90 - -
21 3248880 72.30 4.30 0.50 22.90 - -
Sickle trait
22 3248823 3.40 4.80 16.80 75.00 - -
Double
heterozygous
23 3248797 59.30 3.40 1.10 36.20
HbD
24 3248440 65.10 1.70 .40 - 32.80
HPFH
25 3248447 92.3 2.20 5.50 - - -
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