Expert Review of Hematology

ISSN: 1747-4086 (Print) 1747-4094 (Online) Journal homepage: https://www.tandfonline.com/loi/ierr20

Recent advances in screening and diagnosis of

haemoglobinopathy

Kanjaksha Ghosh, Kinjalka Ghosh, Reepa Agrawal & Anita H. Nadkarni

To cite this article: Kanjaksha Ghosh, Kinjalka Ghosh, Reepa Agrawal & Anita H. Nadkarni (2019):
Recent advances in screening and diagnosis of haemoglobinopathy, Expert Review of Hematology,
DOI: 10.1080/17474086.2019.1656525

To link to this article: https://doi.org/10.1080/17474086.2019.1656525

Accepted author version posted online: 21

Aug 2019.

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Publisher: Taylor & Francis

Journal: Expert Review of Hematology

DOI: 10.1080/17474086.2019.1656525
Recent advances in screening and diagnosis of haemoglobinopathy

Kanjaksha Ghosh1*, Kinjalka Ghosh2, Reepa Agrawal3 and Anita H. Nadkarni1

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1. National Institute of Immunohaematology,13 Th Fl KEM Hospital MS building, Parel,
Mumbai, India
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2.Department of Clinical Biochemistry, Tata Memorial Hospital, Parel Mumbai-400012,
India.

3. BJ Wadia Children Hospital, Parel Mumbai400012, India.

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*Corresponding author:

Kanjaksha Ghosh
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Address: National Institute of Immunohaematology, 13 Th Fl KEM Hospital MS building,

Parel, Mumbai, India

Email: kanjakshaghosh@hotmail.com
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Abstract:

Introduction: Haemoglobinopathies are important causes of inherited disorders with

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substantial mortality and morbidity across the world. Therefore, proper utilisation of
available screening and diagnostic techniques are important for its diagnosis and
management.

Areas covered: In this review the authors attempt to: summarise clinical presentations,
give a brief account of existing techniques and discuss evolving and advanced
techniques for detection and screening of the condition. As prevention of the disease
condition is an important community measure to control the disease, techniques
involving newborn screening, antenatal diagnosis and point of care tests have been
described in addition to more advanced molecular and protein diagnostics. The
literature search in this area is covered between 1980-2018 with pubmed as the main
source along with authors’ own research this area.

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Expert opinion: Screening and detection of haemoglobinoapathy is best accomplished
by a hierarchical approach with the optimum blend of old and newer techniques.

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Starting with point of care techniques through the commonly used HPLC and high

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voltage capillary electrophoresis, or modern and high throughput molecular biology and
mass spectroscopic techniques can be used depending on specific situations. Every
country needs to optimise their techniques depending on the frequency of the problem
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and available resources.
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Keywords: haemoglobinopathy, thalassaemia, electrophoeresis, isoelectric focussing,
capillary electrophoresis, mass spectrescopy, HPLC, sequencing, molecular
techniques, neonatal screening.
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Article highlights
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• Symptomatic haemoglobinopathy is the most important monogenic disease in the

world.
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• Advances in diagnosis of this disorder has taken place in several areas (i). Point
of care tests, tests involving various molecular technologies and protein
fingerprinting using mass spectroscopy.
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• A blend of tests with already existing and modern advances layered as primary
level, secondary level and tertiary level investigation will serve the purpose in
most of the time.
• Genomic studies and its interaction may improve understanding of individual
patients out come with haemoglobinpathy gene and will help in management and
tailor made the therapy.
 • Newborn screening and prenatal diagnosis are important component of
haemoglobinopathy management.
• Marriage of IVF(In vitro fertilisation) and Molecular technology already made
preconception genetic diagnosis possible and will be boon to those who for
various reason does not consideration abortion to be an acceptable as a part of
foetal diagnosis.

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1. Introduction

Haemoglobinopathies constitute one of the most common inherited disorders affecting

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around 3-7 % of world population [1]. There are almost 2000 haemoglobin variants
reported in the literature[2] and many more continues to be discovered [3]. Fortunately
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many of the haemoglobin variants both in heterozygous and homozygous states are
asymptomatic condition. However, this makes them difficult to detect unless detected
during population screening or accidentally like when they interfere with HbA1c
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measurement [4]. Symptomatic haemoglobinopathies like HbS, HbC, beta

thalassaemias, symptomatic variants of alpha- thalassaemias, Hb Lepore, HbD, HbE
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etc. produce disease in double heterozygous or homozygous condition with some

exceptions (Hb D and Hb E). Rarely heterozygotes could also be symptomatic e.g HbS
Oman or Dominant or Inclusiform beta thalassaemias etc.
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Similarly heterozygotes of beta thalassaemias with coinheritance of multiple (more than

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4)copies of normal alpha gene produce the necessary genetic imbalance of alpha &
beta globin chains required for clinical expression of the disease as thalassaemia
syndrome[5]. For more than half of a century many techniques were developedin the
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laboratory to define various haemoglobinopathies.Some of these tests have lost

relevance today, while a few others continue to be usedin various laboratories of the
world [6].
Diagnosis of carrier status i.e inheritance of the single allele of such a disease is an
important public health challenge as also the diagnosis of pathologic
haemoglobinopathies both during neo -natal period and in later part of life.
Several Investigative techniques beginning from simple solubility test, alkali
denaturation test and acid elution test to increasingly more complex tests like
haemoglobin electrophoresis, isoelectric focussing, HPLC, high voltage capillary
electrophoresis, enzyme digestion coupled with mass spectroscopy have been used to
define abnormal haemoglobins.
Modern era of molecular technology encompassing PCR(Polymerase Chain Reaction)

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screening technologies like SSCP(Single Strand Conformation Polymorphism),

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CSGE(Conformation Sensitive Gel Electrophoresis), CRDB(Covalent Reverse Dot Blot
hybridisation), SSP(Sequence Specific Priming), ARMS(Amplification Refractory

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Mutation System), DGGE I Denaturent Gradient Gel Electrophoresis), Gap PCR/

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Multiplex PCR, MLPA (Multiple Ligation of Product Amplification) have allowed us quick
detection of mutation in globin genes including small and large deletions and crossovers
of these genes(Leading to different Lepore and anti- lepore haemoglobins). Finally
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SNP/oligonunleotide(single nucleotide Polymorphism) Microarray,Sanger sequencing
and NGS (Next Generation Sequencing)technologies along with other high throughput
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gene based technologies like dHPLC (denaturant high performance liquid
chromatography),High resolution melting curve analysis using Q-PCR(Quatitative PCR)
machine have speeded up diagnosis of haemoglobinopathies in secondary and tertiary
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care centres. Application of immunological techniques with development of potent

monoclonal antibodies against different pathological and other haemoglobins coupled
with nanoparticle probes and lateral flow technology have made diagnosis of several
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structural haemoglobinopathies as point of care testing an easy affair. Similarly

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combining mobile phone technology with portable microscopy or disposable microfluidic

system for detecting sickling is an innovation for point of care detection. Hence recent
advances in haemoglobinopathy detection involve(i)point of care technologies used at
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primary care facilities and population screening, (ii) the more complicated ones like
haemoglobin electrophoresis (both in acid and alkaline Ph), HPLC(High Performance
Liquid Chromatography), High Voltage Capillary electrophoresis and Isoelectric
focussing and (IEF) along with low level molecular technology for secondary care
facilities, (iii) advance molecular technologies and mass spectroscopic methods at
tertiary and research facilities.(iv) Antenatal diagnosis should preferably be done at a
tertiary care or terminal referral facilities where all techniques, as well as requisite
clinical, laboratory and counselling expertise are available.
Evolution of modern molecular biology and refinement as well as simplification of these
techniques, have allowed us to incorporate them in modern diagnostic laboratories
specializing in haemoglobinopathy diagnosis. Advances in immunological techniques
specially development of various monoclonal antibodies towards common variant and

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other haemoglobins have also added a new dimension in haemoglobinopathy

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diagnosis, particularly as a point of care test (POCT) [7]. These can be used easily in
those remote areas of the world, where both trained manpower and good laboratories

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are lacking but haemoglobinopathies are present in abundant measure.

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Finally but not in the least, prevention of very symptomatic haemoglobinopathies by
prenatal or preconceptional genetic diagnosis [8] has added yet another dimension in
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haemoglobinopathy diagnostics. In the present review of haemoglobinopathy diagnosis
and research, attempts will be made to describe the old & the not so old, well
established technologies in brief followed by elucidation of some of the modern
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advances in this area.

2. Clinical presentations of haemoglobinopathies

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Though many haemoglobinopathies could be largely asymptomatic, the major

symptomatic manifestation of haemoglobinopathies is anaemia due to:
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(i) major ineffective erythropoiesis (Thalassaemia syndromes),

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(ii) Haemolytic anaemia(HbS, HbE in association with beta thalassaemia genes, alpha
thalassaemias, unstable haemoglobinopathies etc.). Homozygous Hb E is an
asymptomatic condition without evidence of haemolysis.
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(iii) Haemoglobinopathies presenting with methaemoglobinemia with or without unstable

haemoglobins.

(iv) Haemoglobins with altered oxygen binding affinity, leading to polycythaemia

(Erythrocytosis) or anaemia.
3. Well established techniques for screening and diagnosis of
haemoglobinopathies

Clinical presentations like anaemia, haemolytic jaundice, failure to thrive,

splenomegaly, presence of similar conditions in other family members, gall stones,
epigastric discomfort or heaviness due to splenomegaly, methaemoglobinemia, sudden
drop in Hb concentrations without any apparent cause, haemoglobinuria, reticulocytosis,

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polycythaemia etc. are the presenting manifestations of hereditary
haemoglobinopathies. Diagnosis of haemoglobinopathy should start following above

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mentioned clinical presentations along with family history of disease.

Investigations should be initiated with a good peripheral blood count with red cell indices

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and examination of well stained peripheral blood smear by a trained morphologist.

The value of routine haematological analysis along with morphology and red cell indices
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should never be under estimated and must for the part of work up in any
haemoglobinoapthy case. In fact combining Red cell Indices, red cell morphology and
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HPLC data helps in detecting many cases where only HPLC would have been
inadequate [9, 10]. Reticulocyte counts (now also available in automatic haematology
counters), HbH preparations, Heinz body preparations etc could be part and parcel of
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haemoglobinopathy work up on blood smears. Similarly solubility test (HbS), unstable

haemoglobin test (Isopropanol or Heat test)(Fig 1C), Dichlorophenol indophenol test
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(HbE screening), one minute alkali denaturation test and its variations, spectroscopic
tests for unstable haemoglobins causing maethaemoglobinaemia, P50 determination for
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Oxygen (i.e. Oxygen affinity test), Oxygen dissociation curves etc for
haemoglobinopathies with altered oxygen affinity have been discussed in detail in a
standard textbook of laboratory haematology along with sickling test or its variations [9].
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Structural variation of haemoglobin molecules can be evaluated by various Hb-

electrophoresis techniques, i.e. cellulose acetate, or cellogel are the most commonly
used matrices on which haemoglobin solutions are made to run on acid or alkaline
pH.(Fig1 A) Depending on variations of electric charges due to variation in amino acid
replacements in different haemoglobinopathies (HbS, HbE, HbF, HbD etc) could be
separated by this technique by combining both acid & alkaline buffer electrophoresis.
HbA2 which is a minor haemoglobin has its importance for the diagnosis of beta
thalassaemia carrier status. In this condition HbA2 levels are increased (>3.5%) and in
electrophoresis sample this haemoglobin can be eluted and photometrically measured.
There are now better and more accurate and consistent techniques to do this test.

Accurate HbA2 measurement along with red cell indices is an important laboratory
exercise to detect beta thalassaemia trait. Haemoglobin electrophoresis in alkaline pH

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(8.4) followed by densitometry of stained electropherogram or spectrophotometric/
colorimetric measurement of eluted HbA2 compared to total haemoglobin have been

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used in the past to detect HbA2 level. As HbA2 has a very narrow normal range (upto
3.5 %) densitometric measurement was found to be inaccurate in substantial number of

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cases [11]. While elution of electropherogram followed by optical (spectrophotometric or
colorimetric) measurement was found to be labour intensive but more accurate and time
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consuming, it was not suitable for mass evaluation of samples. Microcolumn
chromatography with DE-52 column had some popularity in the past as it was accurate,
and it was possible to process upto 50- 200 samples in a day by a technician and when
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manually prepared was found to be cost effective [12].

All these techniques have now been replaced by HPLC (High Performance Liquid
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Chromatography) technique (Fig 1B) which is fast, automated, accurate and in addition
to measuring HbA2 accurately (unless other slow eluting haemoglobins are eluting at
the same window) and with current software is capable of providing rapid results in a
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few minutes time. In addition, some of these HPLC based techniques can detect many
abnormal haemoglobins as well as HbA1c, whose measurement is now imperative in
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assessing good control of diabetes mellitus [13]. Several companies in the world have
made several variations of this instrument. One variation also can be used for newborn
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screening where sickle cell anaemia is extremely common [14]. High voltage capillary
electrophoresis is also as accurate as HPLC based technique.s in detecting structural
haemoglobin variants and quantitation of those haemoglbins as relative percentages.

Traditional haemoglobin electrophoresis on acid or alkaline media is still extensively

used across the world mainly using cellulose acetate or cellogel as the solid stationary
phase through which different hemoglobins (made by lysing red cells) move at different
speed under constant electric current or constant voltage. This process in many
advanced laboratories have been replaced by high voltage capillary electrophoresis [15]
along with detection system and analysing softwares. Similarly FPLC (Fast Protein
Liquid Chromatography) techniques have also been used to detect thalassaemia
traits[16].

HPLC and high voltage capillary electrophoresis are the two techniques which are

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generally used as a primary investigation in the detection of haemoglobinopathies
across the world. As adult haemoglobin consists of two alpha and two beta globin

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chains and defect in one or in both the chains can cause haemoglobinopathy it makes
sense to detect whether alpha or beta chain is abnormal in a given case.. For some

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haemoglobin variants like HbJ, alpha globin chain may be abnormal or in some cases
the beta globin chain may be abnormal. Generally single alpha chain defect produces
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less than 25% of abnormal haemoglobin (usually 15 to 20%). Hence from HPLC or
electrophoresis itself it may be possible to suspect alpha globin chain defect. Short of
molecular studies globin chain electrophoresis provides a way of detecting defective
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migration in alpha or beta chain [17].

Relative abundance or deficiency of globin chain synthesis to detect thalassemia

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syndromes or thalassemic haemoglobinopathies (i.eHbE) was classically detected by

labelling variant haemoglobin chain and normal haemoglobin chain in reticulocytes with
radiolabeled amino acids (3H Alanine/ 3H Leucine) followed by CE/ CM cellulose
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chromatography to separate different globin chains and quantification of the chains in

beta scintillation counters.
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Isoelectric focusing (IEF) is another important but relatively old technique (Fig 1 D) in
detecting hemoglobinopathies[18]. In this technique the haemoglobin in haemolysate is
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made to move in a supporting media through which a pH gradient of the buffers are
created by special buffer mixtures (ampholines). Haemoglobin which differ by their
isoelectric point stop moving when the particular haemoglobin migrates and reaches
into the isoelectric point zone in the ampholine soaked strip. The bands in IEF are
narrow and sharper.It can measure even upto 3 to 5% haemoglobin like Hb-Barts in
newborn carriers of Alpha thalassemia.
4. Recent advances in detection of haemoglobinopathies:

Recent advances have taken place in this field in several areas

1. improved instrumentation and new techniques for diagnosis of abnormal

hemoglobin molecules.
2. Molecular testing of different types.

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3. Prenatal or preconception diagnosis of haemoglobinopathies using a

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combination of above techniques.
4. Newborn screening for common haemoglobinopathies.

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5. Use of computer /internet for improving diagnosis and delivery of results.
6. Improved and newer point of care techniques to detect sickle cell anaemia and

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other common haemoglobinopathies as population study or for diagnosis in the
clinical setting.
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4.1. Improved instrumentation and development of new techniques for detection
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of haemoglobinopathies

HPLC, IEF and electrophoresis techniques are continuously improving with improved
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instrumentation and miniaturization of instruments and improvement in software. HPLC

as a technique has been used in the laboratory for detection of haemoglobinopathies in
fetal blood and we found it quite useful though this technique did not find widespread
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usage in new born screening. However, a modified HPLC technique for screening of
sickle cell related disease using dried blood spots on special paper is used extensively
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in some countries [19].

Of late High voltage capillary electrophoresis (Fig 2A) is competing with HPLC system
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for routine use in haemoglobinopathy detection.

Protein fingerprinting is an old technique but it has been reinvented in combination with
mass spectroscopy to detect various haemoglobinopathies[20]. This technique is
capable of detecting many asymptomatic haemoglobinopahies which may remain
undetected by HPLC, IEF or by High voltage capillary electrophoresis.
4.2. Molecular testing of different types

Common beta thalassemia mutations (5-6 in many communities can detect 85 to 90%
of the cases) and several common pathogenic haemoglobinopathies like HbS, HbE,
HbC are detectable using simple molecular techniques. However, countries where there

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is large mix of different populations and influx of people from other areas of the world

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may require more than one sophisticated technique to find new mutations in
unsuspected locations of the globin genes. (Deep intronic sequence changes or

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mutations in regulatory areas of the genome or in some genes like transcription factor
genes controlling haemoglobin synthesis).

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These user-friendly techniques are ARMS PCR (Fig 2C) [21]. Covalent reverse dot blot
(CRDB)(Fig 2B) hybridization [22],RFLP(Restriction Fragment Length Polymorphism)
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based techniques [23] or PCR-ELISA (Enzyme Linked Immuno Assay) techniques [24].

RFLP based diagnosis using proper restriction endonucleases for amplified product can
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easily detect some of the common haemoglobinopathies like Hb S (Abolition of
restriction site for Dde1) or Hb D Punjab (creation of new restriction site for Eco R1)
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BseR1 for Hb O-Arab or Eae1 for Hb Q India. Many new restriction sites can be
designed for different mutations in reference laboratories and after testing their validity
they can be used in downstream laboratories using this simple technique. The
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Technique requires a PCR machine, Electrophoresis and a gel documentation system.

Simple camera can also replace the costlier Gel documentation system for the purpose.
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With increased availability of quantitative PCR(Q-PCR) machines in many laboratories

particularly for quantitation of HIV or other virus infections for management and follow
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up, these machines can also be fruitfully used to detect various haemoglobinopathies or
its heterozygous states using high melting curve analysis.

For referral centres Sanger sequencing (Fig 2 E) [26] next generation sequencing[27]
should be available. Similarly large deletions can be detected by GAP PCR (Fig 2D) or
MLPA techniques (Fig 2 F) [26] and also should be available in referral centres. Next
generation sequencing with continuously improving software will allow ones to predict
the results of various gene –gene interaction in predicting the outcome of natural history
of haemoglobinopathy in a given patient and help us in tailor mading his management.
For example we know that co inheritance of alpha thalassaemia genes and various
genetic polymorphisms in various locations of human genome controlling the expression
of foetal haemoglobin modulates the severity of sickle cell anaemia. Hence knowing
these poly morphisms/ mutations etc across the genome and use of proper software/

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artificial intelligence will allow us to predict more accurately the hetergenous behaviour

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of this monogenic disorder.

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4.3. Prenatal diagnosis preconception screening

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One of the means to control serious haemoglobinopathy in the population is by
premarital screening or counselling followed by prenatal diagnosis. If couples do not
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accept abortion for affected foetus following prenatal diagnosis, then preconception
diagnosis of the disease condition in the zygote is feasible. The technique detects the
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condition from a single cell from the zygote DNA which is amplified using single cell
PCR [25] to detect the pathogenic mutation in homozygous or double heterozygous
form. Single cell PCR for improved accuracy needs to be suitably a nested PCR
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followed by either sequencing or other techniques as previously described. However,

sequencing is a preferred technique[26]. Present day NGS can also detect other
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unsuspected serious disorders including presence of additional Alpha globin gene in

association with heterozygous beta cell mutation etc. [27].
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For successful prenatal diagnosis it is important to get a representative fetal specimen.

This is obtained by aspirating amniotic fluid (14 to 16 weeks of gestation) or by
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preferred chorionic villus sampling at 10 to 14 weeks. Proper dissection of chorionic

villus to separate out maternal tissue is a skill that needs to be developed to get pure
fetal tissue. All these procedures are invasive in nature and has 0.1-2%foetal loss as a
complication [28,29,30]. There is increasing interest in using maternal blood as a source
of fetal DNA for prenatal diagnosis [31]. One of the difficulties in diagnosis of pathogenic
mutation in foetus is the presence of the same mutation in maternal tissues in
heterozygous/ homozygous state. One approach is to find out whether the paternal
mutation, if different from that of the mother is present in fetal DNA. If that be so then
the next step is to do chorionic villus sampling. In the absence of detectable paternal
mutation in fetal DNA obtained from mother’s blood the next invasive step of chorionic
villus sampling may not be required. The procedure of providing prenatal diagnosis of
haemoglobinopathies using fetal DNA from mother's blood/ vaginal fluid is an evolving
technical field and needs further refinement of detection of additional polymorphism in

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the amplified fetal DNA to make sure that one of the mutations is from the father if the

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same mutation is present in both the father & the mother.

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4.4. Newborn screening

Newborn screening for haemoglobinopathies is developing as an important screening

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tool for diagnosing and managing severe haemoglobinopathies from neonatal period.
Certain haemoglobinopathies like sickle cell anaemia can have significant morbidity and
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mortality even from neonatal period [32]. Hence diagnosing it early can prevent quite a
few of its complications. Though traditional techniques like haemoglobin
electrophoresis, IEF and HPLC techniques are used to diagnose common
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haemoglobinopathies in adults yet there is some imprecision in the diagnosis of

thalassemic hemoglobinopathies, and beta thalassemia during newborn period because
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the beta globin chain synthesis at this period of time is still very low. It becomes very
difficult to discriminate sickle cell trait from sickle beta thalassaemia, at this period of
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life, with beta plus thalassaemia mutation. The presence of even a very small amount of
Hb A at this point in life show that at least one HBB gene is expressed, however in the
presence of mild beta thalassemia mutation a small amount of HbA may also be
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produced[33,34].

Far more precise molecular biological techniques are often needed but may prove very
expensive. However, data from both parents can be extremely useful at this point in
time. Moreover, newborn screen often involves screening for multiple diseases from
blood spots collected on Guthrie card [34,35]. This bloodspot presents some technical
challenge for use in HPLC system. Because the dried blood spot cause partial
degradation of haemoglobin over time and haptoglobin present in the plasma of blood
spot combines with different haemoglobins at a different rate and the combined proteins
produce additional elution peaks in HPLC system. Several companies have newborn
screening reagents to be used in HPLC machines modified for the purpose for

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screening sickle cell disease (Biorad). In future mass spectroscopy with its various

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modifications can be used for this purpose. High throughput molecular protocols are
now developing for different conditions and are also available for haemoglobinopathy

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detection [36].

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4.5. Use of computer/internet/improved software for haemoglobinopathy
diagnosis:
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Many of the standard techniques like electrophoresis IEF, HPLC etc. are nowadays
driven by softwares as additional accessory or in inbuilt computer. Many diagnostic
instruments are currently attached via internet to the company’s main computer
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systems, flow of information from these machines allow the company not only to
evaluate reagent usage by the user but also the results. It also allows them to develop a
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library of different haemoglobin variants with their characteristic patterns of presentation

in that instrument. This information improves the machine’s performance through
improved R&D. This will also allow a more accurate understanding of distribution of rare
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haemoglobinopathies across the globe. Though this kind of information centralisation

and sharing with a company or through clouds accessible by the company can have
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important academic and practical and economic advantages but suitable means to
protect patient identity is important moral and ethical imperative in these cases.
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4.6. Improved and newer point of care techniques for detection of common
haemoglobinopathies:

In this era of monoclonal antibody and nanotechnology antibodies have been produced
against various common haemoglobinopathies, against foetal as well as against adult
haemoglobins. This has led to the development of lateral flow techniques for detection
of these conditions at point of care. These monoclonal proteins are coupled to nano-
particles which allow to change its colour during aggregation[37].

Monoclonal antibody produced against Zeta chain of haemoglobin may allow

development of techniques to diagnose alpha thalassemia [37] due to one or two gene
deletion in adults. At present, without use of molecular biology techniques this condition

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can’t be diagnosed. It may not be out of place to mention that even before the
immunochromatography based test for sickle cell was available, solubility based test for

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similar purpose were available for quite some time.

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5. Conclusion an
Innumerable techniques are available for diagnosis of haemoglobinopathies. Recent
advances have taken place in the area in the form of HPLC, High voltage Capillary
electrophoresis & various mass spectroscopic techniques for detecting variant proteins
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in haemoglobin. Several molecular biology tests, immunological tests and Point of Care
tests(POCT) are constantly improving this area of diagnostics and providing large
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number of technical resources in the hands of a haemoglobinopathy diagnostician.

Hence haemoglobinopath detection at point of care or at secondary care facilities and at
tertiary care facilities as well as at research facilities needs to be prioritised. The
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optimum technique for diagnosis of haemoglobinopathies and its carrier status at

neonate, population, antenatal diagnosis and patient diagnosis levels needs to be
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employed for best result. Table 1 lists and summarises the tests.
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6. Expert opinion

Haemoglobinopathy is an extremely important inherited disorder of haemoglobin

structure and function with diverse clinical presentation from totally asymptomatic state
to an extremely symptomatic morbid and crippling disease compromising functional
ability and life span of the individual. Its importance stems from its widespread
prevalence as a public health challenge. Haemoglobinopathy as a genetic disorder
allowed us to understand some of the great intricacies of gene function and to develop
the concept of balanced polymorphism, a concept which makes it understandable why
some mutations, in spite of its extremely damaging effect in homozygous state persists
in a large number of people by providing resistance against a lethal disease like
falciparum malaria in heterozgotes.

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About 7% of the world’s 6 billion population carry genes for symptomatic
haemoglobinopathies i.e. Hb C, Hb D, Hb E, HbS, beta and alpha thalassemia and

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various combinations of these haemoglobinpathies in double heterozygote and
homozygote states which are mostly symptomatic. In addition haemoglobinopathies can

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present as methaemoglobinaemia, unstable haemoglobins, haemoglobins with altered
oxygen affinity leading to polycythaemia or anaemia. Asymptomatic structural mutations
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are innumerable, more than 2000 have already been described and newer ones
continue to be described.

Another important facet of this condition is its uneven geographical and population class
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distribution. This leads to extremely common occurrence of certain hemoglobinopathies

in certain geographical areas and in certain populations. This phenomenon create
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substantial public health challenges in those areas. With the advent of mass migration,
international travel this distribution of haemoglobinopathy over different geographical
areas have become dynamic and is rapidly changing. So areas where
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haemoglobinopathies were not a big problem is now facing such a problem. At a

community level birth of severe symptomatic haemoglobinopathy babies can be
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prevented by antenatal counselling and prenatal diagnostic techniques. Several

countries have already successfully implemented such a programme. As a majority of
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the symptomatic haemoglobinopathies are autosomal recessive conditions (few are

dominantly expressed also) carrier detection of these asymptomatic population is an
important public health activity for final prevention of birth of symptomatic
haemoglobinopathy babies and for taking precaution for carriers who may become
symptomatic under certain circumstances(ie, carrier of Hb S at high altitude, extreme
dehydration etc). These asymptomatic carriers of HbS could also be blood donors
whose blood if transfused to extremely sick and anoxic patients could create additional
complications. Sickle cell trait (carriers) though asymptomatic could develop severe
symptoms in relatively hypoxic environment like high altitude or in an extremely
dehydrating environment (desert) or with certain diseases like severe diarrhoea un less
precautions are taken.

Diagnosis of certain haemoglobinopathies at a very early stage like in a newborn

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through screening will mitigate many complications and reduce death and morbidity in
these patients allowing them to grow and develop normally with minimum complications.

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In addition to regular red cell transfusion and iron chelation certain symptomatic
haemoglobinopathies could be helped if certain investigation or screening tests could

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tell us whether these patients are likely to respond to alternate modes of therapy which
are convenient and has lesser complication potential than regular red cell transfusion.
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At the individual and family level a patient or his family members with inherited
symptomatic haemoglobinopathy would like to know the prognosis of this condition in
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that particular patient where gene –gene interaction exist and the response of that
genetic endowment in that particular patient can be understood through genomic
studies.
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To address the diagnosis and screening of haemoglobinopathies in its multitude of

dimensions, innumerable investigations discovered over last 70 years are available.
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Certain newer investigations like HPLC,Isoelectric focussing, High voltage capillary

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electrophoresis, Point of care tests(POCT), tests using mass spectroscopy coupled with
HPLC and various techniques to concentrate the material along with high throughput
artificial intelligence based mechanisation and analysis are making significant inroads in
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this area. Finally, molecular diagnostics from simple PCR- RFLP or SSP based
diagnostic to more complex Sanger sequencing, MLPA, GAP -PCR and NGS
sequencing for genomic studies are illuminating this area of diagnostics and research.
Similarly advances in IVF (In Vitro Fertilisation) technology and prenatal diagnostics
including non-invasive sampling and preconception genetic diagnosis (PGD) are making
important contributions in this area. Dried blood samples (DBS) on Guthrie card can
now be used both for diagnosis of haemoglobinopathy as well as genetic and genomic
assessment of the condition in the newborn screening strategy.

All investigations and screening techniques are not suitable for all categories of
population and patients. Different diagnostic algorithms should be developed for the
point of care, primary care facilities, secondary care facilities and at tertiary care or
referral centre facilities. This should be done keeping in mind the objective of these

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facilities and finances available to run them a blend of existing and newer technologies
can be used.

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Funding

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This paper was not funded.

Declaration of interest
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The authors have no relevant affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict with the subject matter or
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materials discussed in the manuscript. This includes employment, consultancies,
honoraria, stock ownership or options, expert testimony, grants or patents received or
pending, or royalties.
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Reviewer disclosures
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Peer reviewers on this manuscript have no relevant financial or other relationships to

disclose.
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 Table 1: List of investigations appropriate at different levels of health care system
to diagnose haemoglobinpathy*.

I. Primary care:

i. Complete Blood count with red cell indices

ii. Good quality peripheral blood smear.
iii. Point of care tests (Immunochromatography based)
iv. Fetal haemoglobin estimation.

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v. Solubility test (HbS)/ Sickling test.

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vi. Dichlorophenol Indophenol test (Hb E)
vii. O2 saturation test (Non-invasive) for methaemoglobinaemia and compromised
oxygen affinity haemoglobins.

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vii. Spectroscopic examination of haemoglobin.

II. Secondary care.

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i. all the above Investigations.
ii. Hb Electrophoresis in acid and alkaline Ph.
iii. Isoelectric Focussing.
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iv. Globin Chain Electrophoresis.
v. HPLC / High Voltage capillary electrophoresis.
vi. Acid elution staining of red cells (for HPFH).
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vii. Urine Hemosiderin.
viii. Neonatal Screening on dried blood spots.
ix. Spectrophotometric quantitation of HBA2 by elution from electrophoresis strips.
x. DNA extraction and storage from blood samples
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xi. Chorionic Villus sampling/ Dissection.

xi. Low level Molecular Technologies: PCR –SSP/ ARMS – PCR/ CRDB etc.

III. Tertiary care/reference lab:

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i. All the above investigations.

ii. Different types of Mass spectroscope with different applications.
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iii. All instruments required for prenatal Dx.

iv. Assisted reproductive technology & PGD.
v. MLPA/ Gap PCR/ Multiplex PCR.
vi. Q PCR and High Resolution Melting Curve analysis.
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vii. Sanger sequencing.

viii. DNA Microarray.
ix. New Generation Sequencing(NGS Platform).
X. Flow cytometer to separate fetal nucleated RBC from mother’s blood as a part of no
invasive antenatal diagnosis

The tests are not comprehensive as many more test platforms are developing every
day.
 Figure legends:

Figure 1.

A. Haemoglobin electrophoresis

B. Four HPLC patterns i.e beta thalassemia trait, Normal, HbS trait and Hb E trait.

C. Unstable haemoglobin test

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D. Isoelectric focussing patterns of different haemoglobins.

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Figure 2.

A: Chromatogram of β- variant hemoglobin (Hb Andrew-Minneapolis

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heterozygous, codon 144 G→C heterozygous) by capillary electrophoresis.
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B: Identification of β-globin gene mutations by CRDB

Strip 1: Normal for CRDB

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Strip 2: Heterozygous for IVS I-5 (G→C) mutation
Strip 3: Homozygous for IVS I-5 (G→C) mutation
Strip 4: Heterozygote for Codon 6 (A→T) mutation (HbS heterozygote)
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Strip 5: Homozygous for Codon 6 (A→T) mutation (HbS homozygous)

C: Detection of the Cap site +1 (A→C) mutation by ARMS technique

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Lane 1: 100 bp ladder, Gene ruler, Thermo Fischer scientific

Lane 2 and 3: Heterozygous for Cap Site +1 (A→C) mutation
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Lane 4 and 5: Homozygous for Cap Site +1 (A→C) mutation

Lane 6 and 7: Homozygous for Cap Site +1 (A→C) mutation
Lane 8 and 9: Normal for Cap Site +1 (A→C) mutation
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D: Detection of α-globin gene deletion by multiplex PCR( Gap –PCR)

Lane 1: Molecular weight marker XVI
Lane 2: -α4.2/αα
Lane 3: -α3.7/αα
Lane 4: -α3.7/-α3.7
Lane 5: -α4.2/-α4.2
Lane 6: -α3.7/--SA
Lane 7: αα/αα

E: Automated DNA sequencing of β-globin gene showing codon 55 (-A) deletion

in heterozygous condition

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F: MLPA analysis of the β-globin cluster showing genomic regions deleted in
patients heterozygous for 4056 bp deletion

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 Fig 1 a
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 Fig 1 B
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 Fig 1b
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 Fig 1 b
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 Fig 1 b
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fig 1 c

Heat stability test

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Stable hemoglobin
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 Fig 1 D
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 Fig 2

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Figure 2 :

A: Chromatogram of β- variant hemoglobin (Hb Andrew-Minneapolis heterozygous, codon

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144 G→C heterozygous) by capillary electrophoresis.

B:Identification of β-globin gene mutations by CRDB

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Strip 1: Normal for CRDB

Strip 2: Heterozygous for IVS I-5 (G→C) mutation
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Strip 3: Homozygous for IVS I-5 (G→C) mutation

Strip 4: Heterozygote for Codon 6 (A→T) mutation (HbS heterozygote)
Strip 5: Homozygous for Codon 6 (A→T) mutation (HbS homozygous)
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C:Detection of the Cap site +1 (A→C) mutation by ARMS technique

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Lane 1 : 100 bp ladder, Gene ruler, Thermo Fischer scientific

Lane 2 and 3 :Heterozygous for Cap Site +1 (A→C) mutation
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Lane 4 and 5 :Homozygous for Cap Site +1 (A→C) mutation

Lane 6 and 7 :Homozygous for Cap Site +1 (A→C) mutation
Lane 8 and 9 :Normal for Cap Site +1 (A→C) mutation

D:Detection of α-globin gene deletion by multiplex PCR( Gap –PCR)

Lane 1:Molecular weight marker XVI
Lane 2: -α4.2/αα
Lane 3: -α3.7/αα
Lane 4:-α3.7/-α3.7
Lane 5: -α4.2/-α4.2
Lane 6: -α3.7/--SA
Lane 7: αα/αα

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E: Automated DNA sequencing of β-globin gene showing codon 55 (-A) deletion in

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heterozygous condition

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F: MLPA analysis of the β-globin cluster showing genomic regions deleted in patients

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heterozygous for 4056 bp deletion

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