A

DISSERTATION ON
“ HEMOGLOBINOPATHIES AWARENESS AND SCREENING AMONG COLLEGE
STUDENTS IN ANAND PEOPLE’S MEDICARE SOCIETY COLLEGE CAMPUS :
OUTCOME A COLLEGE WIDE SURVEY”
COMPLETED AT
DR.I.P. PATEL INSTITUTE OF MEDICAL TECHNOLOGY & RESEARCH,ANAND
WITH COLLABORATION OF
INDIAN RED CROSS SOCIETY
UNDER GUIDENCE OF MS MINAL THAKKAR

CO. GUIDE: DR. KISHOR SONI & DR. ASHOK JAIN

SUBMITED BY
PALLAVI JAYESHBHAI BARIA
(B.Sc. MEDICAL TECHNOLOGY (CLT))

IN PARTIAL FULFILLMENT FOR THE DEGREE

OF M.Sc. (MLT)

ACADEMIC YEAR: (2018-2020)

TO SARDAR PATEL UNIVERSITY

(FACULTY OF MEDICINE)

DR.I.P. PATEL INSTITUTE OF MEDICAL TECHNOLOGY & RESEARCH,ANAND

 Acknowledgement
At this moment of accomplishment, First of all, I am gratefully to God, for helping me
to take the right decision and opting for dissertation, this continual strength and blessing were
always with me throughout my work, this completion of my work would not be possible
without the direction of God. I am grateful to God.
I am sincerely thank full to the Dr. I. P. Patel institute of medical technology and
research who provided such excellent facilities throughout my work, experience and I will
treasure there moments of my experience in this excellent place.
I am sincerely thank full to the Indian Red Cross Society anand who provided such
excellent facilities throughout my work, experience and I will treasure there moments of my
experience in this excellent place.
I would like to express my heart felt gratitude to my Guide, Miss. Minal Thakkar for
her constant guidance, patience, encouragement, advice throughout my dissertation work. I
felt that I am working with my parent in the lab and this is due to the attitude of Miss. Minal
Thakkar.
I express my thanks to Prof. Tammana Ahuja, Head of the department of Dr. I. P. Patel
institute of medical technology and research for allowing me to work and utilize the facilities
of the department.
I will never be complete without thinking Dr. Meghana Patel, who also helped me in some
way or the other. His moral support was always there with me. I am also sincerely and
heartily grateful to DR. Navneetkumar Singh for their moral support, kinds of advice and
encouragement during my work.
Thanks, to the teaching and non-teaching staff of the Dr I.P.Patel Institute of medical
technology and research who helped me directly or indirectly in completing my work.
It’s my fortune to gratefully acknowledge the support of some my friends vidhi, vinayak,
gautam, vikram, disha, dharika, nimisha for their companionship during all ups and downs
that I experienced, I thank you for your constant support during both the good and hard time.
My deepest gratitude goes to my parents for their unflagging love and support
throughout my life; this dissertation is simply impossible without them. I am thankful to other
members of my family for their inspiration, encouragement and sincere prayers without this
training would have not been reality.
My thesis is completed with the prayer and support of so many near and dear ones,
Thanks to the sacrificial love of my family, friends, guide, professor and those who helped
me during my work,
Thank you all. Pallavi Baria
 ABSTRACT:

Haemoglobinopathies is the most common monogenic disease in the whole world.

These disease is arises from the deletion or point mutation of one or both the globin
chain.Haemoglobinopathies comprise of thalassaemias and variation haemoglobins. Bearers
of haemoglobinopathies are in part ensured against horribleness and mortality of falciparum
intestinal sickness and this has brought about their higher pervasiveness in tropical nations. In
India, they are in charge of the biggest number of hereditary issue and subsequently are of
incredible openwellbeing significance. Clinically significant thalassaemic scatter in India is
alpha-thalassaemia while alpha- thalassaemia, albeit increasingly normal in inborn populace,
is free from grimness. Of the few unusual hemoglobin particles, three which are broadly
common in India incorporate : Hb S, Hb E and Hb. The combined quality recurrence of
haemoglobinopathies in India is 4.2%.11 With a populace of more than one billion and a
birth pace of 28 for eachthousand, there are more than 42 million bearers and over12,000
babies are brought into the world every year with a noteworthy and clinical noteworthy
haemoglobinopathy. Out of these, alpha-thalassaemia major and clinically noteworthy
sicklecell issue represent practically equivalent numbers. alpha-thalassaemia is discernible in
pretty much every Indian populace, be that as it may, it is seen with most astounding
recurrence in north-west and far east. Sindhis, Gujaratis, Bengalis, Punjabis and Muslims
represent the vast majority of alpha- thalassaemia. Carrier state for alpha-thalassaemia in
India shifts from 1-17% with a normal of 3.2%.20 alpha-thalassaemia is most generally
common in the innatepopulace with a recurrence of 1-40% in Andhra Pradesh what's more,
Gujarat.Fortunately, the genotype of alpha- thalassaemia pervasive in India is alpha+, which
is clinically quiet both in the heterozygous just as homozygous state. Thus, it is free from
morbidity.Lehman and Cutbush depicted the main instance of Hb S from tribals of southern
India in 1952.8 Subsequently, Hb S has been accounted for from different Indian states,
networks and ethnic gatherings with a normal recurrence of 4.3% (territory : 0-44%).8 Sickle
quality in India is for the most part found among Dravidian and pre-Dravidian clans. Be that
as it may, with movement and blending between the tribals and non-tribals, Hb S is currently
recorded frommost standing gatherings and states. Hb S is dominatingly found in focal India
for example Vidarbh in Maharashtra, Madhya Pradesh, Orissa, Andhra Pradesh, Gujarat and
to a lesser degree in Tamil Nadu, Karnataka, Kerala and Uttar Pradesh. It is uncommon in
Bihar, West Bengal, northeastern states, Punjab, Haryana, Himachal Pradesh, Rajasthan and
Jammu and Kashmir. Five unmistakable haplotypes of Hb S quality have been depicted with
pervasiveness in explicit topographical areas. Four of these for example Benin, Senegal,
Bantu and Cameroon are the African haplotypes while the fifth one is the Saudi
Arabia/Indian haplotype. As the name proposes, this is the haplotype which is pervasive in
India. This haplotype is related with more elevated amounts of Hb F which enhances the
clinical seriousness. High recurrence of alpha-thalassaemia present in the condition of
Gujarat likewise helps in making the clinical seriousness of sickle cell ailment
milder.Unfortunately, the equivalent doesn't apply to the sicklers of Vidarbh in Maharashtra
where alpha-thalassaemia is rare.Thus, the clinical seriousness of sickle cell ailment in India
is milder with contrast with Africa however profoundly factor inside the nation itself. Hb E is
generally appropriated in north-eastern conditions of India It was first revealed among the
Assamese with bearer pace of 23% among the totos. The Ahoms of Assam have the quality
recurrence of 46.4%, one of the most elevated for any unusual hemoglobin detailed from any
populace on the planet. Strangely, the predominance of Hb E bearer state is underneath 1% in
Mizoram. In West Bengal differs from 3-33% while it is nearly nonexisting in southern
India .Hb D is overwhelmingly found in Punjab, Uttar Pradesh, Gujarat and Jammu and
Kashmir. It is most common in Punjabi, Sikh and transient Sindhi populace. Luckily, Hb D in
both heterozygous and homozygous structure is clinically asymptomatic. Indeed at the point
when co-acquired with alpha-thalassaemia, the introduction is like thalassaemia minor. The
main clinically critical type of Hb D is the point at which it is co-acquired with Hb S
delivering a serious sickle cell malady. Luckily, no populace in India has high recurrence of
both Hb D furthermore, Hb S. The general quality recurrence of Hb D in India is underneath
1%. A few other uncommon sorts of thalassaemic issue and hemoglobin variations have been
sporadically detailed from India. These incorporate : alpha-thalassaemia, inherited diligence
of fetal hemoglobin, Hb Lepore, Hb Q, Hb K, Hb J, Hb M, Hb Chandigarh, Hb Sunpraire, Hb
Koyadora and so on..
Content:
1. Introduction

2. Review Of Literature

3. Aims And Objective

4. Methodology

5. Result

6. Discussion

7. Conclusion

8. Reference

9. Masterchart
 INTRODUCTION

Introduction
Haemoglobinopathies are the normal hereditary confusion on the planet. A type of hereditary
issue of hemoglobin is known as heamoglobinopathies,4.5% of the total populace is
influenced by this issue .and 3 needs children are brought into the world with the major
haemoglobinopathies.[1,5]. To keep the event of progressively genuine haemoglobinopathies
in new conceived can done by precise and auspicious identification of various kinds
haemogloib (Hb) variants[5]. in our nation there is families and network which cannot
manage the cost of the costly treatment like bone marrow transplantation.
[6].haemoglobinopathies is one of the enormous general medical issues in India. It has been
seen that with populace of 1 thousand millions at the thousand years year 2000 and birth rate
of new brought into the world 25 for every thousand newborn children, there would be 45
milllions conveys and 15 thousand are brought into the world with haemoglobinpathies in
India every year. In various populace of India the transporter prevelence of
haemoglobinpathies differs from 3 to 17 %.[7] In India most transcendent
haemglobinopathies found are sickle cell Weakness, beta thalassemia ,hemoglobin D and
hemoglobin E that must be assessed 5.35%.[2].the commonness of beta thalassemia attribute
is 3-17% and sickle cell paleness 1-44% in India because of association and position and
region endogamy.[3]. Hemoglobin is compound particle which present inside red platelet,
which transport O2 and CO2 in the body .Any imperfection in hemoglobin quality reason
irregular hemoglobin and weakness. Which produce the condition called as
Haemoglobinopathies. Haemoglobionpathies show up in two fundamental circumstances,1st
diminished combination of one of the globin chain for example thalassemia.2nd irregular
globin chain for example sickle cell disease.[6] Haemoglobinopathies are increasingly
common in Gujarat state as contrast with other province of India Modell and Petrou have
discover that 12% instances of real haemoglobinopathy attributes in Gujarat.[4].It is an
autosomal passive gathering of confusion. The wide variety in the clinical result of
hemoglobin issue could be a scribe to the increments of different acquired modifiers and
ecological variables. Heterogenous conveyance of the haemoglobinopathies and nearness of
high change in the phenotypic sign of explicit transformation are enormous issue the
advancement of projects for the control of the disease.[8]. The event of beta-thalassemia
transporters in the India is 3-4% some populace like Sindhis, Kutchis , Lohanas, Pujabis,
couple of Muslim gatherings and couple of innate populace have higher pervasiveness around
5-17%,alpha thalassemia bearers is about 13% however it can shifts from 3-18% in position
populace wherever it is exceptionally high in couple of inborn gathering about 90%,
hemoglobin S bearers are for the most part observed in ancestral and some in nontribal
assemble around 5-10%,Haemoglobin E transporters are as often as possible found in north-
east with predominance of between 3-64%,Haemoglobin D Punjab is for the most part found
in north-western with commonness of 3-4%. Because of relocation of populace and
admixture of populace hemoglobin variations are seen everywhere throughout the India.
Therefor the complex heterozygous type of Hb like Hb S beta-thalassemia ,Hb E beta-
thalassemia, Hb D-beta-thalassemia are seen in various races.[9,10,11,12] variety in the
qualities that code for the alpha chain or beta chains can do natural effect on capacity of
hemoglobin. a few hundred Present human freak haemoglobins, those in which natural
capacity is change have been quickly talked about here.

The globin present in hemoglobin (Hb) is comprised of 7 unique sorts of polypeptide chains,
changes in number of amino corrosive and course of action of amino acids amid different
phases of human intra-uterine development[14] and are organize by the Greek letters, alpha
(a), beta (b),gamma (g), delta (d), epsilon (e), and zeta (z). Epsilon, zeta and couple of alpha
chains are produce in early embryonic life, alpha and gamma chains are amalgamation in
fetal life, and alpha, beta and delta chains fundamental in the postnatal life. The polypeptide
binds help to decide the sort of which haemoglobin(Hb) particle synthesis.[13]. In soonest
fetal life, zeta and epsilon affixes both join to frame Hb Gower I(z2e2), alpha and epsilon ties
consolidate to shape Hb Gower II (a2e2), and zeta and gamma anchors consolidate to frame
Hb Portland (z2g2). Consummation of first trimester, alpha chains are supplanted by the zeta
chains and gamma chains are supplanted by epsilon chains. In fetal life, the primary
hemoglobin is Hb F (a2g2), which is available around 80 to 90% of the all out hemoglobin
amid birth, while after birth, just less sum are seen up to 6 to a year and fetal hemoglobin is
completely supplanted by the human grown-up hemoglobin Hb A ( Hb A1) which contain a2
and b2, contain about 97% and HbA2 which contain a2 and d2 contain about 1.5 to 3.5% of
the all out grown-up haemoglobin.[15].Haemoglobinopathies can be available in both the
structures homozygous and heterozygous. A change in both alpha and beta chain amino
corrosive grouping called as a two fold heterozygosity.
In my studies all students of APMS campus are screened for haemoglobinopathies by CBC
HPLC electrophoresis methods and give them post councelling who are positive for any of
the different haemoglobinopathies. Students are comes from different state and different
community. Awareness about haemglobinopathies are given to all students by
precouncelling.
 AIMS & OBJECTIVES

AIMS AND OBJECTIVE

Aim
The aim of the study was to know the incidence and prevalence of Haemoglobinopathies by
molecular screening.
Objectives
The objectives of the study were as follows:
1. To find the types of Haemoglobin abnormalities among all cases of heamoglobinopathies.
2. To find the clinical presentation of hemoglobinopathies in adults in APMS campus.
3. To compare the incidence of hemoglobinopathies between adults population in anand and
different districts where from students comes.
4. To explore possibility of screening and proper counselling and suggest protocol to prevent
and decreases the incidence of disease in affected population.
5. To determine the prevalence of different types of haemoglobinopathiess.To correlate
clinical and hematological features of haemoglobinopathies.
 REVIEW OF LITERATURE

REVIEW OF LITERATURE:
The genetic issue of blood contain haemoglibonpathies it is the champion among the most
genuine general therapeutic issue in India, which impact the 7% of the world populations.16.
Haemoglobin structure and function:
Hemoglobin is a tetrahedral particle made out of four sub-units (Figure). Each sub-unit
comprises of a protein called a globin chain and an iron containing haem component.
Recently orchestrated globin chains will normally overlap into a-helical structures. Singular
globin chains are flimsy, yet a-globin like chains (globin created from the qualities of the a-
globin locus) will frame stable dimers with bglobin like chains (globin created from the
qualities of the b-globin locus). The ensuing relationship of two dimer sets results in the
arrangement of a hemoglobin atom (Thom et al. 2013). Hemoglobin is blended inside
erythroid antecedent cells. Amid early fetal advancement hemoglobin combination happens
at first in the burden sac and in this manner in the liver. As the bone marrow creates it
continuously takes over this capacity and is the principle site of hemoglobin combination
when of birth (Sankaran, Xu and Orkin 2010a). The earth inside the red platelet keeps the
iron related with the haem component in a decreased state (Fe2+), which is fundamental for
oxygen official. Hemoglobin will tie oxygen atoms when presented to a high oxygen
fractional weight (pO2) and continuously discharge oxygen as the pO2 falls (Figure 3), in this
way enabling hemoglobin to play out the indispensable capacity of transporting oxygen to
breathing tissues all through the body (Brittain 2002).

Figure : Heamoglobin
The globin gene:
There are eight useful globin qualities just as a few pseudo qualities. The globin qualities are
found in two loci, every one of which has a related upstream administrative component.The
a-globin locus on chromosome 16 (Deisseroth et al. 1977; Mettananda et al.2016) contains
three of the practical globin qualities. Recorded in 5' to 3' request these are Hemoglobin
subunit zeta (HBZ), Hemoglobin subunit alpha 2 (HBA2) and Hemoglobin subunit alpha 1
(HBA1). Articulation of the a-globin locus qualities requires collaboration with four upstream
components called the Multispecies Saved Sequences (MCS), the most significant of which is
MCS locale two (Higgs 2013). The staying five practical globin qualities are found in the b-
globin locus on chromosome 11 (Deisseroth et al. 1978; Noordermeer and de Laat 2008).
Recorded in 5' to 3' request these are Hemoglobin subunit epsilon 1 (HBE1), Hemoglobin
subunit gamma 2 (HBG2), Hemoglobin subunit gamma 1 ((HBG1), Hemoglobin subunit
delta (HBD) and Hemoglobin subunit beta (HBB). An upstream administrative component
known as the b-Locus Control Region (b-LCR) is required for articulation of these qualities
(Kukreti et al. 2010)Hemoglobin structure and capacity:
Hemoglobin is a conjugated protein with an atomic load of around 68,000 includes 95 % of
the RBC's dry weight or 33% of the RBC's weight by volume. Around 65% of the
hemoglobin happens amid the nucleated phases of RBC development and 35% happens amid
the reticulocyte arrange. Typical hemoglobin comprise of (a tetramer of two sets of
polypeptide chain) and four haem gathering, every one of which contains a protoporphyrin
ring in addition to press.

Globin chain switching during development:

Embryonic haemoglobins are framed amid early improvement, these are in this manner
supplanted by fetal haemoglobins, which are thusly supplanted by grown-up haemoglobins.
The highlights of these various haemoglobins are abridged in Table 1. The adjustments in
hemoglobin that happen amid advancement are the consequence of various globin qualities
being communicated at various formative stages; a procedure alluded to as globin quality
exchanging. The b-globin locus change from the HBE1 quality to the HBG1/HBG2 qualities
is named the crude to complete switch and denotes the finish of generation of crude
erythrocytes in the burden sac (Sankaran, Xu and Orkin 2010a). A comparable aglobin locus
change happens from the HBZ quality to the HBA1/HBA2 qualities (Mettananda et al. 2016).
There is additionally a resulting second b-globin locus change from the HBG1/HBG2
qualities to the HBB quality (Sankaran, Xu and Orkin 2010a), bringing about the change
from fetal to grown-up hemoglobin. In any case, this second switch is less outright than the
crude to authoritative switch and therefore follow measures of Hb F (a2g2) can be
distinguished in most solid grown-ups (Thein and Menzel 2009). The procedures controlling
the guideline of the globin qualities have been the subject of extreme examination
(Stamatoyannopoulos 2005). This intrigue stems from the suspicion that improved
comprehension of the components included will give bits of knowledge into formative
science and may prompt treatments for the treatment of acquired issue of hemoglobin
(Stamatoyannopoulos 2005; Sankaran, Xu and Orkin 2010a). Two principle systems control
globin quality exchanging, rivalry for access to the upstream administrative component
(Figure 5) what's more, self-sufficient quality hushing (Figure 6) (Stamatoyannopoulos
2005).
Figure: Globin gene switching is driven by competition for an upstream regulatory
element.
Figure: The role of autonomous gene silencing in the switch from foetal to adult
haemoglobin.
Hemoglobin synthesis:(20)

Typical hemoglobin generation is reliant on three procedures:

1. Adequate iron conveyance and supply

2. Adequate combination of protoporphyrins (the forerunner of haem)

3. Adequate globin combination

Iron Delivery and Supply:

Iron is conveyed to the layer of the RBC precursor(reticulocyte) by the protein transporter
transferrin. Most of the iron that crosses the layer and enters the cytoplasm of the cell is
focused on hemoglobin blend and in this way continues to frame haeme.Abundance iron in
the cytoplasm totals as ferritin, the measure of which is reliant on proportion between the
dimension of plasma iron and the measure of iron required by the erythrocyte for hemoglobin
amalgamation. Two-third of the all body iron supply is bound to haem in the hemoglobin.
Adequet synthesis of Protoporphyrins:

Protoporphyrin union starts in the mitochondria with the development of delta aminolevulinic
corrosive from glycine and succinyl COA, which is the significant rate restricting advance in
haeme biosynthesis the mitochondrial compound delta ALA synthetase, which intercedes this
response in impacted by erythropoietin and requires the nearness of the cofactor
pyridoxalphosphate(vitamin B6).

In the cytoplasm, buildup of two atom of delta ALA, catalyzed by delta ALA dehydrase,
produces the pyrroleporphobiliongen (PBG). Since four particles are associated with the
response, the development of four sorts of isomers is hypothetically conceivable.
Nonetheless, just two sorts of isomers have happened physiologically UPG-1 and UPG-3 and
basically all the UPG is the type3 isomer. Coproporphyrinogen(CPG) is next framed by
means of decarboxylation responses from UPG 3. The last advance of haeme blend are
completed in the mitochondria and include the development of protoporphyrinogen(PP) from
CPG 3. Since PP artificially has three kind of side chains, 15 potential isomers of PP can
frame , contrasted and four UPG and CPG isomer. In any case, ordinary mitochondrial
physiology prompts the arrangement of just one of these isomers, PP9, from CPG3. After
further discussion of protoporphyrin 9, the consolidation of iron outcomes in
haeme(ferroprotopophyrin 9) development..

Globin Synthesis:

Globin chain amalgamation happens on RBC explicit cytoplasmic ribosomes, which are
started from the legacy of different auxiliary qualities, every quality outcomes in the
development of a particular polypeptide chain. Each physical cell including the RBC, contain
four alpha qualities, two beta qualities, two delta qualities and four gamma qualities. The
alpha qualities are situated on chromosome 16, and beta, delta and gamma qualities are
situated on chromosome 11. The subsequent items are called alpha, beta, delta and gamma
chains. In the hatchling, it ought to be recalled that amid the initial couple of long stretches of
life, an alternate polypeptide chain, epsilon, is orchestrated shaping tetramer particles that are
assigned "embryonic" haemoglobin.All grown-up typical hemoglobin are structure as
tetramer comprising of two alpha chains and two Non alpha globin chains.

Each combined globin ties joins with haeme to shape hemoglobin, which principally
comprise of two alpha chains, two beta chains and four haem gathering. Ordinary alpha chain
comprise of 141 amino acids, while typical beta chains comprise of 146 amino acids deposits.
The exact request of amino acids is basic to the hemoglobin particle's structure and capacity.
The substitution of even one amino corrosive can prompts haemoglobinopathy. A sufficient
measure of beta chain amalgamation is additionally significant since diminished creation of
one of the polypeptide anchors prompts gathering of disorders. More regular is beta
thalassemia.
The rate of globin chai production is directly related to the number of porphyrin synthesis and
vice versa; protoporphyrin production is decreased when globin synthesis is less.

Haemoglobinpathies :

The haemoglobinopathies are characterize by creation of fundamentally inadequate

hemoglobin cause because of strange globin moiety arrangement or diminished globin chain
generation. At the point when the organic capacity is change owning to cause a hemoglobin
transformation. This progressions are known as a haemoglobinopathies. Change in qualities
that code for alpha and beta chains can influence the natural capacity of hemoglobin. A few
human hemoglobin freak qualities which have organic changed capacities have been talked
about underneath. Globin present in hemoglobin atom is comprise of 7 distinctive
polypeptide chains they are alpha , beta , gamma , delta , epsilon and zeta they are fluctuating
in number and amino corrosive course of action amid the diverse phases of intrauterine
changes. They are Greek last assigned. Epsilon, zeta and couple of alpha chains are created
amid early embryonic life, alpha and gamma chains are delivered amid fetal life, and alpha ,
beta and delta chains are created amid postnatal life. 17,18 ( figure )
Figure 1.1: globin chain amalgamation amid improvement

In most punctual fetal life, zeta and epsilon chains are union and consolidate together and
structure hemoglobin Gower 1 which contain 2 zeta and 2 epsilon chain, hemoglobin Gower
2 which contain 2 alpha and 2 epsilon chain while hemoglobin Portland contain 2 zeta and 2
gamma chains. End of the first trimester zeta chain supplanted by alpha chain and epsilon
chain is supplanted by gamma chain and structure Hb F. Fetalhemoglobin ( Hb F )(alpha 2
and gamma 2 ) is the dominating hemoglobin present in the intrauterine life and contribute of
90% of all out hemoglobin up to the 34 to 36 weeks of incubation. whileHemoglobin An
(a2b2) contribute around 4-13% of absolute Hemoglobin in the hatchling. At end, the
Hemoglobin F levels decrease to 55-90% and the HemoglobinA dimensions essentially
increment to 20to30% of the absolute Hemoglobin. The transformation from fetal to grown-
up Hemoglobin blend proceeds and by a half year of age, the measure of Hemoglobin F is
typically <2-3% and HemoglobinAn is the critical hemoglobin is seen. A little measure of
delta globin chains are additionally produce amid birth, the measure of Hemoglobin A2
(a2d2) blend is very low but the normal adult levels of Haemoglobin A2 is 2to3.5% are
usually produce completely by 6 months of age.15,19. P50 is the halfway weight of oxygen
that half-immerses a hemoglobin. For human HbA, P50 =26 mm Hg; for HbF, P50 = 20 mm
Hg . This distinction licenses HbF to extricate oxygen from the HbA of placental blood amid
the incubation time frame. Postpartum,however, HbF is unacceptable, since its high fondness
for oxygen directs that it can convey less oxygen to the tissues.During improvement, there is
an organized exchanging of hemoglobin amalgamation, influencing both the site of
erythropoiesis just as the kinds of polypeptide chains 19.The diverse polypeptide chains show
numerous similitudes in their sub-atomic structure, and on examination amino corrosive
organization are plainly partitioned into two gatherings, the alpha and zeta chains and the
beta, gamma,delta and epsilon chains. Of these, one sets of alpha chains is of all inclusive
event. The diverse globin chains have most likely emerged by progressive quality
duplications from a hereditary alpha-like globin structure, and the nearby similitude between
the structures of the beta and the delta chains recommends that they have wandered as of late
as far as development.18. The alpha and zeta chains contain 141 amino corrosive deposits
and the beta, gamma, delta and epsilon chains contain 146 amino corrosive buildups.

Diagnosis and screening of individual are indicated below: [21]

• Premarriage screening
• Antenatal screening
• Preconception screening
• Neonatal screening
• Preoperative/preanesthesia screening.
• Premarriage screening:
This ought to in a perfect world be done to distinguish transporters of b thalassemia and the
auxiliary hemoglobinopathies like sickle cell attribute yet is regularly not adequate because of
social and social reasons. Screening should be possible in universities and colleges, schools,
or network focuses. It is especially important in high-hazard networks where the
predominance is high.
• Antenatal screening:
Pregnant ladies ought to be screened in all antenatal centers independent of the gestational
age, ladies in the second trimester ought to likewise be screened as knowing the transporter
status will help in controlling these disarranges in consequent pregnancies whenever required.
Spouses of transporter ladies with the accompanying Hb variations from the norm should be
screened:
• b thalassemia attribute
• db thalassemia attribute
• Hb S attribute
• Hb E attribute
• Hb D attribute
• HbLepore attribute
• Hb D– bthalassemia
• Hb Q India-b thalassemia.
Pre-birth determination ought to be prompted if the baby is in danger for the accompanying
conditions:
• b thalassemia major
• db thalassemia major
• b‐db thalassemia
• Hb S– b thalassemia
• Hb E– b thalassemia
• Sickle cell weakness
• Hb SD malady
• HbLepore– b thalassemia
• Hb SE malady.
Screening of antenatal ladies even later in pregnancy distinguishes couples in danger who
could get their children's blood checked during childbirth and decide on pre-birth
determination in resulting pregnancies.
• Preconception screening:
In spite of the fact that dif religion in the Indian circumstance, this ought to be done wherever
conceivable as ladies frequently don't register in antenatal facilities previously 12 weeks of
development. Indistinguishable methodology from for antenatal screening ought to be
pursued.
Previously established inclination screening is fundamental for all couples coming to
barrenness centers for helped origination. In the event that the lady is a bearer of a signi
cannot hemoglobinopathy, her accomplice or sperm contributor ought to be screened and in
the event of an ovum benefactor, the giver ought to likewise be screened.
• Neonatal screening:
Infant screening is for the most part prescribed for sickle cell issue among those innate and
nontribal populaces where the commonness of Hb S is high. Preferably, widespread screening
ought to be done where every infant in these high hazard bunches are screened as this would
permit identication of other clinically signicant issue, for example, homozygous b-
thalassemia and all instances of Hb S– b thalassemia.
At the point when assets are limited, a focused on screening approach ought to be utilized
where the moms are rst screened utilizing economical strategies like the solvency test and
children of just those moms who have a positive dissolvability test are screened. This
methodology will miss a couple of instances of sickle b-thalassemia when the mother is a b-
thalassemia bearer and the dad is a transporter of Hb S. Other clinically
signicanthemoglobinopathies will likewise not be gotten. Children ought to be screened either
during childbirth or inside the rst a month after birth. All children with a
signicanthemoglobinopathy ought to be re‐tested utilizing atomic innovation to con rm the
finding inside 3 months after birth. Infants with sickle cell infection (SCD) or sickle b-
thalassemia ought to be followed-up at regular intervals clinically and auspicious penicillin
prophylaxis and pneumococcal immunization given.
• Preoperative/ Preanesthesia screening:
This ought to be done in patients from ethnic gatherings where the pervasiveness of Hb S is
high, as the nearness of sickle Hb preoperative procedures and clinical administration.
• Genetic counselling:
Hereditary directing ought to in a perfect world be given by a restorative authority who has
been prepared in guiding families with hemoglobinopathies. Directing can likewise be given
by a prepared hereditary advocate, a hematologist, or a pediatrician.

Pathophysiology of hemoglobinopathies (20)

Messing around with the amino acid sequence of a globin chain has something of a red
kryptonite effect. While some positions on the protein chain can tolerate a lot of substitutions
without compromising the physiologic integrity of hemoglobin, other positions are very
sensitive to amino acid substitutions. For instance, substitution of valine or lysine for
glutamate at position 6 of the chain produces hemoglobins S and C, respectively, which
form intraerythrocytic tactoids (see below) and crystals (again respectively) that cause
premature destruction of the rbc (hemolysis). On the other hand, substitution of glutamate,
asparagine, and threonine for lysine at position 59 of the chain produces, respectively,
hemoglobins IHigh Wycombe, JLome, and JKaoshiung, all of which are physiologically
indistinguishable from normal Hb A. Without venturing too deeply into tedious
stereochemistry, we can say that abnormal globin structure can functionally manifest itself in
one or more of the following ways:
• Increased O2 affinity
These hemoglobins tend to result when mutations affect the portions of the amino acid
sequence that compose 1) the regions of contact between and chains, 2) the C-terminal
regions, and 3) the regions that form the pocket which binds 2,3-DPG. The hemoglobin
eagerly scarfs up the O2 from the alveoli but then only stingily gives it up to the peripheral
tissues. The kidney, always compulsively vigilant for hypoxia, cranks out the erythropoietin
thinking that a few extra red cells might help out matters. Erythropoiesis then is stimulated,
even though there is no anemia, and erythrocytosis (increased total body rbc mass, increased
blood hemoglobin concentration, increased hematocrit) is the result.
It is important to know that these rare increased O2 affinity hemoglobins exist to prevent
diagnostic errors from occurring in working up patients presenting with erythrocytosis (which
is much more commonly caused by other conditions, including polycythemia vera [a
neoplasm], cigarette smoking, psychosocial stress, chronic residence at high altitudes, and
chronic lung disease). Examples of these include Hb Chesapeake and Hb JCapetown.
• Decreased O2 affinity
This is the other side of the coin. These hemoglobins are reluctant to pick up O2 from the
lung. The result is a decreased proportion of hemoglobin that is oxygenated at a given PO2.
The remainder of the hemoglobin is, of course, deoxygenated and is blue. If the level of blue
hemoglobin exceeds 5 g/dL in capillary blood, the clinical result is cyanosis, a bluish
discoloration of skin and mucous membranes.
Again, it is important to know about these hemoglobins and keep them in the back of your
mind when working up cases of cyanosis, a condition much more commonly caused by
pulmonary dysfunction or right-to-left cardiovascular shunts. Examples of low O2 affinity
hemoglobins include Hb Seattle, Hb Vancouver, and Hb Mobile.
Migration patterns:
Movement designs (Alkaline Electrophoresis):
All in all on basic electrophoresis arranged by expanding versatility are hemoglobins A2,
E=O=C, G=D=S=Lepore, F, A, K, J, Bart's, N, I, and H. All in all a sickling test (sodium
bisulfite) is performed on irregular hemoglobins moving in the S area to check whether the
red cells accelerate in arrangement.
Movement designs (Acid Electrophoresis):
When all is said in done on corrosive electrophoresis arranged by expanding versatility are
hemoglobinsF, A=D=G=E=O=Lepore, S, and C. This is the means by which anomalous
hemoglobin variations are disengaged and recognized utilizing these two strategies. For
instance aHgb G-Philadelphia would relocate with S on antacid electrophoresis and would
move with An on corrosive electrophoresis, individually utilization of isoelectric centering to
decide quantitative contrasts in globin chain blend and superior fluid chromatography that
isolates hemoglobins dependent on their different intensities for the section
NORMAL TYPES OF HAEMOGLOBINS (20)
Typical sorts of hemoglobin:
Hemoglobin A;
Hemoglobin A contains about 97% of the hemoglobin of grown-up red cells. It comprises of
two alpha and two beta chains with the basic equation a2b3. The alpha chain contains 141
amino acids, and the beta chain 146. Limited quantity of Hb An are distinguished in the
hatchling as right on time as the eighth seven day stretch of life. During the initial two
months of post natal life, Hb A totally replaces by Hb F and the grown-up example is
completely settled by the a half year.
Hemoglobin A2:
Hemoglobin A2 is the minor hemoglobin in the grown-up red cell. It has the basic recipe
alpha2delta2 the delta chain containing 146 amino acids. The alpha chain is indistinguishable
from that of the Hb A. Hb A2 is available in modest quantity during childbirth and achieves
the grown-up dimension of 1.5 to 3.2 % during the primary year of life. Rise of Hb A2 is an
element of some kind of thalassemia and incidentally happens in megaloblastic frailty and
flimsy hemoglobin ailment. Hb A2 might be decreased in iron lack.
Hemoglobin F:
Hemoglobin F is the major respiratory shade from early intrauterine life upto term. It has the
basic recipe a2y2, every gamma chain comprising of 146 amino acids. The y chain are
assigned Gy or Ay relying upon whether they have glycine or alanine at position 136. At
term, Hb F represents 70-90% of the all out hemoglobin. It at that point falls quickly to 25 %
at multi month, and 5 % at a half year. The grown-up dimension of around one rate is note
come to in certain kids until pubescence. Hb F is raised in certain haemoglobinopathies and
thalassemia disorders. It might be raised in incidental instances of intrinsic and obtained
aplastic paleness, megaloblastic frailty, paroxysmal nighttime haemoglobinuria , sideroblastic
iron deficiency, and in certain types of leukemia. It is once in a while brought up in early
pregnancy. The corrosive elution test shows that Hb F is unevenly conveyed in the red cells
in these conditions. Hb F is estimated by the salt denaturation system.
Embryonic hemoglobin;
Hb Gower 1 and Hb Gower 2 are limited to the embryonic phase of advancement. They
contain epsilon and zeta chains, Hb Gower 1 being epsilon2zeta2 and Hb Gower 2,
alpha2epsilon2. Hb Portland is found in follow sums all through intra uterine life and in
neonates. It has the auxiliary equation zeta2gamma2. Hb Bart's is likewise found in limited
quantities in rope blood if delicate systems are utilized. Both Hb Portland and Hb Bart's are
expanded in the line blood of neonates with alpha-thalassemia.

CLASSIFICATION OF HAEMOGLOBINOPATHIES: (ABNORMAL HAEMOGGLOBIN

TYPES)
A) HEAMOGLOBIN C:
The quality for Hb C is additionally common in the African-American populace yet
with less recurrence (2-3%) than that of the sickle cell quality. The outcome is
diminished RBC survival time; not withstanding, hemolysis isn't as extreme as in
sickle cell sickness, and the vaso-occlusive wonders, so decimating in sickle cell
ailment, are not commonly noted. Like sickle cell characteristic, the Hb C attribute is
asymptomatic. Homozygotes (and a few heterozygotes) for Hb C frequently have
many target cells in the fringe smear, however the precious stones, despite the fact
that pathognomonic, are just once in a while observed. The forecast of homozygous
Hb C ailment is incredible.

An individual may acquire a Hb S quality from one parent and a Hb C quality from
the other. The consequence of this one-two punch is Hb SC ailment. The clinical
seriousness of this condition is transitional between that of sickle cell sickness and Hb
C malady, then again, actually visual harm because of retinal vascular sores is
typically more regrettable in SC infection than in sickle cell iron deficiency. The
intracellular bodies that happen upon hemoglobin destabilization in SC malady are
interested crossovers of the unpolished finished crystalloids of Hb C and the sharp-
pointed tactoids of Hb S, in that they frequently have one pointed end and one obtuse
end, subsequently ambiguously looking like sharpened stones. 22

Clinical significance:
This changed structure diminishes the typical pliancy of host erythrocytes causing a
hemoglobinopathy. In the individuals who are heterozygous for the transformation,
around 28–44% of all out hemoglobin (Hb) is HbC, and no iron deficiency creates.
In homozygotes, about all Hb is in the HbC structure, bringing about gentle hemolytic
paleness

With different conditions

People with sickle cell–hemoglobin C (HbSC), have the quality for HbS acquired
from one parent and the quality for HbC is acquired from the other parent: they are
"heterozygous". Since HbC does not polymerize as promptly as HbS, there is less
sickling (less sickle cells). The fringe smear exhibits generally target cells and just a
couple of sickle cells. There are less intense vaso-occlusive occasions.
Notwithstanding, people with hemoglobin SC ailment (HbSC) have increasingly huge
retinopathy, ischemic rot of bone, and priapism than those with unadulterated SS
disease.[23] People with hemoglobin C characteristic have red platelets that have
typical hemoglobin An and an anomalous hemoglobin. The strange hemoglobin is
called hemoglobin C. Individuals with hemoglobin C quality have marginally more
hemoglobin A than hemoglobin C. Individuals with Hemoglobin C characteristic
don't have medical issues identified with having the quality. Individuals with
hemoglobin C don't have Hemoglobin C sickness or sickle cell malady. They can't
build up these ailments sometime down the road. They can pass hemoglobin C
attribute to their offspring.[24].

Heredity of Hemoglobin C Disease

Hemoglobin C illness is an autosomal latent confusion that outcomes from the

biparental legacy of the allele that encodes for hemoglobin C.[26] If the two guardians
are transporters of hemoglobin C, there is an opportunity of having a tyke with
hemoglobin C infection. These are the potential results with every pregnancy. 25
percent (1 of every 4) shot of having a kid with hemoglobin C illness 50 percent (1
out of 2) possibility of having a youngster who is a bearer of hemoglobin C 25 percent
(1 of every 4) possibility of having a tyke without infection and isn't a carrier.[24]

Clinical tests

Physical Examination may demonstrate a developed spleen. Tests that might be done
include: Complete Blood Count (CBC), Hemoglobin electrophoresis, Peripheral blood
smear, and Blood hemoglobin.[25]

Treatment
 Generally no treatment is required. Folic corrosive supplementation may help produce
typical red platelets and improve the manifestations of paleness.

B) HAEMOGLOBIN E:

This is a typical chain transformation among Southeast Asians. The Thai and Khmer
bunches have the most noteworthy recurrence, trailed by Burmese and Malays, at that
point Vietnamese and Bengalis. The quality does notoccur in ethnic Han Chinese or
Japanese. The heterozygous state is asymptomatic yet aims microcytosis without
sickliness, accordingly taking after certain instances of thalassemia minor (see
beneath). The homozygous state has progressively extreme microcytosis and
hypochromia, yet little, assuming any, sickliness (this is additionally reminiscent of
thalassemia minor). Hemoglobin E ought to dependably be viewed as stirring up an
unexplained microcytosis in an individual from one of the influenced ethnic
gatherings.

Hemoglobin E or hemoglobin E (HbE) is irregular hemoglobin with a solitary point

change in the β chain. At position 26 there is an adjustment in the amino corrosive,
from glutamic corrosive to lysine. Hemoglobin E has been one of the less notable
variations of typical hemoglobin. It is exceptionally regular in Southeast Asia yet has
a low recurrence among different races. HbE can be recognized on electrophoresis.

The βE change influences β-quality articulation making an other grafting site in the
mRNA at codons 25-27 of the β-globin quality. Through this instrument, there is a
mellow lack in ordinary β mRNA and creation of little measures of odd β mRNA. The
diminished amalgamation of β chain may cause β-thalassemia. Likewise, this
hemoglobin variation has a powerless relationship among α-and β-globin, causing
insecurity when there is a high measure of oxidant.[27]

Heredity of hemoglobin E/β-thalassemia

Hemoglobin E/β-thalassaemia People who have hemoglobin E/β-thalassemia have

acquired one quality for hemoglobin E from one parent and one quality for β-
thalassemia from the other parent. Hemoglobin E/β-thalassemia is a serious sickness,
regardless it has no general fix. It influences in excess of a million people in the
world.[28] The outcomes of hemoglobin E/β-thalassemia when it isn't dealt with can
be heart disappointment, broadening of the liver, issues during the bones, and so on.

There is an assortment of genotypes relying upon the connection of HbE and α-

thalassemia. The nearness of the α-thalassemia decreases the measure of HbE
normally found in HbE heterozygotes. In different cases, in mix with certain
thalassemia changes, it gives an expanded protection from intestinal sickness (P.
falciparum).[29]
C) HAEMOGLOBIN D(30)

Hereditary qualities and Heredity

Hb D is an acquired autosomal latent variety of Hb A that happens in the beta (β)-

globin protein chain of Hb A. The arrangement of Hb D happens by substitution of
glutamic corrosive for glutamin at codon 121 of the β-chain. Hemoglobin D illness
(Hb DD) happens when a baby acquires two duplicates of the Hb D variation quality,
one from each parent. In the event that the two guardians have the Hb D quality, there
is a 25 percent shot with every pregnancy that the kid will be brought into the world
with Hb DD. Infection with no Hb A may have either homozygous Hb DD or
heterozygous Hb D/beta-thalassemia (Hb D/β-thal). Either analysis is related with
couple of clinical signs. The best strategy to recognize the outcomes is to test the two
guardians. There are a few Hb D variations. Hb D-Punjab, otherwise called Hb D-Los
Angelese after the city where it was first found, substitutes glycine for the glutamic
corrosive at codon 121 of the β-chain. Another variation, Hb Ibadan, substitutes lysine
for the typical threonine at condon 7.

Commonness

Hemoglobin D is the fourth most regular hemoglobin variation, which created as a

reaction to the specific weight of jungle fever. It is regularly found in individuals
living in India, Pakistan, England, Ireland, Holland, Australia, China, Iran, Turkey
and their relatives. Homozygous Hb DD is uncommon and a generally mellow
malady. Heterozygous Hb D/β-thal is increasingly normal and progressively genuine.

Pathophysiology

The accompanying signs of hemoglobin D ailment might be watched:

▪ Target cells

▪ Occasional spherocytes

▪ Microcytosis

▪ Hypochromia

▪ Mild hemolytic frailty

▪ Decreased osmotic delicacy

▪ Reduced oxygen proclivity

Homozygous Hemoglobin D:

Hemoglobin D ailment is generally clinically quiet with no exceptional treatment

required. A mellow haemolytic sickliness for the most part creates in the initial couple
of long periods of life as the measure of fetal hemoglobin diminishes. People with
homozygous Hb D malady might be asymptomatic or the next may happen:

▪ Mild hemolytic paleness

▪ Mild to direct splenomegaly

Heterozygous Hemoglobin D/Beta (β)- thalassemia:

Newborn children with heterozygous Hb D/β-thalassemia might be asymptomatic and

have gentle to direct hemolytic pallor relying on the level of β-thalassemia influencing
the A quality. It more often than not creates in the initial couple of long stretches of
life as the measure of Hb F diminishes and Hb D increments. Those with Hb D/β+-
thalassemia have some Hb An and are bound to have mellow to direct iron deficiency
and a non-obvious spleen. Youngsters with hemoglobin D/βº-thalassemia disorder
have no Hb A, showing symptomatic pallor with spleenomegaly and may have a
respectably serious clinical confusion. Since RBC records are strange in Hb D/β-
thalassemia, iron insufficiency may create.

Basic Steps

1. Illuminate the family regarding affirmed Hb D ailment or Hb D/β-thalassemia;

clarify the potential inconveniences and intercessions.

2. On the off chance that hemoglobin D malady is available, guarantee that the
newborn child does not likewise have beta-thalassemia. A CBC with smear at 6 to 9
months of age will distinguish any of the β-thal segments. In the event that therapeutic
concerns emerge or the newborn child is symptomatic, finished the CBC with smear
prior.

3. In the event that iron insufficiency is suspected serum iron dimensions, iron
restricting limit, and percent immersion may should be evaluated.

4. Counsel with a pediatric hematologist in regards to quiet assessment and

conceivable illness the board.

5. Consider family referral to a hereditary guide.

 Hemoglobin D-Punjab is one of the sub-variations of Hemoglobin D, a variation of
hemoglobin found in human blood. It is so named on account of its higher
predominance in the Punjab district of India and Pakistan. It is likewise the most
incessant irregular hemoglobin variation in Xinjiang Uyghur Autonomous Region of
China. Studies demonstrate that Hemoglobin D-Punjab represents over 55% of the all
out hemoglobin variations there.

Hemoglobin D-Punjab was first found in the mid 1950s in a blended British and
American group of Indian beginning from the Los Angeles region; thus it is
additionally at times called "D Los Angeles". Hemoglobin D is the fourth most basic
hemoglobin variation. It created as a reaction to the particular weights of jungle fever
in these locales of Asia.

Hemoglobin D Trait

Hemoglobin is the primary fixing in red platelets. Individuals with hemoglobin D

characteristic have somewhat more hemoglobin A than hemoglobin D. Individuals
with Hemoglobin D quality don't have medical issues identified with having the
attribute. Individuals with hemoglobin D quality don't have Hemoglobin D ailment or
sickle cell infection. They can't build up these sicknesses sometime down the road.
Hemoglobin enables red platelets to convey oxygen from the lungs to different pieces
of the body. Ordinary red platelets have hemoglobin A. Individuals with hemoglobin
D attribute have red platelets that have ordinary hemoglobin An and an unusual
hemoglobin. The unusual hemoglobin is called hemoglobin D. Among the seven
known kinds of Hemoglobin D, just Hemoglobin D Punjab can cause a genuine
hemoglobin issue. While Hemoglobin D can be recognized without a DNA test, one is
expected to learn that an individual who conveys Hemoglobin D conveys hemoglobin
D-Punjab. At the point when an individual has both Hemoglobin An and Hemoglobin
D, one is said to have the Hemoglobin D Trait. Posterity of such people have
expanded danger of having Hemoglobin D Disease, Hemoglobin SD infection or
Beta-thalassemia Disease.

• Symptoms of Hemoglobin D Disease

Hemoglobin D Disease can make gentle haemolytic pallor and mellow moderate
splenomegaly. The pallor generally happens in the initial couple of long stretches of
life, as fetal hemoglobin diminishes and hemoglobin D increments.

D) HAEMOGLOBIN Q:

The primary Hb Q variations to be described were Hb Q-Thailand (α74 Asp → His)

and Hb Q-Iran (α75 Asp → His).37 However, a couple of instances of an alternate Hb
Q variation have been accounted for from India.38 This specific Hb Q variation, Hb
Q-India, is likewise a α chain variation: α64 Asp → His. DNA sequencing thinks
 about have demonstrated the Hb Q India transformation to be AAG → GAG in codon
64 of the α1 gene.39. This variation hemoglobin happens regularly in the
heterozygous structure and isn't related with a thalassaemia phenotype.

Hemoglobin has a lot of variants.31 Hemoglobin (HbQ) is a clinically quiet

hemoglobinopathy that outcomes from adjustment of alpha 1 globin quality
(AAG>GAG) substituting arginine for histidine. HbQ was first depicted by vella et al,
in relationship with alpha thalassaemia in a Chinese patient.32 Three varieties have
been portrayed specifically India, Thailand and Iran dependent on the included codon
(64,74 and 75 respectively).33 This rare issue has been distinguished in the
homozygous and heterozygous states in relationship with alpha and beta thalassaemia.

The principal instance of HbQ-India was accounted for by Sukumaran in 1972 out of
a Sindhi family with related beta thalassaemia and later by Desai.34,35 HbQ-India is
typically found in the heterozygous structure which is clinically quiet not having
harmful phenotypic effect.34 The major clinical criticalness of HbQ is its right
analysis. The distinguishing proof of hemoglobin variations is done by traditional
systems are frequently hypothetical, in view of ethnic starting point of the guardians
and the measurement of electrophoretic versatility of the band.36

On basic electrophoresis, HbQ has a similar versatility as hemoglobin S/D, Hence Hb

F and A2 with alpha chain lead to appearance of adornment groups relating to
anomalous hemoglobin along these lines making the determination of HbQ rather
chromosome 35. Recently numerous labs have received superior fluid
chromatography (HPLC) as their essential method of screening and the purpose
behind this decision might be the situation, cost viability or right analysis for different
hemoglobinopathies.

E) HAEMOGLOBIN S: SICKLE CELL DISEASE

Figure: pathology of sickle cell formation.
SICKLE CELL DISEASE:
Much progress has been made during the last many years in management of Sickle Cell
Disease. Identification of affected infants provides opportunities for educational and
interventions that significantly reduce morbidity and mortality during childhood and
adolescence.
Comprehensive health maintenance with parental education and appropriate prophylactic
measures and monitoring for the development of chronic organ damage are important.
Acute illness in patients with SCD can prove rapidly life threatening; to prevent that,
appropriate care must be provided for the management of complications in a setting where
knowledge and perspective about SCD is available and where physicians have ready access
to baseline information about the patient including results of previous physical examinations,
laboratory reports, and radiographs. It is essential that patients have unimpeded access to
providers who have the expertise necessary to quickly recognize and treat potentially
catastrophic signs and symptoms. Such care not only reduces morbidity and mortality, but it
may also reduce medical costs by preventing some manifestations of the disease and by
limiting the severity or sequel of others. Many acute complications can be managed safely on
an outpatient basis, thus reducing the need for hospitalization.
These guidelines provide information about the diagnosis of SCD, an overview of
comprehensive care, and clinical care and protocols for the management
of some of the more common acute and chronic(40)
Management for sickle cell disease(40)
Acute illness in sickle cell disease : Acute illness requiring immediate medical care, including
emergences that need to be defined. It would include any of the following:
• Temperature >38.5°C
• Pain inadequately relieved by home measures
• Significant respiratory symptoms (e.g. severe cough,
shortness of breath, chest pain)
• Abdominal pain, distention, acute enlargement of
the spleen.
• Any neurologic signs or symptoms even if they are transient

• Significant increase in pallor, fatigue, or lethargy

• Significant vomiting or diarrhea.
Acute illness characterized by any one of the signs or symptoms listed above can prove
rapidly life threatening.
Thus, it is essential that SCD patients have unimpeded access to the health care providers at
the community centers where appropriate measures can be taken. Immediate
contact/consultation with a pediatric hematologist or a primary care physician with expertise
in SCD is necessary. The patient’s baseline data should be available.
Outpatient evaluation and management of febrile illness One should immediately look for the
presentation (e.g. toxic appearance). This should be followed by a physical examination,
taking a brief history and systemic examination, laboratory investigations (e.g. CBC, DLC,
PLT count and reticulocyte count) should then be done.
Sickle-cell crisis
The articulations "sickle-cell crisis" or "sickling crisis" may be used to portray a couple of
free serious conditions occurring in patients with SCD. SCD results in sickliness and crises
that could be of various sorts including the vaso-occlusive crisis, aplastic crisis, sequestration
crisis, haemolytic crisis, and others. Most scenes of sickle-cell crises last some place in the
scope of five and seven days.[41] "despite the way that tainting, drying out, and acidosis (all
of which backing sickling) can go about as triggers, in numerous events, no slanting reason is
identified."[42]
Vaso-occlusive crisis
The vaso-occlusive crisis is realized by sickle-framed RBC that hinder vessels and limit
circulation system to an organ achieving ischaemia, anguish, festering, and consistently organ
damage. The repeat, reality, and length of these crises vary stunningly. Troublesome crises
are treated with hydration, analgesics, and blood transfusion. For continuously extraordinary
crises, most patients require inpatient the administrators for intravenous opiates; tolerant
controlled absense of torment contraptions are conventionally used in this setting. Vaso-
occlusive crisis including organs, for instance, the penis[43] or lungs are seen as an
emergency and treated with RBC transfusions

Splenic sequestration crisis

Because of its tight vessels and limit in clearing imperfect red platelets, the spleen is a great
part of the time affected.[44] It is commonly infarcted before the completion of youth in
individuals encountering sickle-cell delicacy. This spleen mischief manufactures the risk of
illness from encapsulated organisms;[45][46]
Aplastic crisis
Aplastic crises are extreme worsenings of the patient's check shortcoming, conveying pale
appearance, fast heartbeat, and weariness. This crisis is consistently actuated by parvovirus
B19, which honestly impacts age of RBC by assaulting the red cell harbingers and expanding
them.[47]
Haemolytic crisis
Haemolytic crises are serious animated drops in hemoglobin level. The red platelets separate
at a faster rate. This is particularly ordinary in patients with simultaneous G6PD deficiency.
[48]
Sickle cell trait:(49)
This is the benign carrier state and wide majority of peoples have no clinical symptoms are
seen. The occurrence of the trait in U.S. black peoples are about 10%. In tribal areas of Indian
populations its occurrence is approaches to 30%. Sickle cell trait can cause haematuria and a
loss of urine concentrating capacity. Symptoms from intravascular sickling have been
reported with strenuous exercise at high altitudes an flying in un- pressurized aircraft.
Diagnosis:
1. The patient’s clinical history and physical findings.
 2. The presence of hb s shown by the inexpensive and highly sensitive solubility test
even in the carrier state. Haemoglobin electrophoresis confirms the exact phenotype.
3. The presence of hemolytic anemia and morphologic sickling on blood smears.

Other Sickle Cell Disorders

The clinical picture of sickle cell anemia is also seen in individualswith genetic mutations
other than HbSS. The two most common other situations are co-inheritance of HbS
and HbC (also known as SC disease), and the co-inheritance of HbS with beta-thalassemia
(i.e., S/beta-thalassemia).17 Sickling syndromes also occur in individuals who co-inherit HbS
with other hemoglobinopathies, such as HbD. These individuals usually
are less severely affected than individuals with HbSS. SC can generally be diagnosed with
Hb electrophoresis or HPLC, as can other double heterozygote conditions like SD.
The clinical severity of S/beta-thalassemia depends on the type of beta-thalassemia mutation.
50

F) THALASSEMIA:
A hereditary deformity that outcomes underway of a strangely low amount of a given
hemoglobin chain or chains. The imperfection may influence the , or chain, or may
influence a blend of the , and chain in a similar patient (yet never the and chain
together). The outcome is an irregularity underway of globin chains and the
generation of a deficient number of red cells. The phones which are delivered are
hypochromic/microcytic and contain a surfeit of the unaffected chains which can't
stoichiometrically "mate" with the deficient supply of thalassemic chains. These
"single man" chains can create unfavorable impacts on the red cell and lead to
annihilation of the red cell in the marrow (incapable erythropoiesis) and in the flow
(hemolysis). Note that these two definitions are not fundamentally unrelated - a few
hemoglobinopathies may likewise be thalassemias, in that a basically irregular
hemoglobin (hemoglobinopathy) may likewise be underproduced (thalassemia). A
few, yet not all, hemoglobinopathies and thalassemias are hemolytic anemias. These
nosologic ideas are abridged by the Venn outline beneath.

A. Hereditary qualities

Understanding the thalassemias can be encouraged by assessing the beginning of the

typical post-embryonal hemoglobins:

Chromosome 16 contains the qualities for the extremely significant chain. The
qualities for the majority of the other significant globin chains are on chromosome 11,
where they are firmly connected. The linkage implies (on the off chance that you will
quickly mishandle yourself by reviewing essential hereditary qualities) that the
qualities will in general be acquired as a gathering, rather than non-connected (or
remotely connected) qualities which arrange autonomously because of traverse amid
gametogenesis. In light of the linkage, a change that influences the rate of generation
of the chain not exceptionally influences rate of creation of the nearby chain. An
 individual conveying such a change would then have a quality for " thalassemia." He
or she could pass on the thalassemia quality to posterity yet would basically never,
say, pass a thalassemia quality to one tyke and a thalassemia quality to another. On
the other hand, since the genome for the chain is on a totally unexpected chromosome
in comparison to the qualities for the various chains, one would expect no change in a
chromosome 11 chain quality ( , ) to influence chain generation. In addition, if some
poor shlimazel happened to acquire a thalassemia quality from one parent and a
thalassemia quality from another, he would not will in general pass both unusual
qualities on as a unit to his or her posterity. One child (out of a delegate Mendelian
sibship of four) would get the unusual quality, one would get the irregular , one would
get not one or the other, and one would get both.

B. Natural chemistry and pathophysiology

In any case, enough of Mendel! We're in drug school to find out about hemoglobin,
isn't that so? Whatever the hereditary qualities, the clinical issue in the thalassemias is
the failure to keep up a harmony between the blend rate of one kind of globin chain
versus that of its mate. Despite the fact that thalassemias have been depicted for each
of the four of the above chains, we will consider just those that include the chain (the
thalassemias and thalassemias) and the chain ( thalassemias). It will be valuable to
audit what sort of hemoglobins you can work by blending and coordinating globin
chains:

Hemo- Globin Notes

globin chain
compos
ition
A 2 2 The only physiologically important adult hemoglobin in normal
individuals. Includes the post-translational glycosylated hemoglobins
A1a, A1b, and A1c, the last being important in monitoring diabetics.
F 2 2 The major physiologic hemoglobin in post embryonal fetuses. Adapted
best for lowered intrauterine O2 tension because of its left-shifted Hb-
O2 dissociation curve (allowing O2 to be more readily picked up from
maternal circulation). Production normally turns off in early infancy.
Proportion of circulating Hb F fades to insignificance at about 6 months
of age.
A2 2 2 Medical philosopher's proof of the existence of God (and God's love of
physicians). Apparently put here solely as a marker for doctors trying to
figure out whether a patient has iron deficiency anemia or   thalassemia.
Normally less than 3% of circulating hemoglobin (thus physiologically
insignificant), Hb A2is slightly elevated in most   thalassemias, but
normal or decreased in iron deficiency, thus making it a nifty marker for
evaluating microcytic, hypochromic anemias.
Gower 2 2  Very early normal embryonal hemoglobins that disappear after 8 weeks
1 2 2 of gestation. The only one of clinical importance is Hb Portland, which
Gower 2 2 may be seen at birth in cases of the severest form of   thalassemia.
2
Portlan
d
H 4 Abnormal hemoglobin produced in cases of   thalassemia, when excess   
chains decide to get it on with each other, there not being enough   chains
to go around. Intrinsically unstable, Hb H produces Heinz bodies in the
erythrocytes and subsequent hemolysis.
Bart's 4 Delinquent youth gang analogue of Hb H. This abnormal hemoglobin is
found in infants with   thalassemia. Detecting presence of Hb Bart's in
cord blood may be the only practical way to screen for the very large
number of individuals who are silent carriers of one type of   thalassemia
(see below).

C Beta thalassemia

In spite of the fact that this is the great type of thalassemia it isn't the most widely
recognized. The main portrayal was composed by Dr. Thomas Cooley in 1925. The
expression "Cooley's pallor" has been utilized synonymously with clinically extreme
types of thalassemia, in spite of the fact that the rest of Cooley's vocation was so
undistinguished as to make some recommend that his name isn't deserving of
eponymous everlasting status. Cooley's iron deficiency was a lethal microcytic pallor
of offspring of Mediterranean plummet. The name "thalassemia" was instituted to
mirror the first geographic home of the objective populace ( "thalassa" is the
traditional Greek name for the Mediterranean Sea). Throughout the years, it turned
out to be certain that numerous different gatherings (Africans, African-Americans,
Arabs, Indians, and Southeast Asians) are influenced. Truth be told, thalassemias
when all is said in done will in general influence races of individuals that hail from a
tropical belt that supports the Mediterranean and broadens entirely through the Indian
subcontinent to Southeast Asia.

There are an assortment of various thalassemia qualities that offer ascent to a

clinically heterogeneous range extending from asymptomatic articulation to
traditional, dangerous Cooley's weakness. It is helpful to bunch the different
thalassemias into two gatherings, in view of the measure of globin chain generation:

• beta 0 thalassemia

This anomalous quality permits no creation of chains. People homozygous for this
quality produce just Hb A2, Hb F (and almost no of that following a half year of age),
and insecure 4 tetramers that garbage the red cells while they are still in the marrow.
As you may envision, these individuals are in really critical straits except if some
gatekeeper blessed messenger has given them another, autonomous quality for genetic
constancy of fetal hemoglobin (HPFH). This counteracts the Hb F nozzle from going
down to a stream at a half year. Such people can live to ready seniority and still be
youthful on the most fundamental level.

• beta + thalassemia

This anomalous quality permits a few, yet at the same time subnormal, creation of
chains. Individuals homozygous for this quality will make a subnormal measure of Hb
A however will even now experience difficulty with the ruinous impacts of 4
tetramers on the erythrocytes and erythrocyte antecedents in the marrow. The +
qualities can be additionally subdivided into the great + (serious) structure, found in
Mediterranean Caucasians, and the gentle + (Negro) structure found in blacks. These
days this quality has its most noteworthy populace focus in Liberia.

In spite of the fact that these qualities are surprisingly differed in their impact on
chain combination rate, one can make up some helpful dependable guidelines:

1. Individuals heterozygous for any of the thalassemia qualities are either quiet
transporters or have insignificant clinical impacts, normally showed as a marginal
pallor (Hct ~ 35 cL/L) with lopsided microcytosis (MCV ~ 60 fL) and a
proportionally high rbc tally (~ 6 x 106/µL). The Hb A2 is expanded. This clinical
introduction is called thalassemia minor. It makes for fascinating wine sampling party
discussion in the event that you have this condition, and all that your companions can
gather is incessant weariness disorder. Your children ought to have no issues on the
off chance that you simply wed a Teuton, Slav, Balt, or Lapp.

2. Individuals homozygous for the majority of the thalassemia qualities [except

the + (Negro) gene] have extreme iron deficiency and a few or the majority of the
pathophysiological outcomes given in the graph underneath. This is great Cooley's
pallor and is named thalassemia major. This is awful news.

3. Individuals homozygous for the + (Negro) quality and a few different random
kinds of gently acting qualities have a moderately mellow clinical iron deficiency
called thalassemia intermedia. These patients may require transfusion, yet just
sometime down the road than is the situation in the extremely wiped out kids with
thalassemia major.

The pathophysiology of thalassemia major is best comprehended by proceeding to get

yourself a lager (or politically right refreshment), viewing a little TV, completing a
couple of different tasks to defer the unavoidable, and afterward plunking down to
think about the following chart.
 While concentrating the outline, consider the accompanying perceptions concerning
thalassemia:

1. Since there is an abatement in the blend of chains, there is a net diminished

union of Hb A. With less Hb An accessible to fill the red cells, the outcome is
microcytic pallor. Though in iron insufficiency microcytosis happens in light of the
fact that there isn't sufficient heme, in thalassemia something very similar happens on
the grounds that there isn't sufficient globin.

2. Since the body can't make enough chains, it makes a weak endeavor to
remunerate by attempting to produce chains. The outcome is expanded Hb A2, which
can be estimated effectively and reasonably by section chromatography. This is a
quite explicit test for the analysis of thalassemia. Entanglement: both and chains are
diminished in thalassemia, which isn't uncommon and presents like thalassemia, then
again, actually the Hb A2 isn't raised. You would expect this since Hb A2 contains
chains).

3. In a few instances of thalassemia, there is endeavor at remuneration by

keeping up some creation of Hb F. This has some pathophysiologic results (as
appeared) and furthermore gives a research facility marker to aid determination.
Maintenance of Hb F generation isn't as regular as expanded rbc Hb A2 content.

4. In extreme types of thalassemia, the pallor because of inability to make

satisfactory measures of Hb An is aggravated by the hemolysis, insufficient
erythropoiesis, and extramedullary hematopoiesis expedited by precipitation of 4
tetramers (which are precarious). In exemplary Cooley's iron deficiency, the
insufficient erythropoiesis rules the clinical picture by creating huge development of
the marrow space, showed by the alleged "tower skull" with a x-beam appearing
vertical hard striae between the internal and external tables of the calvarium. This
radiographic component is whimsically called the "hair-on-end appearance" by
radiologists, and the "fellow who-incidentally sat-on-a-Van-de-Graaff-generator
appearance" by those wacky electrical architects. Extramedullary hematopoiesis and
hemolysis causes splenomegaly, which produces hypersplenism, and more hemolysis.

5. The high turnover state brought about by the huge erythroproliferative

movement causes wastage of folate and may deliver a confusing megaloblastic iron
deficiency. Another impact of the high turnover state is hyperuricemia (because of
catabolism of the purine substance of cell DNA).

1. Classically in thalassemia major, the treatment is the reason for death. The kids are
kept up by transfusions until about age ten years, at which time they begin to indicate side
effects of abundance iron stacking. This happens on the grounds that the transfusion sidesteps
the body's ordinary gastrointestinal instrument of iron admission and discharge. The iron is
filled the circulation system straightforwardly; the body can't discharge it quick enough. First
iron (as hemosiderin) fills the cytoplasm of the RES phagocytes and after that begins to be
stored in the parenchymal cells of pretty much every organ of the body. The pancreas, liver,
myocardium, adrenals, and gonads are among the organs most delicate to press lethality. The
clincial result is diabetes mellitus, hepatic cirrhosis, congestive heart disappointment, adrenal
deficiency, and inability to experience pubescence. Demise used to happen in the second or
third decade of life, the most widely recognized prompt reason being intricacies of heart
disappointment. These days, thal significant patients live longer due to propels in chelation
treatment. To accomplish such life span, they should submit to every day subcutaneous
infusions of the parenteral chelating specialist, deferoxamine. These infusions are given by
siphon, normally medium-term, and last 9 to 12 hours each .An oral chelating drug in directly
a work in progress in Europe.

Alpha-thalassaemia disorders

The inadequacy of alpha chains prompts an overabundance of gamma chains in the

baby and that of beta chains in the grown-up. In the embryo, gamma chains structure
the tetramer Hb-Bart's (g4), and in the grown-up, the shaky beta chains hasten and
structure Hb-H (b4). The nearness of Hb-Bart's and Hb-H ailments in the red cells has
genuine results as the two haemoglobins have a high oxygen partiality and, in this
manner are inadmissible to convey sufficient oxygen to the tissues 51.

Ordinary subjects have two connected alpha quality loci on theshort arm of
chromosome 16, in this manner giving an alpha quality haplotype of aa and genotype
of aa/aa. The alpha thalassaemias are most basic because of cancellations of at least
one of these qualities. In most influenced populations,two unusual alpha quality
haplotypes are found, to be specific a+(– a/) which emerges from cancellation of one
alpha quality andao(– –/) from erasure of both alpha qualities. The alpha
thalassaemias on the planet have been assessed by Higgs and Weatherall 52.

Work on sub-atomic hereditary qualities has given clear proof that the alpha-
thalassaemia appearing total nonattendance of alpha chain creation results in extreme
formof alpha thalassaemia, called ao thalassaemia, though the mellow type of alpha
thalassaemia having just a halfway deficiency of alpha chain generation is called a+
thalassaemia. Both ao and a+ thalassaemias can result from a few distinctive sub-
atomic imperfections including the alpha globin quality cluster 53. The ao
thalassaemias result from a progression of quality erasures which include both alpha
globin qualities. The a+ thalassaemias result from cancellations of one of the
connected sets of alpha globin sgenes or from a progression of non-erasure absconds
in which the alpha globin qualities are available, however their yield is decreased.
There are basic auxiliary hemoglobin variations which are combined at a decreased
rate and produce the clinical phenotype of a+ thalassaemia.

Alpha-thalassaemia 2(a+ thalassaemia):

This speaks to the heterozygous state for a+ thalassaemia (aa/ – a) (Table 1). Amid
neonatal period, influenced newborn children may have 1–2% Hb-Bart's which they
step by step lose over the following months. In grown-up life, the hemoglobin design
is typical, and Hb-H incorporations are not found at any stage. Haemoglobinlevel and
blood film are typical, in spite of the fact that the mean cell volume (MCV) and mean
cell hemoglobin (MCH) might be somewhat diminished.

Alpha-thalassaemia 1(ao thalassaemia):

This speaks to the heterozygous state for ao (– –/aa) or the homozygous state for a+
thalassaemia (– a/ – a) . Amid neonatal period, 5–6% Hb-Bart's is found, yet the
hemoglobin design is ordinary in later life. Hb-H incorporations are generally present
in extremely little numbers. The hemoglobin level is ordinary or somewhat
diminished, however the red cells are typically gently hypochromic and microcytic,
and the MCV and MCH are reduced 51. HbA2 is diminished now and again.

Hemoglobin-H sickness:

Collaboration of the a+ (– a/) and the ao (– –/) determinants offers ascend to this type
of alphathalassaemia (– a/ – ) Clinically, Hb-H illness is described by a moderate
frailty with a hemoglobin dimension of 8–9 g/dl, mellow jaundice and physical
discoveries like that of beta-thalassaemia major 51. The reticulocytosis goes
somewhere in the range of 5 and 10%. After hatching of red cells with splendid cresyl
blue, many worn out consideration bodies structure because of the redox activity of
the color, causing precipitation of HbH. The hemoglobin design consistsof Hbs A, H
and A2 with variable measures of Hb Bart's,the A2 being decreased to 1.5–2%. It is
entirely flimsy and prone to be hastened at room temperature. On hemoglobin
electrophoresis, Hb-H and Bart's relocate morerapidly than HbA at an antacid pH.
The seriousness ofanaemia vacillates amid pregnancy or intercurrent infection,etc.
Splenomegaly is available in 85% of patients andcholelithiasis is likewise normal.

Blood film indicates stamped red cell morphologicalchanges, including serious

hypochromia, microcytosis andtarget cell development. Red cells are nucleated. There
is a gentle reticulocytosis. Various Hb-H considerations with brilliantcresyl blue stain
and huge Heinz body-like inclusionsare additionally present after the splenectomy.

The hemoglobin design comprises of 2–40% Hb-H, theremainder being HbA, HbA2
(which is diminished) and HbF.HbH ailment has a little measure of an alpha-chain
variant,Hb Constant Spring 51. Neonates with Hb-H diseasehave 25% Hb-Bart's.
Hemoglobin Constant Spring Occurs atextremely low dimensions in heterozygous
carriers,usually under 1% of the absolute hemoglobin. It migratesmore gradually than
HbA2 on antacid hemoglobin electrophoresisand will in general separation into 2–3
separate bands.Family thinks about demonstrate the ao thalassaemia quality in one
parentand the Hb Constant Spring characteristic in the other. Individualshomozygous
for Hb Constant Spring have slightlyhypochromic red cells with typical MCV.

The most punctual instance of Hb-H was recorded in a Bengaleesubject from Calcutta
54. Accordingly, five more suchinstances have been found in Bengalees (4%). The
proportionof Hb-H in these fluctuated from 12 to 25%. In noneof the guardians of
these file patients was the anomalydetected, while Hb-H thalassaemia was found in
the siblings 55.Hb-H was accounted for by Swarup et al. 56 in a 19-year-old
Bengalee Hindu from India. Comparative cases (1%)were announced from
Mumbai55,56. Saha and Banerjee 57reported two instances of Hb-H attributes among
Malayalis, andone each among Tamils, Gujaratis and Sindhis in Singapore.Mishra et
al.58 revealed 15% hemoglobin ConstantSpring (the two heterozygotes and
homozygotes) among thecoastal individuals of Orissa.

Hemoglobin-Bart's hydrops foetalis:

The most extreme indication of the alpha-thalassaemia quality is haemoglobinBart's

hydrops foetalis. The compound heterozygousstate for both ao thalassaemia and a+
thalassaemia,or ao thalassaemia and Hb Constant Spring, results in Hb-H disease5.
The homozygous state for a+ thalassaemiacauses an exceptionally mellow weakness
with hypochromic red cellsand no adjustment in the hemoglobin design. The
homozygousstate for Hb Constant Spring is described by amild haemolytic pallor
with splenomegaly. Affectedinfants are homozygous for the ao determinant (– –/
– ),the two guardians having heterozygous ao-thalassaemia. Thereis all out
concealment of alpha-chain blend with a grossexcess of gamma chains. The gamma-
chain tetramer, Hb-Bart's, has a high oxygen liking, and results in severetissue
hypoxia.

Influenced babies are either brought into the world dead (stillborn between28 and 40
weeks incubation) or pass on inside a couple of hours ofbirth 59. They are
underweight, pale (hemoglobin valuesin the scope of 6–8 g/dl), somewhat embittered,
terribly edematousand have hepatosplenomegaly and ascites. The

hemoglobin is around 6 g/dl, the blood film is horribly strange with

anisopoikilocytosis, hypochromia, target cells, polychromasia and an enormous
number of nucleated red cells. The reticulocyte check is high and serum bilirubin
raised.

The hemoglobin design comprises of 80–90% Hb-Bart's, with a little measure of Hb-
H and Hb-Portland. There is normally no HbA, HbA2 or HbF.

In West Bengal, four out of 100 rope blood tests got from babies demonstrated Hb
Bart's60. In one disconnected case with Hb Barts during childbirth, Hb-H
thalassaemia was recorded at 2 years old years. Frequency of Hb Bart's in2% of rope
blood tests was accounted for by Chouhan et al.55 and 4.2% by Vora et al.61 from
Mumbai. Mishra62reported 7.7% hemoglobin Bart's in the string blood
samplesamong the general population of north-western Orissa and,
subsequently,12.6% among the general population of beach front Orissa58.

METHHAEMOGLOBINOPATHIES:(20)

Methaemoglobinaemia

Methaemoglobin is a subordinates of ordinary hemoglobin where thw iron of the

haem complex has been oxidized from the ferrous to ferric structure the ferrous of the
ferric structure. It doesn't join with oxygen and in this manner does not partake in
oxygen transport. In ordinary red platelet, methaemoglobin is persistently being
shaped by the auto-oxidation of hemoglobin, however it is decreased when it framed;
therefore the convergence of methaemoglobin in the red platelet under typical
condition is under 1 level of the absolute hemoglobin. The decrease of
methaemoglobin is cultivated by the catalyst NADH-methaenoglobin reductase within
the sight of NADH. The NADH is produced by the Embden-Meyerhof pathway of
glycolysis.

The term methaemoglobin is utilized to portray the abundance gathering of

methaemoglobin in red cells. Methaemoglobin does not have the ability to convey
oxygen, and methaemoglobinaemia causes side effects and indications of hypoxia.
The incredible lion's share of cases off methaemoglonaemia are because of the
activity of substance operators that builds the rate of auto-oxidation of hemoglobin in
the red cells.Rare cases because of inherent metabolic deformity of the red cell.

Harmful methaemoglobinaemia:

The reason for lethal methaemoglobinaemia might be named given beneath:

Medication causes:

Medications which may cause methaemoglobinaemia incorporate phenacetin,

acetanilide, sulphonamides, sulphones, prilocaine, primaquine and pamaquinee,
nitrates, nitriets and potassium chlorate.

Word related causes:

Methaemoglobinemia in industry is most generally because of ingestion of nitro and

amino sweet-smelling derivatives,e.g. nitrobenzene,trinitrobenzene, and aniline. The
substance are normally retained through the respiratory tract and skin, and the
confusion is regularly found in laborers in concoction industrial facilities and
dangerous plants.
Family cause:

Synthetic substances equipped for creating methaemoglobinaemia are available in

various family unit substance; these incorporate furnishings and shoe clean, checking
ink, shoe colors and hued pastels , fragrances, and enhancing characters. Intense
methaemoglobinaemia has likewise been accounted for because of the utilization of
aniline-containing inks to check baby's napkins. Well water now and then contain
high centralization of nitrates, and in nation zones the utilization of this water to get
ready milk blend for babies has brought about methaemoglobinaemia.

Clinical highlights;

Cyanosis happens when methaemoglobinaemia comprises about 15% of the complete

shade. Much of the time there are no clinical highlights other than cyanosis, yet when
the convergence of methaemoglobin achieves 30-45%anoxic indications ordinarily
create. These incorporates cerebral pain, discombobulation, tachycardia, dyspnoea on
effort, solid issues, and shortcoming. If there should arise an occurrence of intense
harming, the focus may surpass 60-70% and spewing, dormancy, loss of cognizance,
circulatory disappointment, and passing may happens. In intense cases, the cyanosis
create inside 1-2 hours of the ingestion of the lethal agents.Many of the substance that
causes methaemoglobinaemia may likewise cause a haemolytic sickliness with Heinz-
body development.

Conclusion:

Clinically the conclusion of methaemogloniaemia is proposed by the nearness of

unequivocal cyanosis with practically no dyspnoea. Methaemoglobin can be
distinguished spectroscopically by its ingestion band in the red piece of the range at
630nm;this band vanish on the expansion of yellow ammonium sulfide. When it is
available in enormous sums the blood has a chocolate-darker shading which does not
vanish on oxygenation. Methaemoglobin does not show up in the plasma or pee aside
from in the periodic case with related haemolytic pallor.

Therapeutics:

Following expulsion of the causative operator, methaemoglobin is changed over back

to hemoglobin in a couple of days. Therefore in incessant cases end of the causative
medications or substance results in vanishing of the cyanosis inside a few days.

In instances of intense methaemoglobinaemia because of harming, particularly when

indications are serious, progressively dynamic treatment is fundamental. Treatment
comprise of moderate intravenous infusion of methylene blue; the suggested does is
 2mg/kg body weight for newborn children, 1.2mg/kg body weight for more seasoned
youngsters, and 1 mg/kg body weight for grown-up, 1% clean watery arrangement.

Inherited methaemoglobinaemia:

Inherited methaemoglobinaemia is uncommon. Two principle types are perceived

which contrast in their major impact and method of legacy.

Inherited methaemoglobinaemia related with NADH-methaemoglobin reductase

inadequacy. This issue is transmitted ass an autosomal latent attribute. Influenced
individual are tenaciously cyanotic and as a rule have mellow polycythaemia. Some
have side effects of anoxia. Mental hindrance is an intermittent affiliation. Standard
oral ascorbic corrosive treatment assuages the cyanosis and different indications when
present.

Genetic methaemoglobinaemia related with hemoglobin-M. a few irregular

hemoglobin, alluded to as hemoglobin-M, are related with cyanosis. The confusion is
transmitted as an autosomal overwhelming attribute, and anoxic indications are
generally missing. The cyanosis isn't improved by the organization of methylene blue
or ascorbic corrosive.

Sulphaemoglobinaemia:

Sulphaemoglobin is an anomalous sulfur containing hemoglobin subordinates unified

to methaemoglobin. It doesn't go about as an oxygen transporter and is absent in
ordinary red cells. It is shaped by the lethal activity of the medications and compound
operators that reason methaemoglobinaemia in people who are either obstructed or
who are taking sulfur containing medicinees. A past filled with ingetion of sulfur
containing laxative, for example, magnesium sulfate isn't phenomenal, and in
incidental cases there is a related anatomical variation from the norm of the entrail,
for example stricture or diverticulosis. Methaemoglobinaemia and
sulphaemoglobinaemia are regularly present together.

Quality Assurance for Hemoglobinopathy Screening and Testing (63)

Quality Assurance (QA) is defined as, “a program for the systematic monitoring and
evaluation of the various aspects of a project, service, or facility to ensure that standards of
quality are being met” and is often split into three categories, pre-analytic, analytic and post-
analytic.36 This section focuses on the analytic phase and briefly describes issues in the pre-
analytical and post-analytical phases.
Pre-analytical Phase
For screening, this phase includes variables such as specimen collection devices, quality of
specimen collection and transport to the testing facility. Performance evaluation indicators
may include the percentage of unsatisfactory specimens (as determined by each program)
received and the percentage of specimens that are delayed in transport.
Analytical Phase
 This phase includes all factors related to the laboratory testing. A laboratory quality assurance
program should follow any regulatory guidelines that are applicable. Generally, the laboratory
QA program has components listed below, at a minimum:
 A standard operating procedure detailing the testing method, how to interpret results and how
to resolve problems that may arise
 Flow charts/responsibilities/actions to clarify the role of each person involved in the analysis
 Quality control (QC) materials to monitor the method over time
 Documentation of any corrective actions taken
 Proficiency Testing (PT) an external quality assessment to monitor the laboratory’s
performance
Post-analytical Phase
The post-analytical phase includes the transmittal of the screening laboratory results and
determination of what actions were taken regarding positive screen results.

Characterstics of Structural Hemoglobin Variants detected among Indians

Sr. No Name Substitutio First Mobility Reference

n Report at alkaline
from India pH (8.6)
compared
to HbA
I. β chain variants
1 HbS β 6(A3) Tribals Slow Lehmann
Glu Val from Moving &Cutbush
Nilgiris , 1952
2 HbD- β121(GH4 Sikh Slow Bird, et
Punjab ) GluGln Soldier Moving al., 1955
from
Punjab
3 HbE β26(B8) Bengalis & Slow Chatterjee
GluLys Assamese Moving , et al.,
1956
4 HbJ β126(H4)V Mahar Fast Sukumara
alGlu from Moving n, et al.,
Maharasht 1975
ra
5 Hb β294(FG1) Punjabi Same as Dash, et
Chandigar AspGly Family HbA al., 1989
h
6 HbD-Agri β9(A6)Ser Agri Slow Colah, et
Tyr, communit Moving al., 2001
β121(GH4 y from
) GluGln Maharasht
ra
7 HbMRatn β63 Maratha Same as Kedar, et
agiri (E7)HisT from HbA al., 2005
yr Ratnagiri
8 HbC β6 (A3) Female NA Kumar, et
GluLys from New al., 2007
Delhi
9 HbD-Iran β22(4)Glu Punjabi Slow Colah, et
Gln family Moving al., 2007
10 Hb- β7 (A3) - Slow Edison, et
Vellore GluGln Moving al., 2012
11 HbAcharn β53(D4)Al Female Same as Karmarka
es aThr from West HbA r, et al.,
Bengal 2012
12 Hb-Koln β98(FG5) Female Slow Warang,
ValMet from Moving et al., 2014
Solapur,
Maharasht
ra
13 HbM β-CD 92 Ananthap Slow Upadhye,
Hyde Park (CACTA ur, Moving et al., 2014
C) Andhra
Pradesh
14 HbAgenog β-CD 90 Maratha Slow Colah, et
i (GAGA from Moving al., 2016
AG) Kolhapur
15 Hb British β-CD 101 Punjabi NA Colah, et
Columbia (GAGA from al., 2016
AG) Ludhiana
16 Hb Saint β-CD 28 Nhavi NA Colah, et
Louis from al., 2016
(GTGC Satara
AG)
17 Hb G β-CD 22 Budha Slow Colah, et
Coushatta from moving al., 2016
(GAAG Nashik,
CA) Maharasht
ra
18 HbPyrgos β-CD 83 Brahmin NA Colah, et
from al., 2016
(GGCG Punjab
AC)
II. Fusion hemoglobins
1 Hb- NA A family Slow Chouhan,
Lepore from Moving et al., 1971
North
Canara
2 Hb-P- β-87, δ- Indian NA Prehu, et
India 105 lady in al., 1994
Paris
3 Hb- δ-22, β-50 A boy Slow Shaji, et
LeporeHol from West Moving al., 2003
landia Bengal
4 Hb- δ 287/β116 A boy Slow Shaji, et
Lepore from Moving al., 2003
Boston – Kolkata
Washingto
n
III. α chain variants
1 HbK- N.A. East Fast Ager &
Madras Indian Moving Lehmann,
Family 1957
2 HbJ – N.A. Gujarati Fast Sanghvi,
India from Moving et al., 1958
Bombay
3 HbL N.A. Gujarati Slow Sukumara
from Moving n, et al.,
Bombay 1959
4 HbK- N.A. Bengali Fast Lehmann,
Calcutta Family Moving 1962
5 HbG- α57(E6)Gl Gurkha NA Huntsman
Norfolk yAsp , et al.,
1963
6 HbL- N.A. Lohana Slow Sukumara
Bombay from Moving n&Pik,
Bombay 1965
7 HbJ- α 90(FG2) Tamil Fast Hyde, et
Rajappan LysThr Speaking Moving al., 1971
Lady
8 HbQ- α64(E13)A Sindhi Slow Sukumara
India spHist Family moving, n, et al.,
similar to 1972
 HbLepore
9 HbM [α- CD58 Muslim Slow Bajaj, et
Boston (CACTA Family Moving al., 1973
C)
10 HbJ- α NA Fast Blackwell,
Meerut 120(H3)Al Moving et al., 1974
(Also aGlu
called HbJ
Birmingha
m)
11 HbKoya α 2 142, Tribals Slow Sukumara
Dora TermSer from Moving n, et al.,
Andhra 1975
Pradesh
12 HbRampa α95(G- Tribals Slow Sukumara
2)ProSer from Moving n, et al.,
Andhra 1975
Pradesh
13 HbJ-Paris α212(A10) North Fast Dash, et
AlaAsp Indian Moving al., 1988
Individual
14 Hb α95(G2)Pr Tribals Slow Wajcman,
Godavari oThr from Moving et al., 1998
Andhra
Pradesh
15 Hb Sun- α2 130 Individual Same as Sarkar, et
Prairie (H13) from West HbA al., 2005
AlaPro Bengal
16 HbSallanc α2 Punjabi Same as Dash, et
hes 104(G11) boy from HbA al., 2006
LysTyr North
India
17 Hb α127(H10) Brahmin Fast Nair, et
Jackson LysAsn Male from Moving al., 2010
Maharasht
ra
18 HbO α2116(GH Muslim Slow Nair, et
Indonesia 4) Female Moving al., 2010
GluLys from
Maharasht
ra
19 HbFountai α.21 Female Same as Upadhye,
 nebleau (B2)AlaP baby from HbA et al., 2012
ro Madhya
Pradesh
IV. δ chain variants
1 HbPelendr δ141(H19) A lady N.A. Colaco, et
i LeuPro from al., 2012
Maharasht
ra
2 HbD GlyAsp A girl N.A. Jain, et al.,
c.323GA from 2012
Maharasht
ra
3 HbDSaura δCD100(C Saurashtr N.A. Colaco, et
shtra T), a, Gujrat al., 2014
ProSer

LIMITATIONS OF STUDY:
GENETICS STUDIES ARE IMPORTANT TO COMPARE THE MUTATIONS
PATTERNS OF PATIENTS WITH OTHER STUDIES. GENETIC STUDIES
COULD NOT BE DONE IN OUR STUDY BECAUSE OF NON AVAILABILITY
OF RES5OURCES.

MATERIAL AND METHODOLOGY

METHODOLOGY:
• Inclusion Criteria: All UG and PG understudies (age 18–26 years) of various schools could
be screened overseen by APMS (Anand People's Medicare Society), Anand.
• Exclusion criteria: Already screened and referred to instance of Haemoglobinopathies as
screening test costs 150rs which will be paid by understudies.
PRECOUNCELLING:
First give a precouncelling to the all students to visit in all class separetly.
 LABELLING AND
BLOOD
COLLECTION:
After
precouncelling all
the students are fill
up the forms for their
data for the further
studies, after that
labelling is done
one form and
EDTA bulb barcode labelling is done. And collect the 2 ml of blood in EDTA bulb. And then
transport the blood sample to the Indian Red cross society, Ahemedabad for further
investigation.

CBC: (COMPLETE BLOOD COUNT)

We collect 847 samples, which is further proceed

for CBC, Complete blood count is done in the
sysmex K-4500 machine. In which all
haematological parameters are checkfrom the
EDTA blood sample.
SYSMEX K-4500:

FEATURES

 It provides accurate screening for patients with hematological abnormalities   

 It contributes in the diagnosis and therapeutic monitoring of patients.   
 Bar coding facility is available to enhance system efficiency.   
 It is also equipped with auto sampler unit for processing large number of samples.   
 Two types of QC are used in Sysmex K-4500: Individual QC, which is performed on
individual samples, and statistical QC which uses statistical method.   
 There is a system setup, where the settings can be changed according to each
laboratory?s needs.   
 There is also a criteria for flagging abnormal values obtained.   
 The Sysmex K-4500 has 18 parameters test results plus 3 histograms, 3 Part WBC
Differential, high performance of 80 samples/hr., reliable direct current detection
method. Cap Piercing Sampler & Microprocessor programs to achieve efficient walk-
away automation.   
 Uses reliable Direct Current (DC) method ensuring accurate and stable hematology
testing         
 18 parameters plus histograms for WBC, RBC and PLT (can be printed via thermal
printer)         
 Cap piercing sampler with 50 samples capacity allowing efficient hematology
testing         
 Throughput of 80 samples per hour without operator intervention         
 K-4500 uses cyanide-free, bio-degradable reagent for analysis of HGB         
 Combined with the Cap piercing Sampler, the flexible Barcode Reader* provides
positive sample identification, optimizing laboratory efficiency.   
  The Sysmex automated hematology analyzer offers two types of QC: individual QC,
which is performed on individual samples, and statistical QC which uses statistical
method. Moreover, the system allows for various system settings to suit the needs of
every laboratory and criteria for flagging abnormal values obtained.

Peripheral Blood Smear (PS) :

The polychromatic staining solution (Wright, Leishman, and Giemsa) contain methylene blue
and eosin. These basic and acidic dyes induce multiple colors when applied to cells. The
acidic component (nucleus with nucleic acid) takes blue-purple shades by the basic dye and
they are called as basophilic. The neutral component of the cells is stained by both the
dyes.nuclues stain blue and cytoplasm stain pink colour.
Affected individuals show RBC morphologic changes [microcytosis, hypochromia,
anisocytosis, poikilocytosis (spiculated tear-drop, sickle cell, target cell, smudge cells and
elongated cells)], and nucleated RBC (i.e., erythroblasts). The number of erythroblasts is
related to the degree of anemia and is markedly increased after splenectomy.
Carriers have less severe RBC morphologic changes than affected individuals. Erythroblasts
are normally not seen.

figure : sickle cells , target cells and in HB C peripheral smear.

      HB ELECTROPHORESIS:
 Gel Electrophoresis
In hemoglobin electrophoresis red cell lysates are subjected to electric fields under alkaline
(alkaline gel) and acidic (acid gel) pH. This can be carried out on filter paper, a cellulose
acetate membrane, a starch gel, a citrate agar gel, or an agarose gel. Separation of different
hemoglobins is largely (but not solely) dependent on the charge of the hemoglobin molecule.
A change in the amino acid composition of the globin chains results in alteration of
the charge of the hemoglobin molecule, resulting in a change of the speed of migration. In gel
electrophoresis, different hemoglobins migrate at different speeds. The top lane is called the
H lane and is mainly composed of HbH and HbI, while the point of origin is before
the carbonic acid band (Table 21.5). On the alkaline gel in hemoglobin electrophoresis, the H
is fast-migrating, and the band on the gel should be the same distance from J as A is from J in
the opposite direction. On the acid gel, the H migrates between the S and hemoglobins. The
patterns of various bands in acid gel electrophoresis are summarized in Table 21.5.
Table 21.5. Migration of Various Hemoglobin Bands in Alkaline Gel and Acid Gel
Electrophoresis
Region Hemoglobin Present
Alkaline Gel Electrophoresis
Top band (farthest from origin: H HbH, HbI
Lane)
J Lane HbJ
Hb Bart’s and HbN are between HbJ and HbH
lane
Fast haemoglobin Hb Hope
A Lane HbA
F Lane HbF
S Lane HbS, HbD, HbG, Hb Lepore
C Lane HbC, HbE, HbO, HbA2, HbS/G hybrid
Carbonic anhydrase band (faint) HbG2, HbA2’, HbCS
Acid Gel Electrophoresis
Top band: C Lane HbC
S Lane HbS, HbS/G hybrid; HbO and HbH are between
S and A lane
A lane HbA, HbE, HbA2, HbD, HbG, HbLepore, HbJ,
HbI, HbN, HbH
F Lane HbF, Hb Hope, Hb Bart’s

Cellulose acetate electrophoresis at alkaline pH

Haemoglobin electrophoresis at pH 8.4–8.6 using cellulose acetatemembrane is simple,
reliable and rapid. It is satisfactory for the detection of most common, clinically
important haemoglobin variants.20–24
Principle
At alkaline pH, haemoglobin is a negatively charged protein, and when subjected to
electrophoresis will migrate toward the anode (+). Structural variants that have a change in
the charge on the surface of the molecule at alkaline pH will separate from haemoglobin
A. Haemoglobin variants that have an amino acid substitution that is internally sited may not
separate, and those that have an amino acid substitution that has no effect on overall charge
will not separate by electrophoresis.
Equipment
•Electrophoresis tank and power pack. Any horizontal electrophoresis tank that will allow a
bridge gap of 7 cm. A direct current power supply capable of delivering 350 V at 50 mA is
suitable for both cellulose acetate and acid agarose gels.
•Wicks of filter or chromatography paper
•Blotting paper
•Applicators. These are available from most manufacturers of electrophoresis equipment, but
fine microcapillaries are also satisfactory.
•Cellulose acetate membranes. Plastic-backed membranes (7.6 × 6.0 cm) are recommended
for ease of use and storage.
•Staining equipment
Reagents
•Electrophoresis buffer. Tris/EDTA/borate (TEB), pH 8.5. Tris-
(hydroxymethyl)aminomethane (Tris), 10.2 g; EDTA (disodium salt), 0.6 g; boric acid, 3.2 g;
water to 1 litre. The buffer should be stored at 4 °C and can be used up to 10 times without
deterioration.
•Wetting agent. For example, Zip Zone Prep solution (Helena
Laboratories, www.helena.com): 1 drop of Zip Zone Prep in 100 ml water
•Fixative/stain solution. Ponceau S (www.helena.com), 5 g; trichloroacetic acid, 7.5 g; water
to 1 litre
•Destaining solution. 3% (v/v) acetic acid, 30 ml; water to 1 litre
•Haemolysing reagent. 0.5% (v/v) Triton X-100 (www.helena.com) in 100 mg/l
potassium cyanide.
Method
1. Centrifuge samples at 1200 g for 5 min. Dilute 20 μl of the packed red cells with 150 μl of
the haemolysing reagent. Mix gently and leave for at least 5 min. If purified haemolysates are
used, dilute 40 μl of 100 g/l haemolysate with 150 μl of lysing reagent.
2. With the power supply disconnected, prepare the electrophoresis tank by placing equal
amounts of TEB buffer in each of the outer buffer compartments. Wet two chamber wicks in
the buffer, and place one along each divider/bridge support ensuring that they make good
contact with the buffer.
3. Soak the cellulose acetate by lowering it slowly into a reservoir of buffer. Leave the
cellulose acetate to soak for at least 5 min before use.
4. Fill the sample well plate with 5 μl of each diluted sample or control and cover with a
50 mm coverslip or a ‘short’ glass slide to prevent evaporation. Load a second sample well
plate with Zip Zone Prep solution.
5. Clean the applicator tips immediately prior to use by loading with Zip Zone Prep solution
and then applying them to a blotter.
6. Remove the cellulose acetate strip from the buffer and blot twice between two layers of
clean blotting paper. Do not allow the cellulose acetate to dry.
7. Load the applicator by depressing the tips into the sample wells twice and apply this first
loading onto some clean blotting paper. Reload the applicator and apply the samples to the
cellulose acetate.
8. Place the cellulose acetate plates across the bridges, with the plastic side uppermost. Place
two glass slides across the strip to maintain good contact. Electrophorese at 350 V for 25 min.
9. After 25 min electrophoresis, immediately transfer the cellulose acetate to Ponceau S and
fix and stain for 5 min.
10. Remove excess stain by washing for 5 min in the first acetic acid reservoir and for 10 min
in each of the remaining two. Blot once, using clean blotting paper and leave to dry.
11. Label the membranes and store in a protective plastic envelope.
Interpretation and comments
Figure shows the relative electrophoretic mobilities of some common haemoglobin variants
at pH 8.5 on cellulose acetate. Satisfactory separation of haemoglobins C, S, F, A and J is
obtained (Fig. 14-4). In general, haemoglobins S, D and G migrate closely together, as do
haemoglobins C, E and OArab. Differentiation between these haemoglobins can be obtained
by using acid agarose gels, HPLC or IEF. There are slight differences in mobility between
haemoglobins S, Lepore and DPunjab and also between haemoglobins C and E. Generally,
the Lepore haemoglobins and Hb DPunjab migrate slightly anodal to haemoglobin S (i.e.
they are slightly faster than S); haemoglobin C migrates slightly cathodal to haemoglobin
E (i.e. it is slightly slower than E). These differences are, however, too subtle to avoid the
necessity for a second confirmatory technique.

Sign in to download full-size image

Figure . Schematic representation of relative mobilities of some abnormal haemoglobins.
Cellulose acetate electrophoresis, pH 8.5.
Sign in to download full-size image
Figure: Relative mobilities of some abnormal haemoglobins.
HPLC: (HIGH PERFORMANCE LIQUID CHROMATOGRAPHY)

Mechanized cation trade High Performance Liquid Chromatography (HPLC) is the most
well-known strategy utilized in a few labs associated with distinguishing proof of
hemoglobinopathies in India. The BioRad stages have been utilized, either the Variant
Classic machine or the Variant II analyzer. The Variant II machine permits essential cylinder
inspecting and has the office of standardized tag perusing. The EDTA blood test is
consequently suctioned, lysed and weakened and infused into the silica based section and the
hemoglobins are eluted utilizing a pre-modified inclination of expanding ionic quality framed
by utilizing 2 phosphate cushions of various pH and ionic qualities.
A photometer screens the eluate and measures the adjustment in absorbance at 415nm and
690mn as various hemoglobins experience the stream cell. A chromatogram of absorbance
versus time is shown and the region under the pinnacles gives the level of various
hemoglobins alongside their maintenance times. Explicit windows are set dependent on the
maintenance times for the basic hemoglobins like the HbF, HbA2, HbS, HbD and HbC
windows. Be that as it may, a few other Hb variations could likewise elute in these windows.
The library of variations gives some guide dependent on the maintenance times of various
uncommon variations. Countless examples can be run, each example taking 6.5 minutes for
investigations. Different instruments are additionally accessible for HPLC for hemoglobin
examination.

POSTCOUNCELLING:
After all reports are come we distribute the report to all students and which are positive are
called for postcouncelling.
 RESULT

RESULT:
Table no 1: No.of positive sample out of total no of sample is seen in this table.out of 847
sample 52[6.1%] are positive for different haemoglobinopathies.
Total No.of Normal Positive number Normal Male Normal Female
sample sample no. of sample
847 795[93.8%] 52[6.1%] 319[37.6%] 528[62.3%]

TOTAL NO OF SAMPLE : 847

NORMAL S5AMPLE NO. POS5ITIVE NUMBER SAMPLE

6%

94%
 NORMAL MALE FEMALE RATIO
MALE FEMALE

38%

62%

Table no 2: Table showing the distribution of patients with different type of

haemoglobinopathies.
Haemoglobinopathies No. of positive sample
Hb c/o inheritance with thalassemia 23[2.7%]
Atypical thalassemia 2[0.2%]
Typical thalassemia 22[2.5%]
2[0.2%]
Sickle cell trait (Hb S )
Hb D 1[0.1%]
Hb E 1[0.1%]
Double heterozygous 1[0.1]
Total 52[6.10%]

Table showing that in different haemoglobinopathies there is 2.7% are Hb c/o thalassemia,
0.2% are atypical thalassemia, 2.5 % are typical thalassemia, 0.2 % are sickle cell, 0.1% are
Haemoglobin D, 0.1% are Haemoglobin E and 0.1% shows double heterozygous positive.
 23 22
25
20
15
10
5 2 2 1 1
0

DIFFERENT HAEMOGLOBINOPATHIES

Table no 3: Table showing sex wise distribution of cases among different

haemoglobinopathies.
Haemoglobinopathies Male Female
Hb S c/o inheritance with 10[1.1%] 13[1.5%]
thalassemia
Atypical thalassemia 2[0.2%] 0[0%]
Typical thalassemia 12[1.4%] 10[1.1%]
Sickle cell trait HB S 0[0%] 2[0.2%]
Hb S 1[0.1%] 0[0%]
Hb E 0[0%] 1[0.1%]
Double heterozygous 0[0%] 1[0.1%]
Total 25[2.9%] 27[3.1%]

Table shows that there is 2.9% male and 3.1% female are shown different
haemoglobinopathies. Out of that Hb c/o shows 1.1% male and in female it is 1.5%, in
atypical thalassemia 0.2% male and 0% female. Typical thalassemia 1.4 % males and 1.1%
females., sickle male 0% and female shows 0.2% , HB D 0.1% males and 0% females, HB E
0% male and 0.1% females, double heterozygous 00 % males and 0.1% females.
 Chart Title
MALE FEMALE
13 12
10 10

2 2 1 0
0 0 0 1 0 1

Table no 4 : Table showing district wise distribution of cases among haemoglobinopathies.

District Hbs C/O Atypical Hb D Hb E Hb S Typical Doubl Total
inheritenc thalassemi Thalasse e
e with a mia hetero
thalassemi zygou
a s
Anand 3[0.3%] 1[0.1%] 1[0.1% 1[0.1%] - 5[0.5%] 1[0.1 12[1.4%]
] %]
Tapi 3[0.3%] - - - - - - 3[0.3%]
Narmada 2[0.2%] - - - - - - 2[0.2%]
Kheda 3[0.3%] - - - - 1[0.1%] - 4[0.4%]
Bharuch 1[0.1%] - - - - 3[0.3%] - 4[0.4%]
Surat 2[0.2%] - - - - 2[0.2%] - 4[0.4%]
Vadodara 2[0.2%] 1[01%] - - 1[0.1%] 2[0.2%] - 4[0.4%]
Dahod 2[0.2%] - - - - - - 2[0.2%]
Navsari 2[0.2%] - - - - - - 2[0.2%]
Panchmaha 1[0.1%] - - - - - - 1[0.1%]
l
Chhotaudep 1[0.1%] - - - - 1[0.1%] - 2[0.2%]
ur
Valsad - - - - 1[0.1%] - - 1[0.1%]
Arvali - - - - - 2[0.2%] - 2[0.2%]
DNH - - - - - 1[0.1%] - 1[0.1%]
Junagadh - - - - - 1[0.1%] - 1[0.1%]
Mahisagar - - - - - 1[0.1%] - 1[0.1%]
Amreli - - - - - 2[0.2%] - 2[0.2%]
Table showing that in anand district contain high level of different haemoglobinopathies
about 1.4%, whereas kheda,Bharuch,surat and Vadodara district has 0.4% of different
 haemoglobinopathie which is followed by tapi district contain 0.3% which is followed by
narmada, dahod, navsari,chhotaudepur, arvali and amreli 0.2%, which is followed by
panchmahal,valsad,DNH,junagadh and mahisagar about 0.1%.

DISTRICT WISE DISTRIBUTION

D PI
AN TA A A
A N AD ED UCH AT A D I
RM KH
AR SU
R
DAR HO AR HAL UR
A H S D LI
N B DO DA AV MA AIP LSA VA H H
VA N A D VA R DN AD GAR ELI
H U A G A R
A NC OTA UNA HIS AM
P H J A
CH M

HB S C/O INHERITENCE WITH THALASSEMIA ATYPICAL THALASSEMIA

HB D HB E
HB S TYPICAL THALASSEMIA
DOUBLE HETEROZYGOUS

Community Hb S Hb D Typical Hb E HbSco Atypical Double Total

thalasse inheriten thalalass heterozy
mia ce emia gous
Patel 2[0.2%] - 8[0.9%] 4[0.4%] 1[0.1%] 15[1.7%]
Panchal - - 1[0.1%] 1[0.1%]
Thakor - - 1[0.1%] 1[0.1%]
Kanojiya - - 1[0.1%] 1[0.1%]
Malhotra - 1[0.1%] 1[0.1%]
Chaudhari - - 1[0.1%] 3[0.3%] 4[0.4%]
Valvi - - 3[0.3%] 3[0.3%]
Vasava - - 2[0.2%] 2[0.2%]
Parmar - - 1[0.1%] 1[0.1%]]
Baria - - 2[0.2%] 2[0.2%]
Bareea - - 1[0.1%] 1[0.1%]
Ganvit - - 1[0.1%] 1[0.1%]
Rathod - - 1[0.1%] 1[0.1%] 2[0.2%]
Gamit - - 1[0.1%] 1[0.1%]
Bhil - - 2[0.2%] 2[0.2%]
Rathva - - 1[0.1%] 1[0.1%]
Darbar - - 1[0.1%] 1[0.1%]
Kharadi - - 1[0.1%] 1[0.1%]
Gohil - - 1[0.1%] 1[0.1%]
Mahyavansh - - 2[0.2%] 2[0.2%]
i
Brahmin - - 2[0.2%] 2[0.2%]
Patil - - 1[0.1%] 1[0.1%]
ODE - - 1[0.1%] 1[0.1%]
Pathan - - 1[0.1%] 1[0.1%]
Modi - - 1[0.1%] 1[0.1%]
Soni - - 1[0.1%] 1[0.1%]
Parekh - - 1[0.1%] 1[0.1%]
Table no 5: Table showing community wise distribution of cases among
haemoglobinopathies.
Table shows that high prevalence different haemoglobinopathies are found in the patel 1.7%,
which is followed by chaudhari 0.4%, valvi 0.3%, baria,rathod,bhil,mahyavanshi &
brahmin0.2% and others are contribute about 0.1%.
 CASTE WISE DISTRIBUTION
8
6
4
2
0
L
TE R a
lvi
PA A KO otr a ar ea od
lh V m re il
TH a ar a th Bh ar hi
l
in
M P B Ra rb o E i
Da G
ah m OD od ekh
Br M r
Pa

HB S HB D
TYPICAL THALASSEMIA HB E
HB S C/O INHERITENCE WITH THALASSEMIA ATYPICAL THALASSEMIA
DOUBLE HETEROZYGOUS

Table no 6: Table showing marital status wise distribution of cases among

haemoglobinopathies.

Haemoglobinopathies Marital Premarital

Hb S c/o inheritance with thalassemia 1(M)[0.1%] 22[2.5%]
Atypical thalassemia 0[0%] 2[0.2%]
Typical thalassemia 1(M)[0.1%] 212.4%]
Sickle cell (Hb S) 0[0%] 2[0.2%]
Hb D 0[0%] 1[0.1%]
Hb E 0[0%] 1[0.1%]
Double heterozygous 0[0%] 1[0.1%]
Total 2[0.2%] 50[5.9%]

Table shows that 5.9 % are screen before marriage and 0.2 % are screen after marriage.
 25

20

15

10

0 MARITAL
PREMARITAL
ia ia ia S) D E
ou
s
e m e m e m Hb Hb Hb g
ss ss ss (
oz
y
ala ala ala cell er
th h h t
lt lt kle he
with ica ica Sic le
yp p ub
nc
e
At Ty o
ta D
ri
he
in
/o
Sc
Hb

Table no 7: Table showing haemoglobin wise distribution of cases among

haemoglobinopathies.
Hb value Hb S Atypical Hb D Hb E Typical Hbs c/o Double
Thalassemia thalassemi inheritance heterozyg
a thalassemia os
Normal 2 2[0.2%] 1 1 16 19 -
[0.2%] [0.1%] [0.1% [1.8%] [2.2%]
]
Mild - - 4 3 - - -
[0.4%] [0.3%]
Moderate - - - - 2 1 1
[0.2%] [0.1%] [0.1%]
Severe - - - - - - -
Table showing the haemoglobin values which is differentiate the anemia in mild , moderate
and severe from. But only haemoglobin value is not significant to define
Haemoglobinopathies. Because more number of sample shows normal Haemoglobin value.
 20 19
18 16
16
14
12
10
8
6 4
4 3
2 2 2
2 1 1 1 1
0 000 0 000 000 000 00 0
0

NORMAL MILD MODERATE SEVERE

Table no 8: Table showing CBC wise distribution of cases among haemoglobinopathies.

Haem MCV MCH MCHC RDW
oglob N I D N I D N I D N I D
inopa
thiees
Hb S 2 - - 1 - 1 2 - - - 2 -
[0.1%] [0.1%] [0.1%] [0.2%] [0.2%]
Hb D 1[0.1% - - 1 - - 2% - - - 1 -
] [0.1%] [0.2%] [0.1%]
Hb E - - 1 - - 1 1 - - - 1 -
[0.1%] [0.1%] [0.1%] [0.1%]
Hbs 1 - 20 1 - 20 23 - - 2 2 -
co/in [0.1%] [2.3%] [0.1%] [2.3%] [2.7%] [0.2%] [0.2%]
herite
nce
Doub - - 1 - - 1 - - 1 - 1 -
le [0.1%] [0.1%] [0.1%] [0.1%]
heter
ozygo
us
Typic - - 22 - - 22 19 - 3 - 22 -
al [2.5%] [2.5%] [2.2%] [0.3%] [2.5%]
thalas
semia
Atypi 2 - - 2 - - 2 - - 1 1 -
cal [0.2%] [0.2%] [0.2%] [0.1%] [0.1%]
thalas
semia
In table showing different values of Blood indices(MCV,MCH,MCHC and RDW ) in
different haemoglobinopathies. Here in Hb E,Double Heterozygous anemia, Hb sickle c/o
and in typical thalassemia it shows decrease values. Whereas in others it is normal MCV
values. In Hb S, Hb D , Hbs c/o and in Atypical thalassemia shows MCH values normal
while in others it is decrease. In Double Heterozygous and typical thalassemia MCHC value
is decrease, whereas in others it is normal. In Hb S c/o and Atypical thalassemia RDW is
normal while in other its value is increase.

Table no 9: Table showing HPLC wise distribution of cases among haemoglobinopathies.

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

% % % % % %
(typical thal)

3240076 94.50 4.80 0.70 - - -

3240091 93.80 5.30 0.90 - - -
3240138 93.00 5.40 1.60 - - -
3240176 94.60 5.20 0.20 - - -
3240235 96.00 3.80 0.20 - - -
3240264 94.40 5.30 0.30 - - -
3251305 92.50 5.00 2.50 - - -
3251348 93.80 5.70 0.50 - - -
3251373 94.00 5.60 0.40 - - -
3251376 94.00 4.90 1.10 - - -
3251410 94.30 5.30 0.40 - - -
3251480 95.10 4.60 0.30 - - -
3251481 94.90 4.80 0.30 - - -
3251501 93.80 5.80 0.40 - - -
3251517 94.70 5.00 0.30 - - -
3251522 92.70 6.00 1.30 - - -
3251580 94.50 5.10 0.40 - - -
3251581 94.60 4.90 0.50 - - -
3251583 93.80 5.20 1.00 - - -
3251685 95.80 3.80 0.40 - - -
3251667 91.90 5.30 2.80 - - -
3251724 94.10 5.60 0.30 - - -

Hbs c/o
3240003 64.60 3.30 1.30 30.80 - -
3240047 67.30 2.90 0.90 28.90 - -
3240054 70.70 3.50 0.40 25.40 - -
3240055 70.80 3.60 0.40 25.20 - -
3240056 68.90 3.50 0.50 27.10 - -
3240112 64.20 3.00 0.40 32.40 - -
3240146 98.10 1.60 0.30 - - -
3240217 66.80 3.00 0.20 30.00 - -
3240249 68.00 3.70 0.40 27.90 - -
3240328 62.30 4.40 0.10 33.20 - -
 3240331 65.10 3.50 0.10 31.30 - -
3240364 62.50 4.60 0.70 32.20 - -
3251331 70.20 3.80 0.40 25.60 - -
3251367 71.60 3.30 0.70 24.40 - -
3251384 62.00 3.10 0.80 34.10 - -
3251407 71.30 3.20 0.20 25.30 - -
3251446 69.50 3.80 0.70 26.00 - -
3251598 71.90 3.40 0.50 24.20 - -
3251616 69.60 3.50 0.20 26.70 - -
3251633 71.00 3.60 0.80 24.60 - -
3251639 71.10 3.50 1.00 24.40 - -
3251647 69.40 3.10 0.30 27.20 - -
3251708 62.50 3.10 0.80 33.60 - -
3251743 70.50 3.60 0.50 25.40 - -

Atypical thal
3240006 96.10 3.60 0.30 - - -
3240204 95.50 4.20 0.30 - - -

Sickle trait
3251484 57.90 2.90 1.20 38.00 - -
3251714 60.30 3.30 0.30 36.10 - -
Double heterozygous
3251391 3.40 3.90 8.10 84.60 - -
Hb E
3251438 70.10 28.30 1.60 - - -
Hb D
3251511 65.60 1.70 0.20 - 32.50 -

In patients detected as Sickle cell trait, HBS ranged between 36 to 38 % and HBS value less
than HBA value. Hb ranged between10-12gm%.
In patients detected as typical Beta thalassemia HBA2 was >3.1%, and HB S was > 24%
which is very high, In patients detected to have atypical Beta thalassemia, HbA2 > 3.5%.
Patients detected to have double heterozygous for SA-BTT, showed HbS>84.6%, HbA2
ranging from 3.9 % and HbF 8.10%. Patients detected to have HbD Punjab heterozygous had
unknown peak at D window. HbD ranged between 32.50%. Patients detected to have HbE
showed raised peak in A2 region about 28.30%.

DISCUSSION
DISCUSSION:
India is an ethnically diverse country with marked regional variation. APMS campus contain
different people of varied ethnic groups of different cities and district. Due to migration, there
is constant mixing of people from different regions. We attempted this pilot study to see the
difference in the prevalence of various hemoglobinopathies in APMS campus. Appropriate
laboratory tests are required for diagnosis and confirmation of these disorders.

Hemoglobinopathies are common around the world, in any case, it is progressively common
in some geological territories. In India, According to medical clinic based study, normal
recurrence of sickle cell quality is around 5%. The most noteworthy recurrence of sickle cell
quality in India is accounted for in Orissa (9%), pursued by Assam (8.3%), Madhya Pradesh
(7.4%), Uttar Pradesh (7.1%), Tamil Nadu (7.1%) furthermore, Gujarat (6.4%).The
appropriation of beta thalassemia isn't uniform in the Indian subcontinent. In spite of the fact
that certain networks are recognized to have high predominance, it has been identified in
pretty much every Indian populace. The predominance of beta thalassemia characteristic
changes from 1-17% in various populaces of India.(64)
Our study showed 4.84% patients showing normal pattern of haemoglobin, which is
comparable to study by Balgir etal (34.3%).which is higher than my study.
Present study was conducted on 847 individuals are screened for the haemoglobinopathies.
Among the 847 cases studied, 93.8% were found to be normal and 6.1% had one or the other
forms of Haemoglobinopathies (Table 1). Out of 52 abnormal cases equal incidence in males
(48.0%%) and females (52%) was observed (Table 3). Most common haemoglobinopathies
observed were is 2.7% are Hb c/o thalassemia, 0.2% are atypical thalassemia, 2.5 % are
typical thalassemia, 0.2 % are sickle cell, 0.1% are Haemoglobin D, 0.1% are Haemoglobin E
and 0.1% shows double heterozygous positive.

In this study, HbE trait was found in 0.1% cases out of all haemoglobinopathies. A study
conducted in the rural areas of West Bengal reported the prevalence of HbE trait to be 3.86%
(66) Due to the high prevalence of Hb disorders in various regions of West Bengal premarital
screening must be routinely done for prevention of high risk marriages.

The gene frequency of HbD Punjab is relatively low with highest prevalence in North-
Western India. Balgir et al [65] got only two cases (0.2%) of HbD Punjab trait in a series of
more than 1000 cases. We also got only one case (0.1%) indicating a very low prevalence of
HbD in Gujarat. On CE-HPLC, it gets eluted in the D-window, which is separate from HbS
peak.

Abnormal results are explained, and genetic counseling was done to individual affected.
Those unmarried individual, explained regarding inheritance of disease, and the need to
prevent marriage to traits. Those married individual explained regarding prenatal diagnosis,
nature of inheritance, and selective termination of pregnancy of the affected fetus and
preconceptional counselling for future.

The disorders of Hb frequently encountered in India include beta thalassemia, HbE, HbD,
and sickle cell anemia. In study by Mondal et al beta thalassemia trait was the most common
abnormality found , followed by HbE trait Other variants detected included sickle cell trait,
HbE disease, sickle cell disease, HbD-Punjab trait, double heterozygous state of HbS and
HbE, double heterozygous state of HbS and HbD, Hb Lepore, HbJ-Meerut and HbH.6 In
study of Goswami et al it was found that Hb E trait was the most common
hemoglobinopathies (34.4%) followed by homozygous E (25.3%), beta-thalassemia trait
(17.8%), E-β-thalassemia (15.1%), β-thalassemia major (1.5%), sickle cell-β-thalassemia
(3.4%), sickle cell trait.7 Study done by Mehandi et al in Saudi population found Beta
Thalassemia trait to be the most common hemoglobinopathy detected followed by Sickle cell
trait and sickle cell alpha Thalassemia trait. The Hb variant E and D, which are more
prevalent in Southeast Asia were rarely found among Saudis.8 In study done by Patel U et al
in population of Gujarat, beta Thalassemia trait was most common hemoglobinopathy,
followed by Thalassemia major, sickle cell anemia and sickle cell trait.9 In our study, beta
thalassemia trait was most common, followed sickle cell anemia trait, Hb E, HbD, double
heterozygous.
The largest screening programme for thalassemia using the HPLC system in the population
has been carried out in Gujarat, India by the Indian Red Cross Society in Ahmedabad and
other cities. From 2004 to 2010, they screened 370,117 subjects for carrier status, among
whom there were 173,112 students, 45,000 youths and 8,377 pregnant women. Carrier rate
has varied from 4.3% to 5.0% (67)
Sickle cell trait patients showed HBS values more than 30% along with raised HBF levels., Similar
findings were observed by Kar etal HB S values more than 70%. So in my studies HB S level is
decrease than kar etal (68)
In India HbD is seen in Sindhi, Punjabi and Gujrathi communities. There were 1 patients detected as
HBD trait. They showed band in HB D region and decrease in A2 level (1.7%). Similar findings were
noted by Lele etal(69)

Patterns of hemoglobinopathies in comparison with other studies.

Hemoglobin Variant Our study Balgir etal[65] Uddin etal[70]
Normal 93.8% 34.3% 42.2%
SA 0.2% 29.8% -
BTT 2.7% 18.2% 21.3%
SA-BTT 0.1% 1.7% -
HBD trait 0.1% 0.2% -
HBE trait 0.1% 0.9% 12.1%
Hb c/o inheritance with 2.7% - -
thalassemia
Total 100% 100% 100%

CONCLUSION

CONCLUSION:
Patel community has higher prevalence of hemoglobinopathy, mainly Beta Thalassemia
Trait. The most effective approach to minimize the problem of hemoglobinopathies in India
is to sensitization and screening of individual, genetic counseling, proper health education
regarding the nature of inheritance and prenatal diagnosis in a high-risk community. More
efforts are needed to increase awareness in high risk communities like Sindhi and lohana
regarding thalassemia before the dream to control hemoglobinopathy in India.
HPLC provides rapid and accurate tool for diagnosis of hemoglobinopathies as identification
of abnormal hemoglobin based on electrophoretic mobility is only presumptive and have to
be confirmed by another technique applying different principle.
Furthermore study of prevalence of various hemoglobinopathies, which are endemic in
certain areas, by HPLC helps in detection of rare variants like HbQ, HbD Punjab etc. Further
study for their gene mutations can also be performed.

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