HPLC Hemoglobinopathies patterns atlas HPLC

Variant
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Library
of
Chromatograms
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Bio-Rad
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TM
Variant Hemoglobin Testing System
Library of Chromatograms
Published by Bio-Rad Laboratories Copyright © 2006 Bio-Rad Laboratories, Inc. All right reserved.

VariantTM
Hemoglobin
Testing System
Library of
Chromatograms

Introduction
It gives us immense pleasure to release the first handbook of the reference
chromatograms for the Variant TM
Hemoglobin Testing System. This handbook
is unique as it is a compilation of the most common thalassaemia and
hemoglobinoathy conditions found in India. Also enclosed are the relevant
guidelines for each of the conditions from the well known hematologists of
India.
The enclosed chromatograms have been correlated to the chromatograms of
the “Bio-Rad 's Library ofAbnormal Hemoglobins. ”
We hope this handbook will be a useful aid in the routine interpretation of
chromatograms.
MadhuriMehra
Senior Product Manager,
Bio-Rad Laboratories India Pvt. Ltd.

Foreword
It gives me great pleasure in writing a foreword
for the "Handbook of Chromatograms for
Haemoglobinopathies". There is phenomenal
progress in the last two decades in understanding
and investigating the cases of thalassemia and
other haemoglobinopathies. Dedicated HPLC
system applied to haemoglobinopathies is highly
accurate, sensitive and robust. Being a user of
Variant system for a long time, I could see the good
reasons for such compilation. Realizing the need of the users especially in
India, this manual illustrates the chromatograms on case-to-case basis. The
application of this information is ever more important since this will improve
ultimately clinical services. This book will work like a ready reference. In
addition, the chromatograms along with respective red cell indices of double
heterozygous conditions is also very impressive and useful. Individual case
descriptions have got great educational value. A bit of theoretical knowledge,
for example about the list of Beta gene mutations in India, the prevalence of
sickle cell disease in tribal population of central India given in this manual also
has added value.
I am sure the readers will find it to be a very useful book. While
congratulating the compiler for her sincere effort to transfer the modern
technology to the users, I wish to give due credit to BIO-RAD company for
bringing out such a beautiful manual in print. Simultaneously, I also hope that
the same may be updated from time to time since a lot of new knowledge is
being added in this field rapidly.
Dr.Dipika Mohanty,
M.D, PhD, FRCPath (London),
FACP(USA), FNAMS
Ex-Director, Institute of Immunohaematology,
13 Floor, New MS Building, Parel, Mumbai-12

Acknowledgments
We are extremely grateful to the following eminent hematologists for their expert
comments on various conditions of Thalassaemia and Hemoglobinopathies,
withoutwhichthisdocument could nothavebeen completed.
Dr. S.K. Sood
Ex-Head, Department of Haematology
Sir Ganga Ram Hospital
New Delhi 110060
Dr. Manorama Bhargava
Head, Department of Haematology
New Delhi 110060
Dr. Shirish
Senior Consultant,
Haematology Department,
Gangaram Hospital,
New Delhi 110060
Dr. Anil Handoo
Clinical Assistant, Dept of Hematology
New Delhi 110060
Dr Reena Das, MD, DipNB
Additional Professor,
Department of Haematology
PGIMER, Chandigarh
Prof. G. Garewal
Ex-Head Department of Haematology
PGIMER, Chandigarh
Dr. Debasis Banerjee MD.
Haematologist
Clinical Haematology Service
4,Gorky Terrace, Kolkata - 700 017.
Dr. Kanjaksha Ghosh
Director,Institute of Immunohaematology,
K.E.M Hospital Campus,13th Floor,
New Multistoried Building, Parel,
Mumbai-400012
Dr. Roshan Colah
Deputy Director,
Institute of Immunohaematology,
K.E.M Hospital Campus,13th Floor,
New Multistoried Building,Parel,
Mumbai-400012
Dr. Sarmila Chandra MD.
Haemato-Oncologist,
Clinical Haematology Service
4, Gorky Terrace, Kolkata - 700 017.
A.M.R.I. Hospitals and Kothari Medical
Centre.Kothari Medical Centre, Kolkata.
Dr. Renu Saxena
Professor of Haematology,
All India Institute of Medical Sciences,
New Delhi
Dr. Mrs. A.V, Shrikhande
Project Director, Regional
Hemoglobinopathy Detection and
Management Centre,
Indira Gandhi Medical College, Nagpur
Dr. R.Manchanda
Hemato-pathologist, Director Laboratories,
K.E.M.Hospital, Pune
On behalf of Bio-Rad Laboratories, we would like to acknowledge the special
contribution of Dr. Lal PathLabs for making this library possible.
Our heartfelt thanks to Dr. Puneet K. Nigam (Chief Technical Officer), Rama
Sareen (QC Supervisor, Electrophoresis Lab) and Victor Paul (Technologist)
from Dr. Lal PathLabs for their painstaking efforts and help in making this
document a reality.
Special Thanks...

Index
• Normal Sample
• Beta Thalassaemia Trait ( HbA2 4-7%)
• Beta Thalassaemia Trait ( HbA2 7-9%)
• Beta Thalassaemia Intermedia
• Beta Thalassaemia Major
• HbE Heterozygous
• HbE Homozygous
• Double Heterozygous for HbE and
Beta Thalassaemia Trait
• Sickle Cell Heterozygous
• HbS Homozygous
• Double Heterozygous for HbS and
• Double heterozygous for HbS and HPFH
• Double Heterozygous for HbS and HbE
• HbD- Punjab Heterozygous
11-12 • Double heterozygous for HbD-Punjab and
• Double Heterozygous for HbS and HbD
• HbD-Iran Heterozygous
• HbQ-India Heterozygous
• Double Heterozygous for HbQ-India and
• Hb Lepore
• Alpha Thalassaemia
• HbH
• Elevated P3 peak
• Megaloblastic anemia
• Annexure -1
41-42
43-44
45-46
47-48
49-50
51-52
53-54
55-56
57-58
59-60
61-63
13-14
15-16
17-18
19-20
23-24
25-26
27-28
29-30
31-32
33-34
35-36
37-38
39-40
9 10

Hb 16.4 gm/dl
MCV 92.3 fl
MCH 31.2 pg
RBC 5.25 mill/c.mm
RDW 13.8 %
11
Normal Sample
****Beta Thal Short 30100-A ****
DATE : 18/07/05 TIME : 12:34:36
TECH ID # 0
VIAL # 26
SAMPLE ID # 00000000000003519341
ANALYTE ID % TIME AREA
P2 5.4 1.33 139692
P3 3.5 1.73 91936
A0 88.1 2.50 2287744
A2 2.9 3.63 67327
TOTAL AREA 2586699
30% F 0.0% A2 2.9%
20%
10%
0
0 1 2 3 4 5 6
Normal Sample
Phenotype: Normal
Genotype: Normal α
2
β
2chains. Genes for globin chain synthesis are
located in two clusters on chromosomes 11 & 16.
Ethnicity: Seen in all normal adults, worldwide.
Pathophysiology: Not applicable.
Laboratory findings: There is no anemia or reticulocytosis. MCV, MCH are
usually normal. HPLC reveals~96-98% hemoglobin A (with glycated
hemoglobin), while hemoglobin A2 is < 3.5%. Hemoglobin electrophoresis at
acid pH and alkaline pH reveals migration of hemoglobin to Hb A position in
both of them.
References
12

Hb 13.3 gm/dl
MCV 64.2 fl
MCH 20.2 pg
RBC 6.57 mill/c.mm
RDW 16.6 %
13
Beta Thalassaemia Trait (HbA2 4-7%)
****Beta Thal Short 30100-B ****
DATE : 27/09/05 TIME : 12:05:11
TECH ID # 0
VIAL # 20
SAMPLE ID # 00000000000003750928
F 1.5 1.14 26933
A2 6.0 1.34 107496
P3 3.9 1.73 68997
A0 83.7 2.52 1495167
A2 4.7 3.62 75616
30%
TOTAL AREA 1774209
F 1.5 A2 4.7%
20%
10%
0
0 1 2 3 4 5 6
Phenotype: Normal or mildly anemic
Genotype: Heterozygous for majority of the beta thalassemia
mutations including the common Indian mutations (IVS1-5 (G-C), 619bp
deletion, IVS1-1 (G-T), CD8/9 (+G), CD41/42 (-CTTT), CD15 (G-A),
CD30 (G-C)).The capsite (+1) (A-C) mutation is usually silent with
normal HbA2 levels (3.0 - 3.9%).
Ethnicity: Prevalence of beta thalassemia carriers in the general Indian
population is around 3%. It is much higher in some caste groups from
Gujarat, Punjab, Eastern UP, and the immigrant population from Pakistan
(5 - 15%).
Pathophysiology: The molecular defect causes the absence or
reduced synthesis of beta globin chains leading to ineffective
erythropoiesis and a mild reduction in hemoglobin. Hb may be normal or
reduced to 9-11 g/dl.
Laboratory findings : Peripheral smear shows microcytosis, marked
anisopoikilocytosis, target cells, basophilic stippling and polychromasia.
The MCV is < 80 fl, MCH < 27 pg and MCHC is also reduced. RDW is
increased due to co-existant iron or vitamin B12 / folic acid deficiency.
RBC count is relatively high in relation to the Hb level. Cut off of Hb A2
for carriers lies between >3.6 and 4.0%. However, each laboratory
should establish their own cut off levels.
Borderline HbA2 values could result due to some mild Beta thalassaemia
alleles or co-inheritance of delta thalassaemia. Also, presence of alpha
thalassaemia can result in normal MCV and MCH values.
References
• Collaborative study on Thalassemia. Eds Sood SK, Madan N, Colah R, Sharma S, Apte SV.
Report of ICMR Task Force Study, Indian Council of Medical Research (1993 )
• TIF Publication : Prevention of Thalassaemias and other Hemoglobin Disorders, Vol I,
Galanello R, Elefthereiou A, Traeger-Synodinos J, Old J, Petrou, Angastiniotis M. 14

Hb 9.4gm/dl
MCV 59.2 fl
MCH 18.40 pg
RBC 5.13 mill/c.mm
RDW 16.4 %
15
DATE : 15/07/05 TIME : 11:31:14
TECH ID # 0
VIAL # 10
SAMPLE ID # 00000000000003441747
F 3.2 1.14 45914
A2 5.4 1.35 75015
P3 3.9 1.74 54148
AO 79.9 2.57 1115851
A2 7.5 3.67 91419
TOTAL AREA 1382347
30% F 3.2% A2 7.5%
20%
10%
0
0 1 2 3 4 5 6
Phenotype: Normal or mild anemia.
Genotype: HbA2 levels of >7% are generally seen in beta globin
deletional mutations like the 619bp deletion.
Ethnicity: This mutation is seen mainly among the Sindhis.
Pathophysiology: Genetic mutation causes reduced synthesis of ß
chains leading to ineffective erythropoiesis causing mildly reduced Hb.
Hb is normal or reduced to 9 - 11 gm per dl.
Laboratory findings: The MCV is less than 80 Folic Acid and MCH &
MCHC are reduced. RDW may be increased due to coexistent iron or
Vit B12/ FA deficiency. RBC count is high. Peripheral smear shows
microcytosis, marked anisopoikilocytosis, poikilocytosis, target cell,
basophilic stippling and polychromasia.
References
16

Hb 6.8gm/dl
MCV 64.3 fl
MCH 21.20 pg
RBC 3.21 mill/c.mm
RDW 20.10 %
17
Beta Thalassaemia Intermedia
DATE : 15/07/05 TIME : 11:24:36
TECH ID # 0
VIAL # 9
SAMPLE ID # 00000000000003312327
F 27.5 1.20 573184
A3 3.5 1.76 70808
AO 52.9 2.56 1268678
A2 5.1 3.68 90895
TOTAL AREA 2003565
30% F 27.6 A2 5.1%
20%
10%
0
0 1 2 3 4 5 6
Beta Thalassaemia Intermedia
Phenotype: Physical findings are similar to Beta Thalassemia major.
However, they differ markedly from the latter as the patient is not
dependent on regular blood transfusions for survival, although they may
be required occasionally. The severity of the disease is variable.
Genotype: caused by a genetic mutation in the beta-globin gene. Same
mutations may have different clinical manifestations in different patients.
Some common mutations associated with this disorder are: CAP+1A – C,
IVSI-6 T – C,+33C– G, -101C – T, etc.
Ethnicity: Same as Beta Thalassemia major.
Pathophysiology: In Beta Thalassemia Intermedia there is variable
degree of anemia, ineffective erythropoiesis & extramedullary
hematopoiesis. This condition is associated with microcytic hypochromic
anemia, poikilocytosis and extravascular hemolysis.The manifestations
of the disease usually appear later than 2 years of life.
Laboratory findings:Patients show a variable degree of anemia with
marked variation in shape and size of their red cells with hypochromia
and a mild reticulocytosis. MCV, MCH are usually markedly reduced.
There are marked red cell changes in the peripheral blood film with
normoblastemia. On Bio-Rad cation exchange HPLC, a marked increase
of fetal hemoglobin is seen with a concomitant variable reduction in
hemoglobin A,constituting usually 10-35% of the total hemoglobin. HbA2
levels may be reduced, normal or elevated. Hemoglobin electrophoresis
at acid pH and alkaline pH reveals a major band at HbF position in both
alkaline electrophoresis and acid electrophoresis
References
• Bain BJ. Hemoglobinopathy diagnosis. Blackwell Science Ltd. 2001.
• Huisman Titus H.J.A Syllabus of Human Hemoglobin Variants (1996), Marianne F.H. Carver,
and Georgi D. Efremov, published by The Sickle Cell Anemia Foundation in Augusta, GA, USA.
Copyright © 1996 by Titus H.J. Huisman.
• The Thalassemia syndromes. Weatherall & Clegg. 18

Hb 4.6 gm/dl
MCV 73.2 fl
MCH 22.80 pg
RBC 2.02 mill/c.mm
RDW 35 %
19
Beta Thalassaemia Major
DATE : 07/07/05 TIME : 11:46:43
TECH ID # 0
VIAL # 8
SAMPLE ID # 00000000000003313780
F 91.3 1.22 1267609
AO 4.4 2.50 61574
A2 4.0 3.59 50879
TOTAL AREA 2003565
30%
F 91.3% A2 4.0%
20%
10%
0
0 1 2 3 4 5 6
Beta Thalassaemia Major
Phenotype: Physical findings are related to severe anemia, ineffective
erythropoiesis, extramedullary hematopoiesis, iron overload resulting
from transfusion and increased iron absorption. Skin may show pallor
from anemia and jaundice from hyperbilirubinemia. The skull and other
bones may be deformed secondary to erythroid hyperplasia with
intramedullary expansion and cortical bone thinning. There may be
findings of cardiac failure and arrhythmia, related to either severe anemia
or iron overload.
Abdominal examination may reveal changes in the liver, gall bladder and
spleen. Hepatomegaly related to significant extramedullary hematopoiesis
typically is observed. Patients who have received blood transfusions may
have hepatomegaly or chronic hepatitis due to iron overload; transfusion-
associated viral hepatitis resulting in cirrhosis or portal hypertension also
may be seen. The gall bladder may contain bilirubin stones formed as a
result of the patient's life-long hemolytic state.Splenomegaly typically is
observed as part of the extramedullary hematopoiesis or as a
hypertrophic response related to the extravascular hemolysis. Iron
overload also may cause endocrine dysfunction, especially affecting the
pancreas, testes, and thyroid. Extremities may demonstrate skin
ulceration.
Genotype : This genetic disorder is caused by abnormalities in thebeta-
globin gene, located on chromosome 11. It is not a sex-linked genetic trait.
Same mutations may have different clinical manifestations in different
patients. The following factors are known to influence the clinical
phenotype:
 Intracellular fetal Hb concentrations
 Co-inheritance of alpha thalassemia
20

21
Common mutations in Beta thalassemia in North Western population of India
No. of
patients Beta-genes Alpha-genes
2 IVS 1 nt 5 (G-C)/cap + 1 
2 IVS 1 nt 5 (G-C)/cap + 1 
1 FS 8/9/cap + 1 
1 FS 8/9/cap + 1 
1 FS 41/42/cap + 1 
1 IVS 1 nt 5 (G-C)/IVS 1 3' end 
1 IVS 1 nt 5 (G-C)/FS 47/48 
1 IVS 1 nt 5 (G-C) Normal 
1 FS 8/9/Normal 
1 FS 8/9/Normal 
1 IVS 1 nt 5 (G-C)/-88(C-T) 
1 FS 8/9/-88 (C-T) Not analysed
The prevalence of different mutations varies significantly in different
regions of India. The IVS-1-5 mutation is the commonest mutation found
in the Indian populations and its prevalence varies from 22.8 to 81.4% in
different regions of India, being the highest in Tamil Nadu in south-
eastern India. In the north-western part of India (including the states of
Punjab, Haryana, Uttar Pradesh and Rajasthan, adjoining Delhi ), the 619
bp deletion mutation is the commonest Beta-Thalassemias mutation
observed in patients originating from Sindh, Gujarat or among the families
migrated from Pakistan during partition of the country in 1947.
Ethnicity: reported throughout the world, although more frequently in
Mediterranean, African, and Southeast Asian populations. Patients of
Mediterranean extraction are more likely to be anemic with thalassemia
trait than Africans because they have beta-zero thalassemia rather than
beta-plus thalassemia.
Pathophysiology: In beta thalassemia major there is severe anemia,
ineffective erythropoiesis & extramedullary hematopoiesis. This condition
is associated with severe, microcytic hypochromic anemia, poikilocytosis,
and extravascular hemolysis.The manifestations of the disease may not
be apparent until a complete switch from fetal to adult Hb synthesis
occurs. This switch typically is completed by the sixth month after birth.
Laboratory findings: Patients show a variable degree of anemia with
marked variation in shape and size of their red cells with hypochromia
and a mild reticulocytosis. MCV, MCH are usually markedly reduced.
There are marked red cell changes in the peripheral blood film with
normoblastemia.
On Bio-Rad cation exchange HPLC, a marked increase of fetal hemoglobin
is seen (>85%) with a concomitant marked reduction in hemoglobin A,
constituting usually less than 03% of the total hemoglobin in beta zero
thalassemia and variable in beta+ thalassemia. HbA2 levels which may
be reduced, normal or elevated. Hemoglobin electrophoresis at acid
pH and alkaline pH reveals a major band at HbF position in both alkaline
electrophoresis and acid electrophoresis.
References
and Georgi D. Efremov, published by The Sickle Cell Anemia Foundation in Augusta, GA,
USA. Copyright © 1996 by Titus H.J. Huisman.
• The Thalassemia syndromes. Weatherall & Clegg.
• Balgir RS. Burden of hemoglobinopathies in India and the challenges ahead. Current Science
2000; 79(11):1536-1547.
• Garewal, G., Fearon, C. W., Warren, T. C., Marwaha, N., Marwaha, R. K., Mahadik, C. and
Kazazian, H. H. Jr., Br. J. Hematol., 1994, 86, 372–376.
22

Hb 9.7 gm/dl
MCV 81.2 fl
MCH 25.6 pg
RBC 3.79 mill/c.mm
RDW 17.6 %
23
HbE Heterozygous
DATE : 18/07/05 TIME : 11:41:33
TECH ID # 0
VIAL # 18
SAMPLE ID # 00000000000003494626
F 0.4 1.10 7042
P2 3.4 1.34 6067
P3 3.3 1.74 59652
A0 59.3 2.57 1057398
A2 32.3 3.71 521403
30% TOTAL AREA 1705562
F 0.4% A2 32.3%
20%
10%
0
0 1 2 3 4 5 6
HbE Heterozygous
Phenotype: Normal
Genotype: ß chain mutation α2
ß226GluLys (GAGAAG).
Ethnicity: Most common hemoglobin variant in South East Asia and the
second most prevalent hemoglobin variant worldwide.
Pathophysiology: HbE mutation partially activates a cryptic splice site in
Exon 1, resulting in a proportion of abnormally spliced mRNA. Thus, less ßE
globin is synthesized. In addition, In vitro experiments have shown that HbE
is mildly unstable and may be susceptible to oxidant damage.
Laboratory findings: No anemia or reticulocytosis is noted. Microcytosis
(low MCV) and hypochromia (low MCH) are present. However, MCV and
MCH may be normal in some cases. Hemoglobin-E heterozygotes have
about 30% HbE. HbE elutes in the HbA2 window. The percentage of HbE
may be low in case of coexistent iron deficiency and αthal mutation.
References
24

Hb 11.1 gm/dl
MCV 63.1 fl
MCH 19.9 pg
RBC 5.7 mill/c.mm
RDW 16.7 %
25
HbE Homozygous
DATE :08/10/05 TIME:31:12:41
TECH ID # 0
VIAL # 65
SAMPLE ID # 00000000000003819565
F 2.5 1.12 56895
P3 4.6 1.76 103440
AO 3.4 2.28 76818
A2 84.9 3.68 1734019
TOTAL AREA 1971172
F 2.5% A2 84.9%
30%
20%
10%
0
0 1 2 3 4 5 6
HbE Homozygous
Phenotype: Normal or features of mild hemolytic anemia with mild anemia,
jaundice and splenomegaly.
Genotype: HbE (ß26 GluLys)/ HbE (ß26 GluLys).
Ethnicity: HbE is common in South East Asia.
Pathophysiology: HbE mutation partially activates a cryptic splice site in
Exon 1, resulting in a proportion of abnormally spliced mRNA. Thus, less ßE
globin is synthesized. In addition, In vitro experiments have shown that HbE
is mildly unstable and may be susceptible to oxidant damage.
Laboratory Findings : Prominent microcytosis (low MCV) and hypochromia
(low MCH) with target cells and leptocytes present. Normal or mildly
reduced hemoglobin. Normal reticulocyte count.HbE accounts for 85-95 %
of the hemoglobin. HbF is usually mildly increased or may be normal.
Red cell survival is decreased slightly and osmotic fragility is decreased.
Family studies and clinical correlation will help confirm diagnosis.
References
• Wintrobes Clinical Hematology. 11th Edition
26

Hb 6.9 gm/dl
MCV 75.1 fl
MCH 21.1 pg
RBC 3.26 mill/c.mm
RDW 33.6 %
27
Double Heterozygous for HbE and
DATE : 18/07/05 TIME : 11:01:45
TECH ID # 0
VIAL # 12
SAMPLE ID # 00000000000003464962
F 29.8 1.18 388489
P3 2.4 1.76 30761
AO 5.6 2.26 71107
30% Unknown 1 4.2 2.51 52586
A2 54.8 3.70 627538
TOTAL AREA 1170481
F 29.8% A2 54.8%
20%
10%
0
0 1 2 3 4 5 6
Double Heterozygous for HbE and
Phenotype: HbE -ß - thalassaemia shows remarkable clinical variability. At
its worst it is very similar to the severest forms of Beta thalassaemia. There
may be cases with very mild features like growth retardation, mild anaemia
and mild organomegaly which do not require blood transfusion. A variety of
conditions with intermediate severity are also found in the population.
Genotype: HbE mutation / Beta thalassaemia mutation.
Ethnicity: Common in South East Asia.
Pathophysiology: Less production of ßE
globin, unstable nature of HbE
and less production of Beta globin gene due to Beta thalassaemia mutation
are responsible for thalassaemic features.
Labaratory findings: Haemoglobin is variable depending on the clinical
severity. Sometimes severe anaemia is present from early life. Many
patients with milder features maintain haemoglobin level in the range of
5 – 7 g/dl. Marked anisopoikilocytosis, hypochromia, microcytosis, target
cells, polychromasia, and basophilic stippling present. Nucleated RBC‘s are
almost invariably present and may reach extremely high levels after
splenectomy. The red cell survival time is considerably shortened.
Increased HbF (15 - 50 %) and HbE ( 50 - 80 %) are present in untransfused
cases. However the percentage of HbF decreases in post transfused
patients. The percentage of HbA varies according to the severity ofBeta
mutation. HbA may be upto 25 % in case of ß+
mutation.
References
• Wintrobes Clinical Hematology. 11th Edition
28

Hb 11.4 gm/dl
MCV 76.8 fl
MCH 24.21 pg
RBC 4.66 mill/c.mm
RDW 18.8 %
29
Sickle Cell Heterozygous
DATE : 20/09/05 TIME : 14:56:37
TECH ID # 0
VIAL # 46
SAMPLE ID # 00000000000003718328
F 0.7 1.12 11220
P2 2.7 1.37 43774
P3 1.7 1.72 28291
AO 52.0 2.56 849176
A2 3.6 3.63 52570
S-WINDOW 39.1 4.46 638801
F 0.7 % A2 3.6%
20%
10%
0
0 1 2 3 4 5 6
Sickle Cell Heterozygous
Phenotype: Sickle cell carriers are clinically asymptomatic except when
exposed to low oxygen tension eg. at high altitudes .
Genotype: ßS
ßA
glutamic acid replaced by valine at 6th position of ß chain.
Ethnicity : Details on gene prevalance as per Annexure I
Pathophysiology: When exposed to low oxygen tension, it may lead to
vaso-occulsive crises in the patients. Hematuria and deficient urine
concentration ability are most commonly associated renal abnormalities in
Sickle cell trait.
Laboratory findings: Usually RBC’s are normocytic, normochromic
The hemoglobin is in normal range. The patient may be hypochromic, microcytic
or macrocytic in association with nutritional anemia and Thalassemia. On the
HPLC , HbS ranges between 30- 40%. HbS is reduced and is below 30% in
association with αthalassemia with raised RBC count, low MCV and MCH.
HbA2 may be elevated due to co-elution of glycated HbS. Hemoglobin
electrophoresis shows AS pattern.
References
• A.V. Shrikhande et al (personal communication) Sickle cell disease in central India.
• A.V. Shrikhande et al Hematological profile of sickle cell disease in central India.( sent
for publication)
• A.V. Shrikhande et al Population screening for sickle cell diseasse in various ethnic
groups of Vidarbha (central India). (sent for publication) 30

Hb 6 gm/dl
MCV 82.8 fl
MCH 25.4 pg
RBC 2.35 mill/c.mm
RDW 30.8 %
31
HbS Homozygous
DATE :29/09/05 TIME :12:34:49
TECH ID # 0
VIAL # 23
SAMPLE ID # 00000000000003718386
F 18.1 1.18 286820
AO 1.8 2.33 28604
A2 2.8 3.63 38007
S-WINDOW 77.1 4.46 1208951
TOTAL AREA 1562382
30% F 18.1% A2 2.8%
20%
10%
0
0 1 2 3 4 5 6
HbS Homozygous
Phenotype: Sickle cell anemia is a chronic hemolytic anemia, where
normal adult hemoglobin is replaced by sickle hemoglobin. As a result
RBC’s assume sickle shape and this leads to various clinical manifestations.
Genotype:ßS
ßS
(Glutamic acid is replaced by valine at 6th
position of
ß chain.)
Ethnicity Data – Details on gene prevalence as per Annexure 1
Pathophysiology: Tendency of deoxy HbS to undergo polymerization
underlies the innumerable clinical expression of sickling syndromes.
Clinical symptoms of sickling disorders could be mild or severe. The mild
conditions include HbS/ß+
thalassemia, homozygous HbS (HbSS
associated with Arab-Indian haplotype), HbS/ ß thalassemia and HbSC
disease. The severe sickling disorders are HbS/ß0
thalassemia, HbS trait
associated with ß globin chain variants like HbSD, HbS O-Arab, rarely
doubly substituted variants and HbSS associated Cameroon, Benin, and
Bantu haplotypes. The asymptomatic sickling disorders include HbS trait,
double heterozygous state of HbS with HPFH.
Laboratory findings –Anaemia is moderate to severe with hemoglobin
ranging from 4-8 gm/dl. RBC’s are normocytic normochromic. Associated
nutritional deficiency may give rise to hypochromic, microcytic anemia or
macrocytic blood picture. ISC (irreversible sickle cells) make upto 5-10% of
red cells on peripheral smear. Target cells predominate when there is
association with thalassemias. Reticulocyte count is raised and ranges with
degree of hemolysis. Polymorphonuclear leukocytosis is associated more
with vaso-occulusive crises. Platelet count is normal or raised. Associated
hypersplenism and sometimes vaso-occlusive crises may give rise to
thrombocytopenia. RBC indices will vary as per associated nutritional
deficiency and other hemoglobin abnormalities. HbA2 level is either normal
or slightly raised (HbSS, HbSß) It is observed that HbF value is very high in
central India. HbF ranges between 15% to 25% in central India and 11- 20%
in Orissa. HPLC Pattern (Average values): HbS-77%,HbA2 – 2.1%, HbF – 20%.
Family studies and clinical correlation will help confirm diagnosis.
References
• BC Kar et al. Sickle cell disease in Orissa state, India.Lancet, 1986;22:1198-120
• BC Kar and Satyabhama Devi.Clinical profile of sickle cell disease in Orissa. Indian J Pediatr, 1997; 64:73-77.
• Renzo Galanello et al. Prevention of thalassemias and other hemoglobin disorders, Vol 1, 2003 . 32

Hb 9.2 gm/dl
MCV 77 fl
MCH 23.9 pg
RBC 3.87 mill/c.mm
RDW 21.8 %
33
Double Heterozygous for HbS and
DATE :26/10/05 TIME :14:13:55
TECH ID # 0
VIAL # 40
SAMPLE ID # 00000000000003866941
F 6.5 1.14 109797
P3 0.2 1.68 2753
Unknown 1 0.8 2.14 13293
AO 4.7 2.48 79425
A2 6.3 3.63 100318
S-WINDOW 81.0 4.46 1363085
F 6.5% A2 6.3%
20%
10%
0
0 1 2 3 4 5 6
Double Heterozygous for HbS and
Phenotype: Clinically Symptomatic
Genotype: HbßS
/ Hbß0
Ethnicity: Details as per Annexure 1
Pathophysiology: Clinical manifestations resemble sickle cell anemia &
splenomegaly persists all through adult life.
Laboratory Findings: Laboratory parameters of both sickle cell anemia
and thalassemia are observed. Red cells are microcytic, hypochromic and
target cells predominate blood picture. Red cell indices like MCV, MCH,
MCHC are decreased with increased RBC count. Hb electrophoresis
shows SFA2 pattern.
HPLC Pattern (Average values): Hb S- 89% , Hb A2 - 5%, HbF - 6.5%.
Family Studies and clinical correlation will help confirm diagnosis.
References
• A.V. Shrikhande et al Population screening for sickle cell diseasse in various ethnic groups of
Vidarbha (central India). ( sent for publication)
• Renzo Galanello et al. Prevention of thalassemias and other hemoglobin disorders Vol 1, 2003.
34

Hb 12.1 gm/dl
MCV 74.3 fl
MCH 23.4 pg
RBC 5.19 mill/c.mm
RDW 21.9 %
35
Double heterozygous for
HbS and HPFH
DATE :26/10/05 TIME :14:13:55
TECH ID # 0
VIAL # 10
SAMPLE ID # 00000000000003663590
F 28.0 1.20 605947
AO 2.5 2.33 53106
A2 4.6 3.64 91516
S-WINDOW 64.7 4.47 1399684
TOTAL AREA 2150253
30% F 28.0% A2 4.6%
20%
10%
0
0 1 2 3 4 5 6
Double heterozygous for
HbS and HPFH
Phenotype: Usually asymptomatic or resemble mild sickle cell disease
Genotype: ßS
/ HPFH
Ethnicity : Details as per Annexure 1
Pathophysiology: Higher levels of Hb F in this association leads to a
heterogeneous disorder that is extremely mild.
Laboratory Findings: Red cells are mainly normocytic normochromic,
with few irreversibly sickled cells. Red cell indices will be normal or
reduced when associated with αthalassemia. Hb electrophoresis shows
SFA2 pattern. Kleihaur test will show pan cellular distribution of HbF.
Percentage of HbF will range between 20-30% HPLC Pattern
(Average values) HbS- 62%, HbA2 -2..1%, HbF–35.9%.
It is sometimes difficult to accurately diagnose the phenotypes of HbSS ,
HbS/ ß,0
HbS/ ß-thalassaemia and HbS/ HPFH based on a haematlogical
assessment of the patient and family studies are therefore recommended.
Coexisting Iron Deficiency can result in abnormal indices.
References
• Renzo Galanello et al. Prevention of thalassemias and other hemoglobin
disorders .vol 1, 2003.
• Wintrobe s clinical hematology Eleventh Edition 2004 36

Hb 11.1 gm/dl
MCV 78.8 fl
MCH 25.5pg
RBC 4.34 mill/c.mm
RDW 14.2 %
37
Double Heterozygous for HbS & HbE
DATE :20/07/05 TIME:10:56:31
TECH ID # 0
VIAL # 10
SAMPLE ID # 00000000000003468895
F 4.3 1.12 79762
P3 1.8 1.75 32664
A0 5.2 2.30 95269
A2 33.4 3.68 557750
S-WINDOW 53.9 4.48 992167
TOTAL AREA 1757612
30% F 4.3% A2 33.4%
20%
10%
0
0 1 2 3 4 5 6
Double Heterozygous for HbS & HbE
Phenotype: Asymptomatic. Mild anaemia may be present. Sickling crisis is
uncommon.
Genotype: Co-inheritance of ßs
and ßE
Ethnicity: Uncommon condition. Usually seen among Asians.
Pathophysiology: Presence of haemoglobin E and haemoglobin S is
responsible for mild anaemia and occasional sickling crisis particularly
during pregnancy and air travel.
Laboratory findings: Mild anaemia and microcytosis present. Target cells
are common but sickle cells are rare. Reticulocytes are usually mildly
elevated. Both HbS and HbE fractions are > 30%.
References
• Wintrobes Clinical Hematology. 11th Edition Orphanet
38

Hb 13.4 gm/dl
MCV 83.4 fl
MCH 29.3 pg
RBC 4.57 mill/c.mm
RDW 14.7 %
39
HbD- Punjab Heterozygous
DATE :19/10/05 TIME:12:38:47
TECH ID # 0
VIAL # 24
SAMPLE ID # 00000000000003752390
P2 3.5 1.33 80769
P3 2.4 1.70 54957
Unknown 1 1.6 2.06 37692
AO 51.6 2.52 1196711
A2 1.6 3.61 34572
S-WINDOW 39.2 4.11 907140
F 0.0% A2 1.6%
20%
10%
0
0 1 2 3 4 5 6
HbD- Punjab Heterozygous
Phenotype : Normal / Asymptomatic carrier
Genotype: ß chain mutation at ß121 (GH4) GluGln (GAA-CAA).
Ethnicity: Prevalence of 3% in the general north Indian population is found.
It is the fourth common hemoglobin variant found in the world and there
are reports in the literature from the Mediterranean region.
Pathophysiology: It does not cause any abnormality in the heterozygous
state.
Laboratory findings:The individual is normal with completely normal
hemogram findings. On Bio -Rad cation exchange HPLC, HbD Punjab
elutes in the “D - window” with the retention time window of 4.13 to
4.15, which is clearly demarcated from HbS peak. The abnormal
hemoglobin constitutes usually between 33-43% of the total hemoglobin.
On hemoglobin electrophoresis at alkaline pH, HbA and band in the S/D
region is detected. In an acidic pH, it migrates with the adult hemoglobin.
Molecular diagnosis is made by amplification of DNA at that region and
digesting the PCR product with EcoR1. In HbD-Punjab the mutation causes
the EcoR I site at codon 121 to be abolished. Appropriate controls should
be run with each test batch.
References
• Trent RJ, Davis B, Wilkinson T and Kronenberg H. Identification of ? variant hemoglobins by DNA
restriction endonuclease mapping. Hemoglobin 1984;8:443-462
• Zeng YT, Huang SZ, Zhou LD, Huang HJ, Jiao CT, Tang ZG, Chen CY. Identification of hemoglobin
D Punjab by gene mapping. Hemoglobin. 1986;10(1):87-90.
40

42
References
• Worthington S, Lehmann H. The first observation of HbD Punjab beta zero thalassemia in
an English family with 22 cases of unsuspected beta zero thalassemia minor among its
members. J Med Genet. 1985 Oct; 22(5):377-81.
41
Double Heterozygous for HbD Punjab
and Beta Thalassaemia Trait
Hb 12.2 gm/dl
MCV 65.1 fl
MCH 20.2 pg
RBC 6 mill/c.mm
RDW 16.5 %
Double Heterozygous for HbD Punjab
Punjab and Beta Thalassaemia Trait
Phenotype: Asymptomatic carrier with mild anemia; similar to ß
thalassemia trait.
Genotype: ß chain mutation at ß121 (GH4) Glu Gln along with the
specific beta thalassemia mutation.

Ethnicity- This combination of double heterozygosity for HbD Punjab and
ßTT is found in north Indians.
Pathophysiology- It does not cause any abnormality and behavior is
similar to an individual with ßTT.
Laboratory findings:The individual may be mildly anemic and the red cell
indices are those of a ß thalassemia trait with hypochromic microcytosis
(MCV, MCH)and raised red blood cell count ( RBC). HPLC shows a
major band of HbD of 80 -90% with a mild elevation of HbF 3-6%. HbA
peak is absent. On hemoglobin electrophoresis at alkaline pH the HbA is
seen to be absent and band in the S/D region is detected along with a
raised HbA2. The molecular diagnosis for HbD Punjab by PCR-RFLP shows
a heterozygous pattern with all the mutations encountered in India, except
when the individual co-inherits the 619 bp deletion (found in Gujratis,
Sindhis and Punjabis), a homozygous pattern is encountered. This is
because with the 619 bp deletion the other allele does not contain the HbD
site amenable for PCR amplification and digestion. Family studies are
important to confirm diagnosis.
0
0
1 2 3 4 5 6
10%
20%
30%
DATE :07/10/05 TIME:14:58:29
TECH ID # 0
VIAL # 34
SAMPLE ID # 00000000000003810314
F 1.3 1.12 21488
Unknown 1 0.9 1.23 14715
P3 0.1 1.73 2377
AO 4.8 2.08 76119
Unknow 2 1.0 3.00 15586
A2 1.9 3.64 27623
D-WINDOW 89.9 4.12 1437563
TOTAL AREA 1595471
F 1.3% A2 1.9%

Hb 7.50 gm/dl
MCV 107.50 fl
MCH 33.80 pg
RBC 2.21 mill/c.mm
RDW 25.60 %
43
Double Hetrozygous forHbS & HbD
DATE :02/07/05 TIME:14:43:11
TECH ID # 0
VIAL # 41
SAMPLE ID # 00000000000003468787
F 26.6 1.17 282682
AO 1.5 2.07 15370
Unknown 1 0.8 2.30 8839
Unknown 2 0.5 2.84 5222
A2 1.5 3.61 14728
D-WINDOW 39.5 4.12 414806
S-WINDOW 29.2 4.46 306448
F 26.6% A2 1.5%
20%
10%
0
0 1 2 3 4 5 6
Double Hetrozygous for HbS & HbD
Phenotype: Though individually both HbS and HbD Punjab traits are
asymptomatic, double heterozygosity for HbD with HbS results in a
moderately severe clinical presentation of sickle cell anemia.
Genotype: ß chain mutations at ß121 (GH4) GluGln in HbD Punjab and
ß6 glutamic acid to valine in HbS trait.
Ethnicity: Uncommon condition encountered in Punjabis and a higher
frequency in Muslims in consanguineous marriages.*
Pathophysiology: The ß121 glutamine residue increases the
polymerization of Hb S.
Laboratory findings: Patients present with chronic mild to moderate
anemia and hemoglobin in the range of 60-100 gms/l with increased
reticulocytes. On the HPLC, separate peaks of HbD in the “D window” and
HbS in the “S window” is visualized. HbF peak is significant with
percentage ranging from 10 - 20%. A single band in the SD region on
hemoglobin electrophoresis at alkaline pH is noted with mild excess of HbF.
Separate bands can be seen of HbD and HbS at acidic pH.
References
• Adachi K, Kim J, BallasS, Surrey S,AsakuraT.Facilitationof Hb S polymerizationby the substitution
of Glu for Gln at ßeta 121. J Biol Chem. 1988Apr 25;263(12):5607-10.
• StrutegeonP,Itano HAand Bergen WR.Clinicalmanifestationsof inheritedabnormalhemoglobin.I
The interactionof hemoglobin-Swith hemoglobinD. Blood 1955;10:389-396
• WorthingtonS, Lehmann H.The first observationof Hb D Punjab beta zero thalassemiain an English
familywith 22 cases of unsuspectedbeta zero thalassemiaminor amongits members.J Med Genet.
1985;22(5):377-81.
* As perresearch findingsof Dr.Reena Das , PGI Chandigarh 44

Hb 10.9 gm/dl
MCV 73.5 fl
MCH 24.2 pg
RBC 4.5 mill/c.mm
RDW 17.2 %
45
HbD-Iran Heterozygous
DATE :06/10/05 TIME:13:50:05
TECH ID # 0
VIAL # 36
SAMPLE ID # 00000000000003810291
F 0.3 1.11 5850
P2 2.3 1.33 39554
P3 3.5 1.67 58420
AO 46.1 2.52 776974
A2 46.3 3.56 698132
TOTAL AREA 1578930
30% F 0.3% A2 46.3%
20%
10%
0
0 1 2 3 4 5 6
HbD-Iran Heterozygous
Phenotype: Normal
Genotype: Chain mutation α
2ß2 22 Glu- Gln(GAA-CAA.)
Ethnicity : First described in 1973. Found in Iranian and Pakistani families; a
Jamaican Black, and several families in Northern Calabria, Italy. In India it is
seen in the northwestern region, particularly Punjab.
Pathophysiology: In the heterozygous state there is no abnormality. In the
homozygous state, however, Hb D-Iran is a relatively benign condition with mild
microcytic anemia, poikilocytosis and minimal hemolysis.
usually normal unless associated with an underlying iron deficiency. On Bio -Rad
Cation exchange HPLC, abnormal hemoglobin elutes in the HbA2 retention time
window, constituting usually more than 40% of the total hemoglobin
(c.f. Heterozygous A/E state where the percentage of abnormal hemoglobin is less
than 40%). Hemoglobin electrophoresis at acid pH and alkaline pH reveals
migration of abnormal hemoglobin to Hb S/D/G position in alkaline electrophoresis
and to A position in acid electrophoresis (c.f. On hemoglobin electrophoresis at
acid pH and alkaline pH reveals migration of abnormal hemoglobin to Hb C/E/O
position in alkaline electrophoresis and to A position in acid electrophoresis).
References
• Rohe, R.A., Sharma, V.,and Ranney, H.M.: Blood, 42:455,1973.
• HuismanTitus H.J.ASyllabus of Human HemoglobinVariants (1996), Marianne F.H.Carver, and
Georgi D. Efremov, publishedbyThe SickleCellAnemiaFoundationinAugusta, GA, USA.
Copyright © 1996 byTitus H.J. Huisman.
• Thornburg CD, ZimmermanSA, SchultzWH,WareRE.An infant with homozygous hemoglobinD-
Iran. 46

Hb 13.5 gm/dl
MCV 96.1 fl
MCH 32.9 pg
RBC 4.1 mill/c.mm
RDW 13.2 %
47
HbQ-India Heterozygous
DATE :26/06/05 TIME:13:02:04
TECH ID # 0
VIAL # 26
SAMPLE ID # 00000000000003499409
P2 4.1 1.34 88604
P3 2.6 1.72 55358
AO 71.4 2.56 1549482
A2 2.8 3.64 54233
Unknown 1 19.1 4.79 413575
TOTAL AREA 2161252
30% F 0.0% A2 2.8%
20%
10%
0
0 1 2 3 4 5 6
HbQ-India Heterozygous
Phenotype : Normal in the heterozygotes
Genotype: Mutation in the alpha1 gene: GAC->CAC at codon 64.
Ethnicity: Trincao et al. detected 4 instances of Hb-Q in a survey of 1843
Indians in Goa. Sukumaran et al. recorded a new Hb-Q
α(aspartic acid -
histidine), or Hb-Q (India), in two Sindhi families in Mumbai. Sukumaran
et al. recorded one case of Q-Thalassaemia Major and 2 cases of
Q-thalassaemia minor in Sindhi families in Mumbai.
Pathophysiology: HemoglobinQ-India in heterozygous state with
hemoglobin A does not show any abnormality.
normal or may be marginally reduced. Abnormal hemoglobin is seen as an
unknown peak in the retention time window of 4.77 min + 0.01, constituting
usually 8.7-23.3% of the total hemoglobin on the Variant hemoglobin testing
system. Hemoglobin electrophoresis at acid pH and alkaline pH reveals
migration of abnormal hemoglobin to Hb S/D/G position in alkaline
electrophoresis and between A and S positions in acid electrophoresis.
References
• Huisman Titus H.J. A Syllabus of Human Hemoglobin Variants (1996), Marianne F.H. Carver,
• The Thalassemia syndromes. Weatherall & Clegg. •Balgir RS. Burden of
hemoglobinopathies in India and the challenges ahead. Current Science 2000;
79(11):1536-1547.
• Molchanova, T.P., Pobedimskaya, D.D., and Huisman, T.H.J.: Br. J. Haematol., 88:300, 1994
48

A2
Unknown
F
Hb 10.7 gm/dl
MCV 79.4 fl
MCH 27.2 pg
RBC 3.94 mill/c.mm
RDW 24.5 %
49
Double Heterozygous for HbQ-India
DATE :30/09/05 TIME:14:41:02
TECH ID # 0
VIAL # 34
SAMPLE ID # 00000000000003774108
F 22.3 1.17 308303
P2 6.3 1.33 86755
P3 3.2 1.71 44003
AO 53.0 2.54 731908
6.8 3.66 85742
30% 1 8.0 4.75 110975
TOTAL AREA 1367686
22.3% A2 6.8%
20%
10%
0
0 1 2 3 4 5 6
Double Heterozygous for HbQ-India
Phenotype: Features are like that of Beta Thalassemia trait.
Genotype: Mutation in the alpha1 gene: GAC->CAC at codon 64 with beta
thalassemia mutations.
Ethinicity: Sukumaran et al. recorded one case of Q-thalassaemia major and
2 cases of Q-thalassaemia minor in Sindhi families in Mumbai.
Pathophysiology: Hemoglobin Q-India in heterozygous state with beta
thalassemia trait shows featuresof beta thalassemia trait.
Laboratory findings: There is mild anemia. MCV, MCH, are reduced
(features of beta thalassemia trait). Abnormal hemoglobin is seen as an
unknown peak in the retention time window of 4.77+ 0.01, constituting
usually 8.7-23.3% of the total hemoglobin. In addition to this hemoglobin A2
is >4.0% (4.0-7.5%) Hemoglobin electrophoresis at acid pH and alkaline pH
reveals migration of abnormal hemoglobin to Hb S/D/G position in alkaline
electrophoresis and between A and S positions in acid electrophoresis.
References
• Balgir RS. Burden of hemoglobinopathies in India and the challenges ahead. Current Science
2000; 79(11): 1536-1547.
• Molchanova, T.P., Pobedimskaya, D.D., and Huisman,T.H.J.: Br. J. Haematol., 88:300, 1994
50

Hb 11.8 gm/dl
MCV 69.6 fl
MCH 22.3 pg
RBC 5.3 mill/c.mm
RDW 16.8 %
hi
F 6
51
Hb Lepore Trait
DATE :08/10/05 TIME:13:48:57
TECH ID # 0
VIAL # 30
SAMPLE ID # 00000000000003896299
F 6.3 1.12 86688
P2 4.5 1.32 59780
P3 3.6 1.67 48029
A0 73.9 2.51 981102
A2 11.1 3.40 131446
TOTAL AREA
30% .3%A2 11.1%
20%
10%
0
0 1 2 3 4 5 6
Hb Lepore Trait
Phenotype : Features indistinguishable form beta thalassemia trait.
Genotype: Unequal cross-over during meiosis with deletion of the 3' part of
the gene and 5' part of the ß gene leading to formation of a ß fusion
gene.
Et nicity : Named after the first patient in whom the variant hemoglobin
was recognized. Seen in low frequency in a variety of ethnic groups
including Italians, Greeks and of African ancestry. In India, Agarwal and
Mehta have reported few cases from Mumbai.
Pathophysiology : The Hb Lepore disorders show feature of deltabeta
thalassaemia. These conditions result from the production of deltabeta fusion
genes which direct the synthesis of deltabeta fusion chains. They arise by
unequal crossing over between the delta and beta globin genes, depending
upon the position and structure of abnormal crossing over. Three different
types of Lepore Hemoglobins have been identified; namely Hemoglobin
Lepore Boston, Hemoglobin Lepore Hollandia and Hemoglobin
LeporeBaltimore. The point of fusion of the two polypeptide chains and the
proportion of the and ß in the ß chain varies in different types of Lepore
Hemoglobins.
Laboratory findings : There is anemia and reticulocytosis. MCV, MCH are
reduced. Heterozygotes with hemoglobin Lepore trait have abnormal
hemoglobin in the HbA2 retention time window, constituting usually 10-15%
of the total hemoglobin. Hemoglobin electrophoresis at acid pH and alkaline
pH reveals migration of abnormal hemoglobin to Hb S/D/G position in
alkaline electrophoresis and to A position in acid electrophoresis. Family
studies and molecular studies will help confirm diagnosis
References
• Huisman Titus H.J. A Syllabus of Human Hemoglobin Variants (1996), Marianne F.H.
Carver, and Georgi D. Efremov, published by The Sickle Cell Anemia Foundation in
Augusta, GA, USA. Copyright © 1996 by Titus H.J. Huisman.
• The Thalassemia syndromes. Weatherall & Clegg. 52

53
Alpha Thalassaemia
Phenotype: Alpha thalassaemia is the result of defective production of the
alpha chains of hemoglobin.
Genotype: The alpha gene is duplicated and one individual inherits two pairs of
genes from both parents. The normal alpha genotype is written as alpha alpha /
alpha alpha ( α
α
/
α
α).The following genotypic variations are known:
Ethnicity: It is mainly found in the South-east Asian countries, Middle East,
Mediterranean countries and centralAfrican countries
i) Deletion of one alpha globin geneof one of the chromosomepairs produces
the haemotologically silent carrier state,genotype : alpha -/ alpha alpha
(-α
/αα).
ii) Deletion of two alpha globin genes produces the alpha thalassaemia trait.
This is associated with mild hypochromic anaemia and may result from
either loss of both pairs of genes fromone chromosomegiving --/alpha
Alpha (- -/
α
α
)genotype or loss of one gene fromeach chromosomegiving
rise to a - alpha/- alpha (-α
/-α
) genotype.The formercondition is prevalent
amongAsians especially in S.E.Asia and India.Thelatter condition is most
prevalent amongAfricans.
iii) Deletion of three alpha globin genes results in Hemoglobin H disease with
genotype of - -/ - alpha (--/-α).Alpha chain synthesis is severely suppressed
so thatunstable tetramersof excess beta globin, HbH (ß4) are formed.
Clinically, it resembles Beta Thalassaemia Intermedia.
iv) Deletion of all four alpha globin genes produces haemoglobin Bart's
Hydrops syndrome with genotype --/--. It is incompatible with life with foetal
death occurring at about 38 weeks.
Ethnicity:Itis mainly foundin theSouth-eastAsian countries,Middle East,
Mediterranean countriesand centralAfrican countries.In India, this
condition is seen mainly in tribals and sporadically in caste population.
Pathophysiology - Imbalance of globin chain ratio with excess of ß chain
production with resultant precipitation of ß chains and haemolysis and
ineffective haemopoiesis..
Laboratory findings:
i
) αcarrier state: Microcytic hypochromic anaemia. Normal HPLC with low
HbA2 level.
ii) Hb H Disease: Microcytic hypochromic anaemia with anisocytosis and
poikilocytosis & Hb H inclusion bodies in peripheral blood .In HPLC for
VariantTM
ßthalassaemia shortprogramme,HbH and Hb Bartselute before
thepoint of integration showing peaks beforethe HbF peak.
References
• The Thalassaemia syndromes : Weatherall & Clegg.
54

Hb 6.60 gm/dl
MCV 87.5 fl
MCH 21.1 pg
RBC 3.13 mill/c.mm
RDW 37.2 %
55
HbH
DATE :27/10/05 TIME:11:43:20
TECH ID # 0
VIAL # 7
SAMPLE ID # 00000000000003409782
F 0.5 0.97 586
P2 2.2 1.36 2521
P3 4.0 1.74 4615
AO 92.1 2.62 105548
A2 1.2 3.33 1200*
TOTAL AREA 1144730
30% F 0.5% A2 1.2%
20%
10%
0
0 1 2 3 4 5 6
HbH
Phenotype : Variable, mild anemia to chronic hemolytic anemia
Genotype : ( - - / - α),( - - / α
α
),(- - /
αcs
α),(
αα
T
/αα
T
)
Ethnicity : Southeast Asia, the Mediterranean islands, parts of Middle East
rarely in people ofAfrican descent.
Pathophysiology - Defect in alpha chain synthesis results in an accumulation
of gamma and beta chains. Tetramers of αchains (Hb Bart's) and ß chains ( Hb
H ) are unstable . They have high oxygen affinity, thus are useless for oxygen
transport.Theyoxidize easilyand precipitatewithcellaging.
Laboratory findings : Variable anemia , Hb between 7-10 g / dl, Reticulocytes
range from 5-10 % .Red cells are microcytic, hypochromic, targeting, small
red cell fragments seen. HbH precipitation can be seen with 1% brilliant cresyl
blue. At birth 20-40 % Hb Bart's is found. Later Hb H is seen between
5 - 40 %. Hb A 2 is low (average 1.55 %) ß : αchain synthetic ratio is between
1.5-3.5.Fresh samples may show anormalMCV.
Note : On the VariantTM
Hemoglobin Testing system, a significant peak appears
in the first minute of elution. However, such a peak is only suggestive of alpha
thalassaemia / HBH condition. Other tests like inclusion body tests and DNA
studiesare requiredforconfirmation.
References
56

Hb 15.2 gm/dl
MCV 84.6 fl
MCH 28.7 pg
RBC 5.31 mill/c.mm
RDW 13.6 %
57
Elevated P3 peak
DATE :27/10/05 TIME:15:37:17
TECH ID # 0
VIAL # 37
SAMPLE ID # 00000000000003857407
F 1.6 1.02 33087
P2 4.9 1.32 102325
P3 26.4 1.78 552228
AO 64.9 2.52 1355951
A2 2.0 3.60 40403
TOTAL AREA 1240214
30% F 1.6% A2 2.0%
20%
10%
0
0 1 2 3 4 5 6
Elevated P3 peak
Phenotype: The peak within the P3 retention time window is usually due to
number of alpha and/or beta variants including J-Norfolk[alpha57(E6)Gly-
>Asp] or J-Anatolia/J-Mexico/J-Meerut), Camden,Grady, N-Baltimore, Fannin
Lubbock,J-Baltimore, J-Bangkok which elute at that position.
Genotype: caused by genetic mutations in either the alpha-globin gene in
alpha variants or the beta-globin gene in beta variants. Mutations differ in
patients with different variants.
VARIANT NAME RETENTION TIME ASSOCIATED MUTATION
Hb J-Oxford 1.60  15Gly3Asp
Hb J-Anatolia 1.75  61Lys3Thr
Hb J-Mexico 1.74 54Gln3Glu
Hb J-Meerut 1.88  120Ala3Glu
Hb J-Toronto 1.94  5Ala3Asp
Hb Hope 1.39  136Gly3Asp
Hb Camden 1.50 131Gln3Glu
Hb Austin 1.68  40Arg3Ser
Hb N-Baltimore 1.70  95Lys3Glu
Hb Fukuyama 1.72  77His3Tyr
Hb Fannin-Lubbock 1.75  119Gly3Asp
Hb J-Bangkok 2.02  56Gly3Asp
Ethinicity: reported throughout the world.
Pathophysiology: Usually asymptomatic.
Laboratory findings: Patients may show a mild anemia. MCV, MCH are
normal to reduced. J-Meerut shows a mild increase in Oxygen affinity
compared to the normal control in addition to an increase in RBC count.
On the Bio-Rad cation exchange HPLC, a significant peak is seen in the P3
window at a retention time of 1.3 minutes. Hemoglobin electrophoresis shows
a fastmoving band anodal toHbAatalkalinepH.
References
• Huisman Titus H.J.A Syllabus of Human Hemoglobin Variants (1996), Marianne F.H.
Carver, and Georgi D. Efremov, published by The Sickle Cell Anemia Foundation in
Augusta, GA, USA. Copyright © 1996 by Titus H.J. Huisman.
• Balgir RS. Burden of hemoglobinopathies in India and the challenges ahead. Current
Science 2000;79(11):1536-1547.
• JoutovskyA, et al. HPLC retention time as a diagnostic tool for hemoglobin variants and
hemoglobinopathies: A study of 60,000 samples in a clinical diagnostic laboratory. Clinical
Chemistry Vol 50(10), 2004:1736-1747
58

Hb 2.8 gm/dl
MCV 142.10 fl
MCH 47 pg
RBC 0.58 mill/c.mm
RDW 43.40 %
59
Megaloblastic anemia
DATE :02/11/05 TIME:11:16:23
TECH ID # 0
VIAL # 6
SAMPLE ID # 0000000000000300732
F 2.3 1.12 36203
P2 3.2 1.32 50085
P3 3.8 1.69 59650
AO 86.9 2.51 1373416
A2 3.7 3.60 53443
TOTAL AREA 1572797
30% F 2.3% A2 3.7%
20%
10%
0
0 1 2 3 4 5 6
Megaloblastic anemia
Phenotype : Anemia
Incidence : Common cause of anemia in India.
Pathophysiology: Deficiency of vitamin B12 and Folic acid causes reduced
DNA synthesis, Thus, nuclear maturation lags behind cytoplasmic maturation
leading to megaloblastosis and dyserythropoisis
Laboratory findings: MCV is high and generally >110 fl, MCH is normal, RBC
count is reduced & RDW is increased . Peripheral smear shows macrocytes,
polychromatic cells, macro ovalocyte, hypersegmented PMN, cabot ring,
megaloblast, basophilic stippling. HBA2 values may be borderline or in
abnormal range. The values correct after trial of Vitamin B12/ Folic acid,
subsequent to which theHPLC testshould be repeated.
References
60

62
61
Kunbi 1.89
Mali 0.45
Sutar 0.45
Sonar, sahu,yadav 0.39
Kalar 0.27
Lodhi, Koshti 0.22
Bhadai 0.14
Maratha 0.12
Komti 0.02
Sahu 0.1
Thakur 0.02
Pawar 0.06
Powar 0.04
Kumbhar 0.02
Kasar 0.04
Kayastha 0.06
Gaur 0.02
Dhobi 0.08
Open 5.91% Muslim 4.33%
Christian 0.37
Sikh 0.21
Brahmin 0.32
Sindhi 0.16
Kshtriya 0.10
Baniya 0.02
Berga? 0.02
Patel 0.02
Pathan 0.02
ANNEXURE 1
SCD in various ethnic groups in central India, Nagpur division.
Data of 51145 cases screened RHDMC, IGGMC, Nagpur,
(sent for publication by Dr. (Mrs.) A.V. Shrikhande)
Caste Prevalence Subcaste Prevalence
SC 30.14 Mahar 29.08%
Mang 0.29%
Chambhar 0.18%
ST 15.66 Gond 10.95%
Madiya 2.46%
Paradhan 0.80%
Gowari 0.62%
Thakur 0.22%
VJ 6.31 Pardesi 3.68%
Banjara 1.05%
Pardhi
Kabirdas, 0.52%
Bhamate
Pradhan 1.56%
NT 6.45 Dhiver 0.93%
Lohar 0.93
Bhoi 0.41
Nathjogi 0.41
Bairagi 0.41
Bahripi 0.31
Beldar 0.31
Rajput 0.31
Barai 0.20
Ahir 0.20
Koli 0.1
Kurmar 0.1
Gwari? 0.1
OBC 8.16% Teli 2.38

Disclaimer
The information provided for various thalassaemia and hemoglobinopathy conditions is
on the basis of sampling and testing done in India . These are general guidelines and as
there are several genotypic and phenotypic variations possible, the user is advised to
referto detail studies available forbetter interpretation.
Bio-Rad LaboratoriesIndia Private Limited.
63
ßs
-gene prevalence in India Data by Dr B C Kar
State Place Caste/community Incidence Author
M.P. Indore 2 cases
Chhatisgarh Murmi 25.2%
Maharashtra Nagpur Mahar 22.2%
Kunbi 9.4
Teli 11.3
Bhil 15.5
Dhodia 20.6
Dubia 9.5
Naika 22.2
Pardhans 33.3
Bhill 18.5
Pawara 17.8
Tadwi 8.3
Kolam 8.3
Gond 6.6
Banjara 5.6
Andha 1.9
Mahadeo koli 1.9
Others 4.3
Palghar (in guj) Tribal 30.6
U.P. danukh One family
Bengal Midnapore Santhals 2 families
A.P. Koya doras 19.4
Godavari Hill Reddies 9.7
hyderabad Tribals 15.5
Hindus & muslims 0.5
Gujarat Surat Bhils
Bharuch Vasavas
Chaudhury 19.2
Gamit
Karnataka Nilgiri Hill Bhils 4.3
Badagas 6.6-8.4
Todas 3.3
Irulas 18.3
Kurumba 27
Tolap 14
Mysore Rajput One case
harijan One case
Assam Griza Orihas 29
Bihar Kumar One case
Chota nagpur Oraon 9.5
Munda 4.76

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