This study analyzed 48,000 samples from Nanded, Maharashtra, India to characterize the spectrum of hemoglobin disorders in the region. High performance liquid chromatography (HPLC) identified 2,742 cases of abnormal hemoglobin, including thalassemia major, sickle cell disease, sickle cell trait, and double heterozygous cases. The prevalence of sickle cell disease was 0.81% and thalassemia trait was 2.17%. Caste distributions of cases matched other local studies. Early detection of hemoglobin disorders through expanded newborn screening could help reduce disease burden through prevention and management strategies.

1. Spectrum of haemoglobin disorders at
Nanded region of Maharashtra : study of
48000 cases by HPLC
Authors:
Dr. Sundar Shewale Dr. S.A.Deshpande,
Dr. M.A.Sameer Dr. Raj Hanmante
Institute: Dr. Shankarrao Chavan Govt.
Medical College Nanded,Maharashtra
H:01

2. • Hemoglobinopathies and thalassemia are the commonest
genetic disorders in the world. In India also they constitute
major public health problem. [1] .
• The Government is aware of this problem so sickle cell
prevention and control program has implemented in 19 high
prevalent districts in Maharashtra since 2008.[2,3]
• To arrive at the exact diagnosis of these abnormal
haemoglobins , a battery of screening tests should be
performed since, presumptive identification usually requires
minimum two techniques ,based on different principles.[4]
BACKGROUND

3. • We screened samples by solubility test, NESTROFT test and
electrophoresis. The positive samples were further evaluated
by HPLC and also confirmed by family studies..
• The present study aims at evaluating haemoglobin
disorders in Nanded region of Maharashtra during the
period of September 2011 to May 2014.

4. PERIPHERAL SMEAR+ COMPLETE
BLOOD COUNT
SOLUBILITY TEST+NESTROFT
ELECTROPHORESIS TEST
HPLC (ALONG WITH FAMILY STUDY)
SAMPLES FROM
GOVT. MEDICAL COLLEGE
OPD+WARD
SAMPLES FROM
PRIMARY HEALTH
CENTERS
ELECRTOPHORESIS CENTERS
SOLUBILITY/SICKLING +
NESTROFT TEST
GOVT. MEDICAL COLLEGE NANDED
GOVT. MEDICAL COLLEGE

5. C) ALKALINE GEL
ELECTROPHORESIS
S
A
S
A
A) SOLUBILITY TEST
B) NESTROFT TEST

6. MATERIALS AND METHODS:
• We conducted prospective and retrospective study from
September 2011 to May 2014. We set inclusion and exclusion
criterias.
• Inclusion criteria:
• 1)Samples showing Hb < 10 gm % ,MCV < 78fl
• 2)Sample of patients having clinical and laboratory suspicion
of hemolytic anaemia
• 3)Samples which showed positive picture of
haemoglobinopathies on Hb electrophoresis at Rural Health
Centre
• Exclusion criteria:
• Patients who received blood transfusion in the last 6 months
were excluded from the study.

7. BIO-RAD HPLC VARIANT.

8. Table1
Analyte Name
[5]
Retention time
(minutes)
Band
(minutes)
Window
(minutes)
F -window 1.15 0.15 1.00-1.30
P2 1.45 0.15 1.30-1.60
P3 1.75 0.15 1.60-1.90
A0 2.60 0.40 2.20-3.30
A2-window 3.83 0.15 3.68-3.98
D-window 4.05 0.15 3.98-4.12
S-window 4.27 0.15 4.12-4.42
C-window 5.03 0.15 4.88-5.18

9. RESULTS:
• Total 48000 patients screened for haemoglobin disorders

10. Table2
Sample source Positive
on
screening
Samples
positive
on HPLC
Sensitivity
Total samples positive
by solubility test (GMC)
645 418 64.80%
Total samples positive
by NESTROFT(GMC)
1008 645 63.98%
Total samples positive
for both NESTROFT
and solubility (GMC)
241 170 70.54%
Total electrophoresis
positive samples came
from RH
1540 1509 97.98%
Total sample run on
HPLC
3434 2742 --

11. Table 3 : Abnormal spectrum of Hb disorders.
Haemoglobin disorder Number
of cases
Prevalan
ce
Common age group
Thal major 199 0.47% < 5 year
Thal Intermedia 027 0.05% 10-20 year
Thalassemia trait 1044 2.17% 16-45 year
Sickle cell Disease 385 0.81% 5-15 year
Sickle cell trait 713 1.48% 16-45 year
Double heterozygous 374 0.77% 16-45 year
TOTAL 2742 5.7%

12. Table 4: Caste wise distribution of cases
Caste
Total
N=2742
Percentage of
cases
Buddha (Mahar) 682 24.87 %
Muslim 587 21.40 %
Banjara 283 10.32%
Matang 282 10.28%
Chambar 234 8.53%
Teli 209 7.63%
Kunbi 163 5.95%
Others 302 11.02%

13. Variant hemoglobin No. of cases
Hb D trait 6
HPFH trait 6
Hb SD 2
HbE trait 2
Hb E disease 1
Hb E - β thal 1
Hb D-β thal 1
(?)Hb Lepore – HbS 1
Delta β thal 1
Total 21
Table 5:Uncommon variant haemoglobin cases
found in Nanded region

14. Sample Data
Results
Chromatogram

15. Fig 2: Thalassemia trait
[HbA2 > 4%]
HbA2 > 4%
Fig:3 Thalassemia major
[HbF >80%]
HbF >80%

16. Fig 4 : Sickle trait
[HbS 25-40%]
HbS <40% >40%HbS <40%
Fig 5: Sickle cell disease
[ HbS>40%]

17. Fig 6: Sickle disease with
thalssemia trait
HbS > 40%
HbA2> 4%
HbS < 4
HbA > 4
Fig 7: Sickle trait with
thalassemia trait

18. Fig 8: HbD [Retention window
3.98-4.12]
HbD - 30.7%
Fig 9:HbSD
HbSD

19. Fig:10 HbEβ thalassemia
HbA2-51.6%
HbF - 43%

20. Fig11: (?)HbS-Lepore
HbS - 59 %
HbA2 - 15 %

21. SICKLE CELL MOLECULAR LABORATARY
ODISHA SICKLE CELL PROJECT (NHM)
V.S.S. INSTITUTE OF MEDICAL SCIENCES
AND RESEARCH BURLA, SAMBHALPUR,
ODISHA, INDIA

23. MUTATION STUDY-ARMS PCR
Mutation No. of cases(B thal major)200
IVS-1 POSITION5(G-C) 63
619bp deletion 1
IVS1-POSITION1 (G-T) 2

24. Prenatal diagnosis
Families counselled
for diagnosis
Families not willing
for prenatal
diagnosis
Foetuses diagnosed
as Hb disorder
Diagnosed
Negative for Hb
disorder
30 19 06 5
Prenatal diagnosis done by Chorionic villous sampling
(10-14 wk) at National institute of immunohematology
13 floor KEM Hosp, Mumbai
Time period: September 2011 to May 2014.

25. Discussion
Table 6:Prevalence of Sickle Disease
Study Prevalence
Kate et al (2000) 0.5%
Vasaikar et al(2009) 1.09%
Present study 0.81%

26. • Frequency of β-thalassemia in India ranges from 3.5-15 % in
general population[3]
• Roshan Colah et al found prevalence of thalassemia trait 2%
in our region
• We found its prevalence as 2.17%
• This study is pilot study contributing data of prevalence of
Thalassemia major ,Sickle trait and Double heterozygous in
Nanded region of Maharashtra.
• Caste wise distribution of our study matches with study
conducted by M.A. Sameer et al in same region in 2014[7].

27. Conclusion
• Early detection of haemoglobin disorder is of paramount
importance in treatment, management and prevention of
genetic transmission. Also carrier identification of these
diseases would be useful to create awareness regarding partner
selection, prenatal diagnosis thereby reducing future disease
burden in the society
• The present study is useful to generate detailed data of
haemoglobin disorders so that health care resources can be
successfully targeted at them.

28. References
1. Weatherall DJ, Clegg JB. Inherited haemoglobin disorders:
an increasing global health problem. Bulletin of the World
Health Organization 2001;79:704-12
2. http://www.nrhm.maharashtra.gov.in/sickle.htm
3. Agarwal MB, Mehta BC. Sickle syndrome - A study of 44
cases from Bombay. Indian Paediatrics. 1980;17:793.
4. Kate Ryan,Barbara J Bain, Significant hemoglobinopathies
guidelines for screening Anddiagnosis. British journal of
hematology, 14: 35-49,(2010). Blackwell publishing Ltd.

29. 5. Biorad: Instruction manual.Variant Beta-thalassemia short
program2006 L70018803: Table-4.2;12.
6. Kate SL. 2000. Health problems of tribal population groups
from Maharashtra Immunohematol. Bull. Vol.pp31.1-10.
7. M. A. Sameer et al 2014 study of double heterozygous cases
by using cation exchange high performance liquid
chromatography in nanded region of maharashtra
8. Hanmante et al Neonatal Screening for Sickle Cell Disorders
International Journal of Recent Trends in Science And
Technology, E-ISSN 2249-8109, Volume 1, Issue 3, pp 104-109
,2011

30. *Take Home Message*
Thank you.
Let it pinch you ,before it pinches your little
one…