SQU Med J, November 2011, Vol. 11, Iss. 4, pp. 462-469, Epub. 25th Oct 11
Submitted 2nd May 11
Revision Req. 30th May 11, Revision recd. 9th Jul 11
Accepted 7th Sep 11

1Department of  Haematology, College of Medicine & Health Sciences, Sultan Qaboos University, Muscat, Oman; 2Department 
of Haematology, Sultan Qaboos University Hospital, Muscat, Oman; 3Department of Medicine, Sohar Hospital, Sohar, Oman; 
4Department of Obstetrics & Gynecology, Sultan Qaboos University Hospital, Muscat, Oman; 5Hôpital Robert Debré, INSERM 
U763, Paris, France.
Corresponding Author email: sskindi@yahoo.com

فرز )حتري( حديثي الوالدة
 املعدالت املرجعية ملؤشرات اخلاليا احلمراء ومتوسط اهليموغلوبني يف دم احلبل السري

 عند األطفال الُعمانيني حديثي الوالدة
�ضلم الكندي، انيل بثاري، علي املدحاين، �ضعيب الزدجايل، حمود الهدابي، قمرية العربي، ديفيد جرافل، مرمي 

ماثيو، راجو جوبال كر�ضنا مورثي
يف  الأداء  عايل  اِئل  ال�ضَّ اُب  ِت�رسرْ وال�ضرْ الكامل  العددي  الدم  فح�س  يف  احلمراء  الدم  خليا  معدلت  موؤ�رسات  من  التحقق  الهدف:  امللخ�ص: 
عينات من دم احلبل ال�رسي عند الُعمانيني حديثي الولدة. الطريقة: مت فح�س 7837 عينة من دم احلبل ال�رسي للأطفال حديثي الولدة 
وذلك بالقيام بفح�س الدم العددي الكامل وفح�س ال�ضت�رساب ال�ضائل عايل الأداء با�ضتخدام الربنامج الق�ضري للثال�ضيميا "بيتا". مت اإجراء 
اجلودة.  عايل  ال�ضائل  ال�ضت�رساب  نتائج  من  للتحقق  و)�ضي(  و)اأي(  و)دي(  )ا�س(  للهيموغلوبني  الطبيعية  غري  للعينات  املبا�رس  الت�ضل�ضل 
اإ�ضافة لذلك فقد مت اإجراء الفح�س اجليني يف احلالت التي وجد فيها كمية هيموغلوبني )اأي( اأقل من 10% لتاأكيد وجود طفرة. النتائج: 
 – هيموغلوبني   ،8.03±22.88 – اأي  )هيموغلوبني  طبيعيا  ال�ضائل  ال�ضت�رساب  حتليل  عندهم  كان   )%51.58( 4042 وليدا  اأن  ثبت 
حالة  اأية  على  نح�ضل  ومل   ،)%48.42( البقية  يف  األفا   – بالثل�ضيميا  بالإ�ضابة  توؤ�رس  النتيجة  كانت  بينما   ،)  8.04±77.02 اف 
لهيموغلوبني – اأت�س. كانت نتائج املجموعة ال�ضابقة كما يلي: متو�ضط الهيموغلوبني )15.38±2.04 جرام/ لرت(، وتعداد كريات الدم 
هيموغلوبنُي   ،(7.75±107.66( الَو�َضِطّي  الُكَريِويُّ  ُم  احَلجرْ  ،)%7.18±50.5( الدَّم  َدا�ُس  ِمكرْ لرت(،   /1012  x  0.68±4.69  ( احلمراء 
اخلليا  توزيع  عر�س  لرت(،  جرام/دي�ضي   3.44±30.98( الكريوي  الهيموغلوبني  متو�ضط  بيكوجرام(،   4.07±33.31( الَو�َضِطّي  ِة  الُكَريَّ
بينما كانت يف املجموعة الثانية امل�ضابة بالثال�ضيميا )األفا( كما ياأتي وعلى التوايل: )14.79±2.90  احلمراء )%2.17±17.01(، 
 30.39±3.6(; بيكوجرام(   29.74±11.80(;  )97.29±13.8(;  )%49.7±7.40(; لرت(   /1012  x  5.09±0.77(; لرت(  جرام/ 
الدم  فح�س  نتائج  يوؤكد  اأن  ميكن  الطبيعي  غري  للهيموغلوبني  للعينات  النووي  احلم�س  ت�ضل�ضل  جرام/دي�ضي لرت( ;)%18.09±2.56( 
اِئل عايل الأداء يف جميع احلالت. اخلال�صة: هذه هي اأول درا�ضة ملقارنة الهيموغلوبني وموؤ�رسات خليا  اُب ال�ضَّ ِت�رسرْ العددي الكامل وال�ضرْ
الدم احلمراء يف دم احلبل ال�رسي حلديثي الولدة الُعمانيني مع مثيلتها من بلدان اأخرى يف املنطقة، والتي تبني نتائج مماثلة لتلك التي 
اُب  ِت�رسرْ ظهرت عند حديثي الولدة ال�ضعوديني. كما قمنايف هذه الدرا�ضة اأي�ضا بالتحقق من �ضحة تف�ضريات فح�س الدم العددي الكامل وال�ضرْ
اِئل عايل الأداء ملوؤ�رسات دم احلبل ال�رسي عند حديثي الولدة الُعمانيني. كان وقوع الثل�ضيميا )األفا( التي مت ت�ضخي�ضها عن طريق  ال�ضَّ

الهيموجلوبني يف دم احلبل ال�رسي حلديثي الولدة %48.42.
مفتاح الكلمات: وليدي، فرز، حتري، تق�ضي، هيموغلوبني، متغريات، ثتل�ضيميا األفا، ُعمان.

abstract: Objectives: The aim of this study was to validate the interpretation of red blood cell indices in 
complete blood count (CBC) and high performance liquid chromatography (HPLC) results on cord blood samples 
in consecutive Omani neonates. Methods: Cord blood samples from 7,837 neonates, were analysed with CBC and 
HPLC using the β-thalassaemia short programme. Direct sequencing of abnormal samples with HbS, HbD, HbE 
and HbC was performed to validate the HPLC results. Additionally, in cases with HbA <10%, the β-globin gene 
was directly sequenced for β-thalassaemia mutation analysis. Results: Overall, 4,042 subjects (51.58%) had normal 
HPLC (HbA 22.88±8.03; HbF 77.02±8.04), whereas the presence of Hb Barts in the remaining 3,795 cases (48.42%) 
indicated the presence of α-thalassaemia. No case of HbH was detected. In the former subgroup respectively, the 
mean Hb (15.38±2.04 g/dl) red blood cell (RBC) count (4.69±0.68 x 1012/l), Hct (50.5±7.18%), mean corpuscular 
volume (MCV) (107.66±7.75 fl), mean corpuscular haemoglobin (MCH) (33.31±4.07 pg), mean corpuscular 
haemoglobin concentration (MCHC) (30.98±3.44 g/dl), red cell distribution width (RDW) (17.01±2.17%) whereas, 
in the latter group with α-thalassaemia, it was (14.79±2.90 g/dl); (5.09±0.77 x 1012/l); (49.7±7.40%); (97.29±13.8 fl); 
(29.74±11.80 pg); (30.39±3.6 g/dl), and (18.09±2.56%) respectively. DNA sequencing of samples with abnormal 

Neonatal Screening
Mean haemoglobin and red cell indices in cord blood from  

Omani neonates
*Salam Alkindi,1 Anil Pathare,2 Ali Al-Madhani,3 Shoaib Al-Zadjali,2 Hamood Al-Haddabi,2 Qamariya Al-

Abri,2 David Gravell,2 Mariam Mathew,4 Rajagopal Krishnamoorthy5

CLINICAL & BASIC RESEARCH



Salam Alkindi, Anil Pathare, Ali Al-Madhani, Shoaib Al-Zadjali, Hamood Al-Haddabi, Qamariya Al-Abri, David Gravell, Mariam 
Mathews and Rajagopal Krishnamoorthy

Clinical and Basic Research | 463

Several studies have shown that inherited haemoglobinopathies are widespread in Oman and are at a sufficiently 
high level to be of considerable national concern.1,2 
Specifically, Oman has a relatively high prevalence 
of α and β-thalassaemia, G6PD deficiency and 
sickle cell syndromes compared to other Arabian 
Gulf Countries.3-6 A community based survey 
carried out in 1995 in Oman showed a significantly 
high prevalence of haemoglobinopathy.5 
The reported prevalence of sickle cell trait was 6%  
and β-thalassaemia trait was 2%, whereas 
the prevalence of homozygous sickle cell and 
β-thalassaemia was 0.2% and 0.07% respectively. 
The prevalence of other abnormal haemoglobins 
namely HbD, HbE and HbC were 0.6%, 0.3% and 
0.02% respectively. Enzyme deficiency of G6PD 
was also prevalent (18%). Our own report in 
2010 also showed comparatively high prevalence 
rates.6 Interestingly, a high incidence of inherited 
haemoglobinopathies has also been reported in 
Mediterranean and Middle Eastern contries.3,7,8 

The prevalence of α-thalassaemia was reported as 
39.99% and the sickle cell gene was seen in 23.4% 
of cord blood neonatal samples from Saudi Arabia.8

It has been postulated that the likelihood of 
children being born with a major haemoglobinopathy 
in Oman would be about 3 per 1,000 births.5 Thus, 
with an annual birth rate of 35.76 births/1,000 
population,9 there would approximately be 106 new 

cases every year. Therefore, with this burden of 
haemoglobinopathy, the establishment of neonatal 
reference ranges is extremely important.

This study was undertaken under the auspices of 
His Majesty’s Research directive to screen neonates 
by implementing a universal newborn screening at 
the Sultan Qaboos University Hospital (SQUH), 
Oman. Cord blood samples from newborns at 
SQUH (representing the Muscat Governorate), and 
from the Sohar Hospital (representing the Batinah 
costal region) were collected. 

The objectives of our study were twofold. The 
first objective was to screen the newborn Omani 
subjects to establish the current prevalence of 
haemoglobinopathy and to see the effect of the 
decade long measures that were implemented 
following the first community study in 1995. The 
second objective was to validate the CBC and HPLC 
interpretations of the cord blood red cell indices in 
the Omani neonate. HPLC is a powerful tool for 
the simultaneous screening of newborn samples for 
haemoglobinopathies and to detect the presence 
of haemoglobin Barts.10,11 It is extremely accurate, 
reliable with reproducible results and has thus 
become the method of choice due to its speed and 
precision. The objective of this study was therefore 
to validate the CBC and HPLC interpretations of 
the cord blood red cell indices in the Omani neonate 
and by a universal newborn cord blood screening in 
the Omani population. 

Advances in Knowledge
1. This study validates the cord blood red cell indices obtained from Omani newborn subjects without any underlying haemoglobinopathy.
2. It validates the cord blood red cell indices in Omani newborn subjects with α-thalassaemia.
3. Finally, it validates the abnormal haemoglobins in Omani newborn subjects.

Application to the patient care 
1. The results of this study will benefit patient care by comparative analysis of Omani cord blood red cell indices in subjects without any 

underlying haemoglobinopathy with those from other countries in the region.
2. It will improve patient care by comparative analysis of Omani cord blood red cell indices with α-thalassaemia with those from other 

countries in the region.
3. It will also improve patient care by comparing the influence of α-thalassaemia on cord red cell indices in the different subsets with 

variant haemoglobins amongst Omani newborn subjects.

haemoglobin could validate the CBC and HLPC interpretations in all cases. Conclusion: This is the first study 
comparing the hemoglobin and red cell indices in the cord blood from newborn Omani subjects with those from 
other countries in the region, showing comparable results to those seen in Saudi neonates. The study also validates 
the CBC and HPLC interpretations of the cord blood red cell indices in the Omani neonate. The incidence of 
α-thalassaemia diagnosed by the presence of Hb Barts in cord blood of neonates was 48.42%.

Keywords: Neonatal; Screening; Reference range, Haemoglobin; Variants; Alpha-thalassaemia.



Neonatal Screening 
Mean haemoglobin and red cell indices in cord blood from Omani neonates

464 | SQU Medical Journal, November 2011, Volume 11, Issue 4

Methods
This study was conducted under the auspices of His 
Majesty’s Strategic Research Project initiated and 
funded between the years 2005 to 2008. The study 
was approved by the Institutional review board 
and conformed to the Declaration of Helsinki. The 
study enrolled a total of 3,740 subjects from SQUH 

in Muscat and 4,097 neonates from Sohar Hospital, 
Oman. All data was archived in a Microsoft Excel 
database. Statistical analysis was performed using 
the student’s t test with a value <0.05 considered 
significant.

Between April 2005 and March 2007 and 
after informed consent was obtained, a total 
of 7,837 consecutive cord blood samples were 

Table 1: Comparative analysis of cord blood red cell indices (mean ± SD) in newborn Omani neonates compared with 
Saudi neonates8

Haemoglobins n Hb  
gm/dl

RBC 
1012/L

HCT 
%

MCV 
fl

MCH
pg

MCHC 
g/dl

RDW 
%

HbA 
%

HbF 
%

Ab. Hb 
S,D,E,C

Omani HbAF 3, 765 15.38 
2.04*

4.69 
0.68*

50.5 
7.18*

107.66 
7.75*

33.31 
4.07*

30.98 
3.44*

17.01 
2.17*

22.88 
8.03*

77.02 
8.04*

--

Saudi HbAF8 243 15.1 
1.65

4.5  
0.52

47.4 
5.32

106.0  
8.0

33.6 
2.36

31.8  
1.7

17.9 
1.7

27.2 
7.1

72.8    
7.7

Omani HbAF 
Barts  

3,505 14.79 
2.9*

5.09 
0.77* 

49.7 
7.4* 

97.29 
13.8* 

29.74 
11.8*

30.39 
3.6* 

18.09 
2.56*

25.74 
9.05* 

73.96 
8.91* 

--

Saudi HbAF 
Barts8 

136 14.3 
1.98

5.03 
0.68

46.4 
5.9

92.5 
9.9

28.6 
2.9

30.7  
2.2

19.2 
2.0

28.1 
8.2

67.8    
8.3

--

Omani HbAFS 188 15.09 
1.56

4.91 
0.62

48.59 
5.5

103.34 
9.6*

32.92 
2.9*

30.5 
2.96*

16.65 
1.5

13.13 
7.8*

76.7 
11.4*

8.18 
2.9*

Saudi HbAFS8 57 14.97 
1.47

4.66  
0.5

47.1 
5.2

101.2 
6.2

32.3 
2.1

31.9  
1.7

17.7 
1.4

18.5 
3.8

73.6 
5.4

7.9 
2.57

Omani HbAFS 
Barts

218 15.02 
2.0 

4.73 
0.66 

47.0 
7.6 

96.51 
8.04* 

30.12 
4.2* 

30.26 
3.51* 

17.33 
2.1 

14.28 
5.2 * 

78.73 
8.3* 

6.97 
3.4* 

Saudi HbAFS 
Barts8

50 13.88 
1.37

5.03 
0.57

45.2  
5.3

89.3 
8.24

27.7 
2.2

31.1 
1.94

19.6  
4.2

18.7 
5.56

70.1 
5.8

6.8 2.02

Omani HbFS 9 16.45 
0.21*

5.06 
0.37

48.5 
8.09*

104.52 
5.9*

32.55 
2.8*

30.81 
3.26

14.95 
0.49*

-- 89.55 
3.78*

12.9 
4.7*

Saudi HbFS8  3 14.13 
0.1

4.26  
0.1

44.8 
3.6

105.2 
7.8

33.2 
0.57

31.7 
2.7

17.5 
0.5

-- 89.2 
3.8

10.8 
3.8

Omani HbFS 
Barts

14 12.89 
2.4*

4.40 
1.04

45.57 
5.5*

97.5 
4.4*

30.63 
6.8*

30.85 
3.24

17.16 
0.97*

-- 86.85 
5.2*

10.8 
5.2*

Saudi HbFS 
Barts8 

4 12.9  
1.6

4.7 
0.4

41.1 
9.1

87.2 
14.8

27.5 
1.9

31.8 
2.76

20.03 
2.31

-- 83.7 
5.6

11.87 
5.9

Omani HbAFD 45 14.86 
1.3

4.4  
0.39*

43.3 
3.8

104.7 
7.6*

33.84 
3.5

30.77 
2.66

16.75 
2.01

13.91 
7.41

78.76 
8.3*

8.66 
3.6

Omani HbAFD 
Barts

28 15.0  
1.5

4.76 
0.64*

49.47 
7.5

101.4 
10.23*

31.8 
3.4

30.22 
2.9

18.24 
3.57

12.7 
4.8

87.24 
4.8*

8.42 
3.8

Omani HbAFE  29 16.63 
1.8*

5.31 
0.91

57.6 
5.07

104.77 
7.88*

31.67 
3.4

30.89 
3.05

17.95 
2.26

20.3 
9.91*

75.36 
6.5*

6.78 
3.21

Omani HbAFE  
Barts

30 15.17 
1.57*

4.9 
0.99

50.9 
11.0

99.6 
6.8*

30.57 
8.2

30.15 
3.55

18.85 
6.93

13.29 
5.32*

86.7 
5.32*

6.54 
2.84

Omani HbAFC 3 15.7 
1.19

4.24 
0.78

42.12 
6.78

101.5 
7.8*

32.55 
4.5

30.99 
2.34

15.35 
1.67

13.5 
3.4

77.9 
5.33*

7.8 
2.67

Omani HbAFC 
Barts

3 15.05 
1.62

4.62  
0.8

44.9 
9.32

99.75 
5.6*

32.2 
5.6

30.2 
3.89

16.65 
0.56

11.31 
4.6

85.13 
6.45*

6.21 
3.24

Legend: * P <0.05 between red cell indices, with and without α-thalassemia (Hb Barts) in Omani neonates; RBC = red blood cell;  
HCT = haematocrit; MCV = mean cell volume; MCH = mean corpuscular haemoglobin; MCHC = mean corpuscular haemoglobin concentration; 
RDW = red cell distribution width; HbAF = Hb A+F; HbAFS = Hb A+F+S; HbFS = Hb F+S; HbAFD = Hb A+F+D; HbAFE = Hb A+F+E; HbAFC = 
Hb A+F+C.



Salam Alkindi, Anil Pathare, Ali Al-Madhani, Shoaib Al-Zadjali, Hamood Al-Haddabi, Qamariya Al-Abri, David Gravell, Mariam 
Mathews and Rajagopal Krishnamoorthy

Clinical and Basic Research | 465

screened prospectively, for the presence of possible 
haemoglobinopathies by a full blood count followed 
by HPLC using the Biorad Variant ΙΙ β-thalassaemia 
short programme. 

The CBC was performed using EDTA cord 
blood samples on Cell Dyn 4000TM automated 
blood cell counter (Abbot Diagnostics, Abbot 
Laboratories, IL, USA) within 4–12 hours of 
collection. HPLC was performed within 12–24 
hours of collection using the β-thalassaemia 
short programme on the Bio-Rad VARIANT IITM 

instrument (Bio-Rad Laboratories, Hercules, CA, 
USA) using the manufacturer’s instructions and 
controls. The samples were refrigerated from 
the time of collection up to the time of analysis. 
All samples were then processed to isolate and 
store mononuclear leukocytes for subsequent 
confirmatory molecular diagnostics. 

Direct sequencing of abnormal samples with 
HbS, HbD, HbE and HbC was performed on the ABI 
PrismTM 3100 genetic analyser (Applied Biosystems, 
Foster City, CA, USA) to assign the genotype status 
to these subjects and validate the HPLC results. The 
DNA sequencing was performed by polymerase 
chain reaction (PCR)-amplified β-globin gene 
segment to look for the following mutations, 
namely HbS (β6 Glu-Val), HbD (β121 Glu-Gln), HbE (β26 
Glu-Lys) and HbC (β6Glu-Lys) as per the manufacturer’s 
instructions and PCR conditions. Additionally, in 
samples with HbA below 10%, the β-globin gene 
was directly sequenced including the promoter, all 
exons and introns in these samples to look for all 
the known mutations reported for β-thalassaemia.

Results
On the basis of a CBC and HPLC, all samples were 
characterised as either normal (HbA+HbF), 4,042 
cases; or α–thalassaemia, (HbA+HbF+Hb Barts) 
3,795 cases. In the former group, 200 cases also had 
HbS, 45 cases had HbD, 29 cases had HbE, and 3 
cases had HbC. Whereas in the latter group, 229 
cases additionally also had HbS, 28 cases had HbD, 
30 cases had HbE,  and 3 cases had HbC. 

Table 1 shows the comparative analysis of cord 
blood red cell indices (mean ± standard deviation 
[SD]) from newborn Omani neonates compared 
with Saudi neonates.8 In a subset of the Omani 
neonates without any abnormal haemoglobin 
(HbA+HbF) (n = 3,765), the mean (±SD) Hb(g/
dl), red blood cell count (RBC) count (x 1012/L), 
haematocrit (Hct) (%), mean cell volume (MCV) (fl), 
mean corpuscular haemoglobin (MCH) (pg), mean 
corpuscular haemoglobin concentration (MCHC) 
(g/dl), red cell distribution width (RDW) (%), were 
15.38±2.04; 4.69±0.68; 50.5±7.18; 107.66±7.75; 
33.31±4.07, 30.98±3.44, and 17.01±2.17 respectively. 
Whereas in the subset of  subjects with HbA, HbF, 
Hb Barts (n = 3,505), the study observed a 48.42% 
incidence of α-thalassaemia, based on low MCV 
and MCH on the CBC and significant amounts of 
Hb Barts on HPLC based on the manufacturer’s cut-
off limit. Their mean (±SD) Hb(g/dl), RBC count (x 
1012/L), Hct (%), MCV (fl), MCH (pg), MCHC (g/dl), 
RDW (%), were 14.79±2.90; 5.09±0.77; 49.7±7.40; 
97.29±13.8; 29.74±11.80; 30.39±3.6, and 18.09±2.56 
respectively. There was a statistically significant 
reduction in the MCV, MCH, MCHC, HCT, Hb 
and an increase in the RBC count. Furthermore, 
MCV was the best discriminator between the two 

Table 2: Incidence of abnormal haemoglobinopathies observed in this study (n = 7,837)

Haemoglobins Number (%) High performance liquid 
chromatography (HPLC)

Direct Sequencing Concordance

Numbers % Numbers % %

Normal (A+F) 7,837
(100)

4042 51.57

α-Thalassemia(A+F+Barts) 3795 48.42

HbS (F+S+A)

773 
(9.86)

429 5.47 428 5.46 99.8

HbD (F+D+A) 73 0.93 73 0.93 100

HbE (F+E+A) 59 0.75 59 0.75 100

HbC (F+C+A) 6 0.08 6 0.08 100

Low HbA,<10% 206 2.62 202 2.57 98.1



Neonatal Screening 
Mean haemoglobin and red cell indices in cord blood from Omani neonates

466 | SQU Medical Journal, November 2011, Volume 11, Issue 4

groups (P = 7.0e-118).
Since HPLC cannot diagnose the presence of 

the β-thalassaemia gene at birth, in samples with 
HbA below 10%, the β-globin gene was directly 
sequenced including the promoter, all exons 
and introns in the abnormal samples (as per the 
manufacturer’s technical report) [Table 2]. In 105 of 
the 206 cases, an underlying known and previously 
reported mutation described in β-thalassaemia 
cases could be documented in this cohort of 
newborn cases.6 On complete analysis of the 
remaining cases, amongst the 206 cases we could 
demonstrate a known β-thalassaemia mutation in 
a total of 202 cases (98.1%). Additionally, in cases 
with an abnormal haemoglobin on HPLC, direct 
sequencing of abnormal samples with HbS (n = 
429), HbD (n = 73), HbE (n = 59), and HbC (n = 6) 
was also performed on an ABI Prism 3100 genetic 
analyser to assign the genotype status to these 
subjects and validate the HPLC results [Table 2 
and Figure 1]. In 428 of the 429 cases with HPLC 
showing HbS, the β6Glu—˃Val mutation could be 
documented by direct sequencing of the relative 
region of the β-gene, (99.76%) whereas, in the single 
sample with HbS in cord blood, but no β6Glu—˃Val 
mutation, we found an abnormality in codon 16 
of the delta chain. Furthermore, direct sequencing 
results in all cases of HbD, HbE and HbC showed 
complete concordance (100%) [Table 2].

Population reference range for various 
haemoglobins (%) in cord blood from newborn 
Omani subjects (n = 7,837) are shown in Table 3. 
The mean HbA, HbF, HbS, HbD, HbE, HbC (±SD) 
were 22.88±8.04; 77.02±8.04; 13.6±6.96; 8.88±4.87; 
6.64±2.98, and 6.21±3.41 respectively. The overall 
incidence of other haemoglobinopathies was 9.86% 
(n = 773), with 5.46% (n = 428) incidence of sickle 
haemoglobin. 

Discussion
Oman is a country with a population comprising 
a wide range of ethnic groups, and high 
rates of consanguinous marriages.12 This is a 
significant reason for the increased prevalence 
of haemoglobinopathies, which is of growing 
importance as knowledge of the population 
structure can be a unique aid in planning genetic 
services. 

HPLC is a powerful tool to screen newborns 
for haemoglobinopathies. Cord blood sampling 
offers an easy, simple and practical method for 
demonstrating the coexistence of α-thalassaemia by 
detecting the presence of Hb Barts. We were able 
to identify these subjects very well with the Biorad 
Variant II© system, using the β-thalassaemia short 
programme which works on the principle of cation 
exchange high pressure liquid chromatography. 
Since each haemoglobin has a characteristic 
retention time, Hb Barts being a fast moving 
haemoglobin, is easily separated and eluted from 
the other haemoglobins present in the neonatal cord 
blood samples. The precision is further improved 
by running a chromatographic calibrator with an 
assigned value for Hb Barts at the beginning of each 
run. Thus, using the presence of Hb Barts as an 
indicator of α-thalassaemia, we found that 51.57% 
subjects had normal HPLC (absence of Hb Barts). 

Before the availability of molecular diagnostic 
methods for the diagnosis of α-thalassaemia, there 
was good evidence that the presence of Hb Barts 
in the neonatal period indicated the presence 
of α-thalassaemia.13,14 However, the relationship 
between the amounts of Hb Barts and the 
underlying molecular defect was not clear. Most 
surveys using assays that detect 0.5% to 1% Hb Barts 
in cord blood detect a large proportion of neonates 
with α-thalassaemia, but not all; hence, surveys 
solely based on the presence of Hb Barts in cord 

Table 3: Reference range for various haemoglobins (%) in cord blood from newborn Omani subjects (n = 7,837)

HbA % 
(n = 7837)

HbF % 
(n = 7837)

HbS % 
(n = 429)

HbD % 
(n = 73)

HbE % 
(n = 59)

HbC % 
(n = 6)

Mean 22.88 77.02 13.6 8.88 6.64 6.21

Standard Deviation 8.04 8.05 6.96 4.87 2.98 3.41

Median 22.0 78.0 12.6 9.15 5.8 5.7

Range 4–50 25.1–96 2.1–21.9 3.7–19.1 2.2–14.6 2.7–11.6

95% confidence interval 22.62–23.13 76.75–77.29 12.7–14.5 6.94–9.83 5.47–8.05 2.64–9.79



Salam Alkindi, Anil Pathare, Ali Al-Madhani, Shoaib Al-Zadjali, Hamood Al-Haddabi, Qamariya Al-Abri, David Gravell, Mariam 
Mathews and Rajagopal Krishnamoorthy

Clinical and Basic Research | 467

blood consistently under-reported the frequency 
of α-thalassaemia.15 Moreover, although the levels 
of Hb Barts are related to the degree of α-chain 
deficit, there is no way to distinguish the various 
α-thalassaemia syndromes solely on the basis of 
HPLC.16

A comparative analysis of the red cell indices 
in these two groups revealed that α-thalassaemia 
resulted in a lower mean Hb, Hct, MCV, MCH, 
MCHC and HbF whereas it resulted in a higher 
mean red cell count and HbA concentration 
[Table 1]. Furthermore, all these differences 
were statistically strongly significant. The best 
discriminator was found to be MCV followed 
by red cell count, MCH and Hb concentration. 
Similar observations have been also made by other 
investigators; however, the degree of abnormality 
varies amongst these parameters.7,8 The greatest 
difference reported has been seen in MCH as 
individuals with α-thalassaemia clearly make less 
haemoglobin per cell than their normal counter-
parts. However, subjects with α-thalassaemia trait 
maintain adequate haemoglobin levels, the main 
compensatory mechanism being via the increased 
red cell numbers. 

The presence of α-thalassaemia was found 
to reduce the amount of HbA both in HbS 
heterozygous as well as homozygous HbSS subjects 
[Table 1]. Thus the presence of α-thalassaemia not 
only resulted in anatomically smaller RBCs, but also 
a cell which carried less HbA making it rheologically 

more adapted to flow through the small capillaries, 
thereby increasing the disease severity.7,8,17

In subjects with HbD, the presence of 
α-thalassaemia also resulted in lower HbA, HbD, 
MCV, and MCH; whereas the Hb, red cell count, 
Hct and HbF were higher. The differences in RBC 
count, MCV and HbF were statistically significant 
[Table 1]. 

In subjects with HbE the presence of 
α-thalassaemia resulted in lower HbA, HbE, MCV, 
MCH, Hb, RBC counts and Hct, whereas only the 
HbF was higher [Table 1]. The differences in Hb, 
MCV and HbF were statistically significant.

Overall, α-thalassaemia appears to influence all 
the red cell indices when it is the only abnormality. 
In the presence of abnormal haemoglobin, its 
influence was marginal unless the abnormal 
haemoglobin was present in a homozygous state, 
as in subjects who showed homozygous HbS. Thus 
with the presence of α-thalassaemia leading to a 
reduction in α-chains, the additional presence of a 
β structural variant will lead to a variable situation 
the outcome of which depends on the net globin 
chain synthesis rate. Some β-globin variants like 
HbE are synthesised less efficiently than HbA and 
represent less than 50% of the haemoglobin in the 
heterozygote. Furthermore, the rate of assembly 
of the αβ-chain complexes also would affect the 
final product. Therefore, the formation of the  
αβ-dimer is the rate limiting step in the assembly  
of haemoglobin.

Sickle cell trait β6
A>T [heterozygous]

Sickle cell disease
β6 A>T [homozygous]

Hb E trait β26 
G>C [heterozygous]

Hb C trait β6 G>
A A>T [both hetero]

Hb D trait β121 C>G
[heterozygous] 

β Thal. trait β IVS
1-5 C>G [hetero]

β Thal. trait β IVS
1,-2 T>G [hetero]

β Thal. trait β44(-c)
[heterozygous]

Figure 1: Capillary electropherogram results of DNA sequencing by PCR-amplified β-globin gene segment to 
document HbS (β6 Glu-Val) (a,b), HbD (β121 Glu-Gln) (e), HbE (β26 Glu-Lys) (c), HbC (β6 Glu-Lys) (d) and the common β-thalassemia 
mutations (f,g,h).



Neonatal Screening 
Mean haemoglobin and red cell indices in cord blood from Omani neonates

468 | SQU Medical Journal, November 2011, Volume 11, Issue 4

Direct sequencing of samples with abnormal 
haemoglobin was used to validate the interpretation 
of the CBC and HPLC results and was found to 
be quite accurate [Figure 1]. All cases with HbD, 
HbE, HbC were documented to show HbD (β121 
Glu-Gln), HbE (β26 Glu-Lys) and HbC (β6Glu-Lys) mutation 
respectively. All cases with HbS were shown to 
carry the HbS (β6 Glu-Val) except in one case which had 
the delta chain codon 16 mutation, and is known to 
show a small abnormal haemoglobin band in the 
HbS window on HPLC in the β-thalassaemia short 
programme HPLC runs. Furthermore, in cases 
with low HbA (below 10%), 98.1% of subjects were 
documented to have one of the known mutations 
described as causative of β-thalassaemia, consistent 
with the recommendations of the manufacturer 
(Bio-Rad Laboratories, Hercules, CA, USA ).

Thus, the significantly high prevalence of 
haemoglobinopathies in newborns from Oman 
emphasises the value of neonatal cord blood 
screening. This should be implemented as the 
first step in the national strategy towards total 
management of haemoglobinopathies—including 
early diagnosis, comprehensive clinical care and 
counselling of the affected families. In the light of 
the results of the current study, this initiative is 
being taken forward to encompass all the regions 
of Oman. The results of this large study would 
indicate that using HPLC is a cost effective method 
(<2 US $ per sample).18

Conclusion
In this study, for the first time, we were able to 
establish and validate the neonatal cord blood 
red cell indices and the prevalence of underlying 
abnormal haemoglobin by a universal newborn cord 
blood screening in the Omani population. The study 
observed that approximately 10% of the population 
still carries an abnormal haemoglobinopathy with 
significant clinical consequences, like Hb S, Hb D, 
Hb C and the β-thalassaemia gene. Furthermore, 
we have also established the prevalence of 
α-thalassaemia in this cohort and its potential to 
alter the red cell indices as well as its ameliorating 
effect when co-associated with the presence of other 
haemoglobin variants, which are highly prevalent in 
Oman. The comparative analysis of cord blood red 
cell indices and mean haemoglobins showed similar 
results to those seen with neonates from other 

countries in the region.

Therefore, as a direct result of this study, 
it is suggested that “targeted screening” with 
prescreening of both parents, and then selecting 
only the samples of neonatal cord blood from 
newborns with one parent having an underlying 
genetic trait for haemoglobinopathy, would result 
in a huge cost saving compared to the universal 
neonatal cord blood screening undertaken in this 
study. 

c o n f l i c t o f i n t e r e s t
The authors reported no conflict of interest.
a c k n o w l e d g e m e n t s

This study was supported by a grant from His 
Majesty’s Research Project IG/MED/HAEM/05/01. 
We would like to thank the parents who consented 
to the use of the cord blood samples. We would also 
thank the nurses, midwives and attending doctors 
who helped in the sample collection and in making 
this project a success. 

References
1. White JM, Byrne M, Richards R, Buchanan T, 

Katsoulis E, Weerasingh K. Red cell genetic 
abnormalities in Peninsular Arabs: Sickle 
hemoglobin, G6PD deficiency, and alpha and beta 
thalassemia. J Med Genet 1986; 23:245–51.

2. White JM, Christie BS, Nam D, Daar S, Higgs DR. 
Frequency and clinical significance of erythrocyte 
genetic abnormalities in Omanis. J Med Genet 1993; 
30:396–400.

3. El-Hazmi MA, Al-Swailem AR, Al-Faleh FZ, 
Warsy AS. Frequency of glucose-6-phosphate 
dehydrogenase, pyruvate kinase and hexokinase 
deficiency in the Saudi population. Hum Hered 1986; 
36:45–9.

4. Al-Awamy BH, Al-Muzan M, Al-Turki M, Serjeant 
GR. Neonatal screening for sickle cell disease in the 
Eastern Province of Saudi Arabia. Trans R Soc Trop 
Med Hyg 1984; 78:792–4.

5. Al-Riyami AA, Suleiman AJ, Afifi M, Al-Lamki 
ZM, Daar S. A community-based study of common 
hereditary blood disorders in Oman. East Mediterr 
Health J 2001; 7:1004–11.

6. Al Kindi S, Zadjali S, Al Madhani A, Daar SD, 
Al Haddabi H, Al Abri Q, et al. Forecasting 
hemoglobinopathy burden through neonatal 
screening in Omani neonates. Hemoglobin 2010; 
34:135–44.

7. Nasserullah Z, Al-Jame A, Abu Srair H, Al-Qatari 
G, Al-Naim S, Al-Aqib A, et al. Neonatal screening 



Salam Alkindi, Anil Pathare, Ali Al-Madhani, Shoaib Al-Zadjali, Hamood Al-Haddabi, Qamariya Al-Abri, David Gravell, Mariam 
Mathews and Rajagopal Krishnamoorthy

Clinical and Basic Research | 469

for sickle cell disease, glucose-6-phosphate 
dehydrogenase deficiency and α-thalassemia in Qatif 
and Al Hasa. Ann Saudi Med 1998; 18:289–92.

8. Quadri MI, Islam SIAM, Nasserullah Z. The effect 
of α-thalassemia on cord blood red cell indices and 
interaction with sickle cell gene, Ann Saudi Med  
2000; 20:367–70.

9. Birth rate in Oman. From: http://www.nationmaster.
com/country/mu-oman/peo-people  Accessed: Apr 
2011.

10. Rogers BB, Wessels RA, Ou CN, Buffone GJ. High-
performance liquid chromatography in the diagnosis 
of hemoglobinopathies and thalassemias. Report of 
three cases. Am J Clin Pathol 1985; 84:671–4.

11. Lorey F, Cunningham G, Shafer F, Lubin B, Vichinsky 
E. Universal screening for hemoglobinopathies using 
high-performance liquid chromatography: Clinical 
results of 2.2 million screens. Eur J Hum Genet 1994; 
24:262–71.

12. Rajab A, Patton MA. A study of consanguinity in the 
Sultanate of Oman. Ann Hum Biol 2000; 27:321–6.

13. Weatherall DJ. Abnormal hemoglobins in the 
neonatal period and their relationship to thalassemia. 
Br J Haematol 1963; 9:265–77.

14. Wasi P, Na-Nakorn S, Pootrakul S. The 
α-thalassemias. Clin Haematol 1974; 3:383–410.

15. Higgs DR, Pressley L, Clegg JB, Weatherall DJ, Higgs 
S, Carey P, et al. Detection of beta-thalassemia in 
negro infants. Br J Haematol 1980; 46:39–46.

16. Higgs DR, Vickers MA, Wilkie AOM, Pretorius I-M, 
Jarman AP, Weatheral DJ. A review of the molecular 
genetics of the human α-globin gene cluster. Blood 
1989; 73:1091–108.

17. Weatheral DJ. The Thalassemias. In: Beutler E, 
Litchmen MA, Coller BS, Kipps TJ, Eds. William’s 
Hematology. 5th Ed, (International ed.). New York: 
McGraw-Hill Inc, Health Professions Division, 1995. 
Pp. 581–615.

18. Pathare AV, Al Kindi S, Al Zadjali S, Al Abri Q, 
Al Haddabi H, Muralitharan S, et al. Newborn 
hemoglobinopathy carrier screening: Towards 
comprehensive and improved patient care of sickle 
cell disease in Oman, a cost effective analysis (Abstr. 
806) Hematol J 2006; 91:296–7.