Home About Us Contact Us

Official Journals By StatPerson Publication

Table of Content-Volume 12 Issue 2 - November 2019


 

 


Comparison of haemoglobin electrophoresis alkaline with scanning densitometry and high performance liquid chromatography in diagnosis of haemoglobin disorders

 

Piyush Narkhede1, H R Kokandakar2*, R S Bindu3

 

1Assistant Professor, Department of Pathology, JIIU’s IIMSR Medical College, Jalna, Maharashtra, INDIA.

2Professor, Department of Pathology, Government Cancer Hospital Aurangabad, Maharashtra, INDIA.

3Ex-Professor and HOD, Government Medical College, Aurangabad, Maharashtra, INDIA.

Email: khemantr@gmail.com

 

Abstract               Background: Inherited Haemoglobin Disorders constitutes major bulk of genetic diseases in India. They cause high degree of morbidity, moderate to severe haemolytic anaemia and death in affected individuals. Alkaline Electrophoresis is rapid reproducible method and capable of separating common Hb variants. Automated cation-exchange High Performance Liquid Chromatography (HPLC) has emerged as an excellent screening tool for diagnosing these abnormal haemoglobins/ thalassemia states. Present study is aimed at finding sensitive, specific easy reproducible method among these two techniques used. Also limitations of these techniques will be known Aims and Objectives: To study haemoglobin patterns by HPLC and Electrophoresis alkaline with scanning densitometry and comparison of results by two techniques. Materials and methods: Present study was carried out in Department of Pathology of a tertiary care hospital, after the approval of the hospital ethical committee. Patients attending OPD and Indoor patients, Patients referred from Peripheral Health Centres, Rural Health Centres were studied. The present study is observational prospective study in which diagnostic evaluation of these two methods is done. Study period is 2 years. Results:- In the present study, total 200 suspected cases of Haemoglobin Disorders were studied by HPLC and Haemoglobin Electrophoresis Alkaline with scanning Densitometry in Department of Pathology of Tertiary Care Hospital from July 2012 to October 2014.Both methods were equally good for quantification of HbS [p>0.05], in Sickle cell trait, Sickle-beta thalassemia, sickle cell anaemia and HbF [p>0.05] in Sickle cell anaemia, Sickle-beta thalassemia, Beta-Thalassemia Major. [Equally good for quantifying fractions present in large amount]. HbE and HbA2 were not separated by both methods. Both methods are complementary in detection of Hb Lepore trait. HbA2 % in sickle cell syndromes was higher on HPLC than Hb-Electrophoresis due to effect of Sickle adducts.[p<0.05].HPLC found to be less labour intensive, Rapid, more reliable than Hb-Electrophoresis. Precision of Hb-Electrophoresis with Scanning Densitometry was adequate for Hb-fraction present in large fraction such as HbS [around 36%], HbF[>90%].Overall, Precision of HPLC was excellent for quantification of Hb-variants. Poor precision of Hb-Electrophoresis with Scanning Densitometry [intra-run precision CV -17.71, inter-run precision CV- 19.55, for HbA2 quantification than HPLC[intra-run precision CV -2.57, inter-run precision CV- 2 ]. Over-estimation and under-estimation of HbA2% by Hb-Electrophoresis with scanning densitometry in diagnosis of beta thalassemia trait was recorded. Conclusion: HPLC and Hb-electrophoresis [alkaline] are both complimentary to each other as Haemoglobin variant is confirmed by two methods. Result given by both methods should be interpreted in view of clinico-haematological profile, family study and if available molecular studies. Hb-Electrophoresis with scanning densitometry has limitations for HbA2 quantification in diagnosis of beta-thalassemia trait. Though, HPLC is not without limitations, HPLC has found to be Rapid, less labour intensive, accurate, more reliable than Hb-Electrophoresis for quantification of haemoglobin variants. Hb-Electrophoresis with scanning densitometry should be used as adjuvant method for confirmation of haemoglobin variants.

 Key Words: HPLC, Hb-Electrophoresis, Scanning Densitometry,

INTRODUCTION

Inherited Haemoglobin Disorders includes Haemoglobinopathies and thalassemia. Haemoglobinopathies are characterized by structurally defective haemoglobin due to abnormalities in formation of globin moiety of molecule such as S,C,D,E. The term, thalassemia‟ is characterized by reduced rate of synthesis of normal haemoglobin due to absence or decrease in synthesis of one or more type of globin polypeptide chains. It has been estimated that 7% of the world populations are carriers of such disorders. The cumulative gene frequency of haemoglobinopathies in India is 4.2%. Inherited Haemoglobin Disorders constitutes major bulk of genetic diseases in India. They cause high degree of morbidity, moderate to severe haemolytic anaemia and death in affected individuals.1,2 Laboratory Diagnosis of Haemoglobin disorders is required for 3

a) Confirmation of provisional Diagnosis of significant sickling disorders and beta thalassemia major.

b) Explain haematological abnormalities.

c) Identify an abnormality in pre-symptomatic phase as in neonate.

d) To predict serious globin chain synthesis disorders in foetus and offer option of termination of pregnancy.

e) Permit genetic counselling of prospective parents.

Alkaline Electrophoresis is rapid reproducible method and capable of separating common Hb variants such as HbA,HbF,HbS,HbC3. Commercial, rapid electrophoretic methods have been developed that allow for separation at pH 8.4 (alkaline) on agarose gels. These provide a clear background, allowing for quantification of the Hb present by densitometric scanning. Visualization of the Hb bands is made possible by staining with Amino Black and Acid Violet (or similar stains) 4&5. Automated cation-exchange High Performance Liquid Chromatography (HPLC) has emerged as an excellent screening tool for diagnosing these abnormal haemoglobins/ thalassemia states. The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional haemoglobin electrophoresis for the detection and identification of haemoglobin variants. 6,7,8 The definite identification of disorders of haemoglobin synthesis can be achieved only by DNA analysis. Exact diagnosis of these diseases is of paramount importance in therapy and prevention of genetic transmission. Currently, HPLC and Hb-Electrophoresis are commonly used in diagnosis. 9&10 Present study is aimed at finding sensitive, specific easy reproducible method among these two techniques used. Also limitations of these techniques will be known.

MATERIALS AND METHODS

Present study was carried out in Department of Pathology of a tertiary care hospital, after the approval of the hospital ethical committee. Patients attending OPD and Indoor patients, Patients referred from Peripheral Health Centres, Rural Health Centres were studied. The present study is observational prospective study in which diagnostic evaluation of these two methods is done. Study period is 2 years from July 2012 to October 2014. Haematological profile of cases was done, if required, bone marrow was done. Two ml blood collected in K2EDTA anticoagulant bulb was used for HPLC and Hb-Electrophoresis. HPLC requires whole blood while Hb-Electrophoresis has to be performed on RBC Concentrate. Haemoglobin Electrophoresis Alkaline was performed to know the haemoglobin patterns and quantification was done by scanning densitometry. Differential diagnoses were put forwards. Haemoglobin variants were confirmed and again quantified by HPLC, final Diagnosis was given. Results given by these two techniques were compared and correlated. Study of family members accompanying the cases was done to confirm the diagnosis.


 

RESULTS AND OBSERVATIONS

In the present study, total 200 suspected cases of Haemoglobin Disorders were studied by HPLC and Haemoglobin Electrophoresis Alkaline with scanning Densitometry. HPLC was considered as standard method.

 

Table 1: Diagnosis of cases by HPLC and Hb-Electrophoresis

No.

DIAGNOSIS

HPLC

Hb-Ele

1.

Sickle Cell Trait (SCT)

43

43

2.

Sickle Cell Anaemia (SCA)

9

9

3.

Sickle Cell - Beta Thalassemia (SBT)

18

18

4.

Beta Thalassemia Trait (BTT)

32

35 [30+5**]

7.

Beta Thalassemia Major (BTM)

16

16

8.

HbE - Beta Thalassemia

1

1

9.

Hb Lepore Trait

1

1

10.

Normal Pattern

80

77[75+2*]

 

Total no. of cases

200

200

Table 2: No. of cases diagnosed as Beta thalassemia trait by Electrophoresis

Electrophoresis Result

No. of Beta thalassemia trait cases confirmed by HPLC and CBC indices

No. of cases with Normal Pattern confirmed by HPLC and CBC indices

Total

Beta Thalassemia trait

30

5

35

Normal

2

75

77

Total

32

80

112

 

Table.3.Quantification of HbA2 by HPLC and Hb-Electrophoresis in Beta thalassemia trait [n=32]

Techniques

HbA2[%]

[Mean±SD]

HPLC

5.15 ±0.518

Alkaline ELE

4.9±0.830

HbA2:-% of HbA2 was greater by HPLC than by Hb-Electrophoresis. Correlation coefficient of HPLC-Hb-Electrophoresis for HbA2 was r=0.23901. Student t test was applied, p value greater than 0.05 [p>0.05].

 

Table 4: Quantification of haemoglobin variants in Sickle cell trait [n=43] by HPLC and Hb-Electrophoresis.

Techniques

HbA [%]

[Mean±SD]

 

HbS [%]

[Mean±SD]

 

HbA2 [%]

[Mean±SD]

 

HPLC

 

53.87 ±4.285

 

35.72±4.713

3.76±0.47

Alkaline Electrophoresis with scanning Densitometry

60.53±4.436

36.83±4.510

2.6±0.65

 

1] HbA:-% of HbA was greater by Hb-Electrophoresis than by HPLC.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbA was r=0.8247.

Z-score test was applied, p value less than 0.05 [p<0.05].

2] HbS:-% of HbS was slightly greater by Hb-Electrophoresis than by HPLC.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbS was r=0.9008.

Z-score test was applied, p value greater than 0.05 [p>0.05].

3] HbA2:-% of HbA2 was greater by HPLC than by Hb-Electrophoresis.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbA2 was r=0.051.

Z-score test was applied, p value less than 0.05 [p<0.05].

Table 5: Quantification of haemoglobin variants in Sickle cell anaemia [n=9] by HPLC and Hb-Electrophoresis

Techniques

HbF[%]

[Mean±SD]

 

 

 

 

[Mean+SD]

 

HbS [%]

[Mean±SD]

 

[Mean+SD]

 

HbA [%]

[Mean±SD]

 

[Mean+SD]

 

HbA2 [%]

[Mean±SD]

 

[Mean+SD]

 

HPLC

 

18.58±9.28

77.05±8.17

1.98±0.385

3.14±0.576

Alkaline ELE

21.25±7.25

76.88±6.67

------------

1.84±0.754

 

1]HbS:-% of HbS was slightly greater by HPLC than by Hb-Electrophoresis.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbS was r=0.8029.

Student t test was applied, p value greater than 0.05 [p>0.05].

2]HbF:-% of HbF was greater by Hb-Electrophoresis than by HPLC.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbS was r=0.8527

Student t test was applied. p value greater than 0.05 [p>0.05].

3]HbA2:-% of HbA2 was greater by HPLC than by Hb-Electrophoresis.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbA2 was r=0.7530.

Student t test was applied, p value less than 0.05 [p<0.05].

4]HbA:-Mean HbA by HPLC was 1.98%.No HbA% was detected by Hb-electrophoresis.

 

Table 6: Quantification of haemoglobin variants in sickle beta thalassemia [n=18] by HPLC and Hb-Electrophoresis

Techniques

HbF [%][Mean±SD

HbS [%][Mean±SD]

HbA [%][Mean±SD]

HbA2 [%][Mean±SD]

HPLC

20.7±9.67

66.68±9.321

6.02±3.54

5.65±1.028

Alkaline ELE

21.6±8.98

65.85±10.23

8.15±5.53

4.38±0.891

1]HbS:-% of HbS was slightly greater by HPLC than by Hb-Electrophoresis.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbS was r=0.9411.

Student t test was applied, p value greater than 0.05 [p>0.05].

2]HbF:-% of HbF was greater by Hb-Electrophoresis than by HPLC.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbS was r=0.9082.

Student t test was applied,p value greater than 0.05 [p>0.05].

3] HbA:-% of HbA was greater by Hb-Electrophoresis than by HPLC.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbA was r=0.9605.

Student t test was applied, p value less than 0.05 [p<0.05].

4]HbA2:-% of HbA2 was greater by HPLC than by Hb-Electrophoresis.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbA2 was r=0.8506.

Student t test was applied, p value less than 0.05 [p<0.05]

Table 7: Quantification of haemoglobin F in Beta thalassemia major [n=16] by HPLC and Hb-Electrophoresis

Techniques

HbF [%]

[Mean±SD]

 

[Mean+SD]

 

HPLC

 

95.51±3.29

Alkaline ELE

95.68±3.91

 

HbF:-%of HbF was slightly greater by Hb-Electrophoresis.

Correlation coefficient of HPLC-Hb-Electrophoresis for HbF was r=0.8563.

Student t test was applied, p value greater than 0.05 [p>0.05].

 

Table 8: Quantification of haemoglobin variants in HbE-Beta Thalassemia[n=1] by HPLC and Hb-Electrophoresis

Techniques

HbF

HbA

HbA2

HPLC

33.2%

5.4%

62.8%

Alkaline  ELE

35.6%

6.4%

58%

Case diagnosed provisionally as HbE-beta thalassemia on basis of severity of symptoms, HbF% and HbA2 %.

 

Table 9: Quantification of haemoglobin variants in Haemoglobin Lepore trait [n=1]

Techniques

HbA

Lepore

HbF

HbA2

HPLC

73.1 %

------------

2.8%

16.7%

Alkaline ELE

 85.2%

11.8%

------------

3%

Case was diagnosed provisionally as haemoglobin Lepore trait based on HPLC and Electrophoresis pattern, HbA2 Level 16.7% on HPLC.

 

Table 10: Human labour and time consumption for HPLC

Procedures

Time consumption in minutes

*Warm up period+ Sample loading

18 minutes 30 sec

Time for run of 7 samples

45 minutes 30 sec

Human labour and time consumption per human required for 7 samples were compared. [As each gel of Hb-electrophoresis has 7 tracks.]

 

Warm-up period is period taken from start of machine to actual run. [6 minute for display+12 minutes 30 sec. cycle before actual run].During that period sample loading can be done. 64 minutes were required each time to run 7 samples per human on HPLC

Table 11: Human labour and time consumption for Hb- Electrophoresis

Procedures

Time consumption in minutes

Preparation of RBC Concentrate

30

Buffer preparation

4

Gel soaking and placement on Applicator

2

Preparation of haemolysate and sample

5

Electrophoresis Run

15

Fixation and drying

12

Staining

5

Destaining

10

Scanning + Adjustment on Scanner

4

87 minutes were required each time to run 7 samples per human on Hb-Electrophoresis. Also number of procedures were greater in Hb-Electrophoresis than HPLC.

DISCUSSION

1.Out of total 200 suspected cases of Haemoglobin Disorders,118 cases were found to have Hb-Disorder pattern on HPLC as well as Hb-Electrophoresis, 2* cases were found to be Beta thalassemia Trait pattern by HPLC but missed on Hb-Electrophoresis [Alkaline] with Scanning Densitometry, also the CBC Indices were consistent with Beta Thalassemia Trait. Of the remaining 80 cases, 75 cases were found to have normal Haemoglobin pattern by HPLC as well as Hb-Electrophoresis. Remaining 5** cases out of 80 cases, on Hb-Electrophoresis were found to be having Beta-Thalassemia Trait but CBC Indices were not consistent with Beta-Thalassemia Trait and also that, on HPLC these cases were found to have normal pattern. So, after complete haematological work up, HPLC and Hb-Electrophoresis Results, 120 cases were classified as Haemoglobin Disorders and 80 cases were classified as Normal Pattern. Dogaru Monica et al,[2007]11compared two analytical methods (electrophoresis and HPLC) to detect thalassemia and hemoglobinopathies. Both methods (Haemoglobin Electrophoresis and HPLC) gave almost similar results. Densitometric evaluation of HbA2 after electrophoresis is not an accurate and reliable method for quantifying HbA2 minimizing the usefulness to detect patients with β thalassemia minor. The HPLC (High Performance Liquid Chromatography) method is a sensitive and precise method and has become the preferred method for thalassemia screening because of its simplicity, superior resolution, rapid assay time and accurate quantification of Hb fractions.2. From table 2, Total 30 cases diagnosed by both methods as Beta thalassemia trait based on HbA2 level and indices. 2 cases showed high HbA2 %[>4%] by HPLC, normal HbA2 % by Electrophoresis, but indices were consistent with beta thalassemia trait, so classified as beta thalassemia trait.5 cases showed high HbA2[HbA2>4%] by Electrophoresis, normal HbA2 value by HPLC, but indices were not consistent with Beta thalassemia trait, so classified as normal.75 cases show normal pattern by both methods. . Considering HPLC as standard for HbA2 measurement [as it is highly precise and accurate method available for HbA2 measurement].Sensitivity and specificity of Hb-Electrophoresis were found to be 93.5% in present study. 3. From table 3 and 4, Results given by Hb-Electrophoresis were well correlated with HPLC. Correlation for HbA2 % in Sickle cell trait [r=0.051] and Beta-Thalassemia trait [r=0.23901] was weak.[FIG 10-12] 4. From table 4,5,6 HbA2 values on HPLC were found to be higher than Hb-Electrophoresis with Scanning Densitometry in Sickle cell Syndromes[SCT,SCA, Sickle-Beta Thal]. Difference was statistically significant [P<0.05]. Estimates of haemoglobin A2 by high performance liquid chromatography are inaccurate in the presence of haemoglobin S. This is because some haemoglobin S that has undergone post-translational modification has the same retention time as haemoglobin A2, and thus is measured along with it. Diagnosis of Sickle cell trait and sickle-beta thalassemia are not affected as it is % of HbS which is important. [HbS>50% with HbS>HbA,-sickle beta thal. HbS<50% with HbA>HbS- SCT]. Family studies are helpful in distinction of Sickle cell Anaemia and Sickle-beta thalassaemia12. Head C E et al[2004]13 found that HPLC measurements of HbA2 in presence of HbS were significantly higher than scanning densitometry (p , 0.01) measurements. 5. Statistically significant difference was found for HbA % by two methods in Sickle cell trait and Sickle-Beta Thalassemia [p<0.05].HbA % was found to be higher on Hb-Electrophoresis with Scanning Densitometry than HPLC. Above finding was due to fact that ,In Hb-Electrophoresis non-glycated and glycated fraction of HbA are not separated, while in HPLC fractions are separated [HbA0,P2,P3].12,14 6. In Sickle cell Anaemia, as both genes are occupied by βs, Normal HbA chain synthesis is not expected, but on HPLC small fraction of HbA[Mean 1.98%] was detected. This is glycosylated S which has same Retention window as HbA.12 7. From table 7, Both the methods are equally good for measurement of HbF value in beta thalassemia major. No statistically significant difference was found.[p>0.05].8. From table 8, HbE and HbA2 were not separated by both methods. 9. From table 9, HPLC pattern for Haemoglobin Lepore trait was HbF,HbA,HbA2. Electrophoresis pattern was Band HbA, Band at S,D,G, Lepore region,HbA2 Band.HbA2 is separated from Lepore in Alkaline Electrophoresis while in HPLC, HbA2 and Lepore was not separated. Both methods are complementary in detection of Hb Lepore trait.10. From table 10 and 11, Hb-Electrophoresis Alkaline with scanning densitometry is time consuming, labour intensive. HPLC is less time consuming and with more automation. Shrivastav Atul et al [2013]8 demonstrated that HPLC has high degree of reproducibility and precision. The simplicity of sample preparation, accurate quantification of Hb concentration combined with complete automation, makes HPLC an ideal methodology for the routine diagnosis of Hb disorders. Sachdev Ritesh et al [2010]15detected Hb-variants and Haemoglobinopathies using HPLC. They found that HPLC forms rapid accurate and reproducible tool for early detection and management of Haemoglobinopathies.11. Precision study Hb-Electrophoresis alkaline with scanning densitometry was found to be imprecise for HbA2 Measurement, Coefficient of variation[CV] of intra-run precision ,was 17.71 around mean of 2.71% and Coefficient of variation[CV] of inter-run precision was 19.55% around mean of 2.66%.HPLC precision was found to have greater precision than Hb-Electrophoresis in measuring haemoglobin variants.   Clarke Gwendolyn M. et al4 in their study mention that A recent College of American Pathologists hemoglobinopathy survey showed a CV of 33.6% for densitometric scanning of electrophoretic gels at a HbA2 concentration of 2.41%. For column chromatography, the CV was 14.6% at a mean HbA2 concentration of 3.21%, and for HPLC, the CV was 4.3% at a mean Hb A2 of 3.47%. Precision of Hb-Electrophoresis with Scanning Densitometry was adequate for Hb-fraction present in large fraction such as HbS [around 36%], HbF[>90%].Overall, Precision of HPLC was excellent for quantification of Hb-variants. 12. Limitations of Both Techniques:-1] Haemoglobin A2 and Haemoglobin E were not separated from each other, by both methods.2] Diagnosis of Beta-Thalassemia trait depends on HbA2 level >4% on HPLC and Hb-electrophoresis, but HbA2 level can be raised in 1. Megaloblastic Anemia 2. HIV positive individuals on antiretroviral therapy. 3. Hyperthyroidism.4. Some unstable Hb variants. These conditions should be ruled out before diagnosis.3] In Sickle cell anaemia and sickle-beta thalassemia only provisional diagnosis can be made, family study /genetic study is required for confirmation. In Indian scenario, Family studies are chief and best for confirmation.


 

Images:

1
Figure1:
Processed gel plate

 

HPLC AND HB-ELECTROPHORESIS PATTERNS

 

23

Figure 2: Sickle cell trait                                                                 Figure3: Sickle cell anaemia

45

          Figure 4: Sickle-Beta compound Heterozygous                                Figure 5: Beta-Thalassemia trait

67

    Figure 6: Beta-Thalassemia Major                                                        Figure 7: HbE-Beta Thalassemia

89

Figure 8: Hb-Lepore Trait                                                                                   Figure 9: Normal Pattern

 

 

  • Correlation graphs

101112


CONCLUSION

1. HPLC and Hb-electrophoresis [alkaline] are both complimentary to each other as Haemoglobin variant is confirmed by two methods.

2. Result given by both methods should be interpreted in view of clinico-haematological profile, family study and if available molecular studies.

3. Places where HPLC is not available, haemoglobin electrophoresis alkaline with scanning densitometry can be used for primary identification and quantification of most of the variants but Hb-Electrophoresis with scanning densitometry has limitations for HbA2 quantification in diagnosis of beta-thalassemia trait, [low precision, low accuracy], results should be interpreted with the help of CBC indices, sample should be send to higher centres where HPLC facilities are available, if CBC indices are consistent with beta-thal trait.

4. Though, HPLC is not without limitations ,HPLC has found to be Rapid, less labour intensive, accurate, more reliable than Hb-Electrophoresis for quantification of haemoglobin variants. Hb-Electrophoresis should be used as adjuvant method for confirmation of haemoglobin variants.

 

REFRENCES

  • Weatherall DJ, Clegg JB. Inherited haemoglobin disorders:an increasing global health problem. Bulletin of the world health organization 2001; 79(8): 704-12.
  • Balgir RS. The burden of haemoglobinopathies in India and the challenges ahead. Current Science 2000 Dec;79(11):1536-47.
  • Agrawal MB. The burden of haemoglobinopathies in India-Time to wake up? J Assoc Physicians India 2005 Dec;53:1017-8.
  • Clarke GM Higgins TN. Laboratory Investigation of Haemoglobinopathies and Thalassaemias: Review and Update. Clinical Chemistry 2000; 46(8B): 1284-90.
  • Ching-Nan Ou, Cheryl L. Rognerud. Rapid Analysis of Haemoglobin Varients by Cation Exchange HPLC. Clinical Chemistry 1993;39(5):820-4.
  • Colah RB, Surve R, Sawant P, D‟Souza E, Italia K, Phanasgaonkar S et al. HPLC Studies in Haemoglobinopathies. Indian J Pediatr 2007 July; 74 (7) : 657-62.
  • Riou J, Godrat C, Hurtel D, Mathis M, Bimet C, Bardakdjian-Michau J et al. Cation-exchange HPLC evaluated for presumptive identification of haemoglobin variants. Clinical Chemistry1997,43(1): 34-39.
  • Shrivastav A, Patel U, Joshi JR, Kaur A, Agnihotri AS. Study of haemoglobinopathies and Hb variants in population of Western India using HPLC: A report of 7,000 cases. Journal of Applied Haematology 2013 July-September;4 (3): 104-109.
  • Joutovsky A, Hadzi-Nesic J, Nardi MA.HPLC Retention Time as a Diagnostic Tool for Haemoglobin Variants and Haemoglobinopathies: A Study of 60000 samples in a Clinical Diagnostic Laboratory. Clinical Chemistry 2004 Oct;50(10):1736-47.
  • Rangan A, Handoo A, Sinha S, Saxena R, Verma IC, Kumar S et al. Utility of family studies in diagnosing abnormal haemoglobins/ Thalassaemic states. Indian Journal of Pediatrics 2009 June;76(6): 615-21.
  • Dogaru M, Coriu D, Higgins T. Comparison of two analytical methods (electrophoresis and HPLC) to detect Thalassaemias and haemoglobinopathies. Revista Romana de Medicina de Laborator 2007Decembrie;9(4): 39-48.
  • Bain BJ. Laboratory techniques for the identification of abnormalities of globin chain synthesis. In: Haemoglobinopathy diagnosis 2nded. England: Oxford, Blackwell Science;2006.p26-62.
  • Head C E, Conroy M, Jarvis M, Phelan L, Bain BJ. Some observations on the measurement of haemoglobin A2 and S percentages by high performance liquid chromatography in the presence and absence of a thalassaemia. J Clin Pathol 2004;57: 276-80.
  • SEBIA: Instruction manual. Hydragel Haemoglobin [E] K-20.2014.
  • Sachdev R, Dam AR, Tyagi G. Detection of Hb variants and haemoglobinopathies in Indian population using HPLC: report of 2600 cases. Indian J Pathol Microbiol 2010 Jan-Mar;53(1):57-62.