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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2021  |  Volume : 64  |  Issue : 3  |  Page : 518-523
Evaluation of role of HPLC (Merits & Pitfalls), in the diagnosis of various hemoglobinopathies & thalassemic syndromes


1 Specialist Hematologist, Department of Hematology, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA
2 Prof. and Head Department of Anatomy LUC Malaysia, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA
3 Head Cytogenetics, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA
4 Prof. Cytogenetic Department, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA
5 Laboratory Head and Director, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA
6 Deputy Lab. Director, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA
7 Supervisor Laboratory, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA
8 Senior Staff, Department of Special Chemistry, MMCH (Tertiary Care Hospital and Thalassemia Referral Centre), Madinah Munawarah, KSA

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Date of Submission19-Jun-2020
Date of Decision20-Jul-2020
Date of Acceptance09-Dec-2020
Date of Web Publication28-Jul-2021
 

   Abstract 


Background: HPLC is one of the most important tools for accurate diagnosis of hemoglobinopathies and thalassemias. The advantage of the HPLC system is the excellent resolution, reproducibility &quantification of several normal and abnormal hemoglobin. Results: BIO RAD Variant II analyzer was used. Sickle cell syndromes including double heterozygous states accounted for 56.13% of total cases. HbSS, HbS/β0-th, HbS/β+–th β-thal trait comprises 29%, 6.5%, 5.1%& 10% of total cases respectively with mean MCV (fl) = 84, 68,71,64 respectively. The Mean HbA2 for β-thal trait, HbE trait &HbE-β thal showed 5.1 ± 1.1, 19 ± 9 & 24 ± 8 respectively. HbF is increased in 8.6% case (excluding SC syndromes & β-thal disorders), of these 5.5% were infants & 12 cases of Aplastic Anemias. Peak P2 >7% (2.4% cases) was seen in uncontrolled diabetes mellitus which on quantification showed HbA1C = 8 ± 2.1 mmol/L. Discussion: HPLC in correlation with CBC parameters & family studies can aid in the diagnosis of majority of Hemoglobinopathies and thalassemic syndrome. The CBC & HPLC parameters of the present study are in good correlation with the research conducted by Tejinder Sing, RiouJ & Alla Joutovsky. Present study showed HPLC comprehensively characterizing HbS, A, A2, F, S, C, D from each other & was also applicable for the quantification of HbA1c for the monitoring of Diabetes Mellitus. Conclusion: The merits of HPLC are small quantity of sample required, economical, less TAT, accurate categorization of HbS, HbA2 & F. But one has to be aware of the limitations and problems associated with this method due to variant hemoglobin within the same retention windows. The present findings show HPLC as an excellent & powerful diagnostic tool for the direct identification of hemoglobin variants with a high degree of precision in the quantification of normal and abnormal hemoglobin fractions.

Keywords: Hemoglobinopathies, High-performance liquid chromatography, sickling test, thalassemia

How to cite this article:
Baig MA, Swamy K B, Baksh AD, Bahashwan A, Moshrif Y, Al Sawat A, Al Mutairi N, Alharbi N. Evaluation of role of HPLC (Merits & Pitfalls), in the diagnosis of various hemoglobinopathies & thalassemic syndromes. Indian J Pathol Microbiol 2021;64:518-23

How to cite this URL:
Baig MA, Swamy K B, Baksh AD, Bahashwan A, Moshrif Y, Al Sawat A, Al Mutairi N, Alharbi N. Evaluation of role of HPLC (Merits & Pitfalls), in the diagnosis of various hemoglobinopathies & thalassemic syndromes. Indian J Pathol Microbiol [serial online] 2021 [cited 2022 Dec 6];64:518-23. Available from: https://www.ijpmonline.org/text.asp?2021/64/3/518/322415





   Introduction Top


Hemoglobinopathies are inherited single-gene disorders affecting globin chains. SCD is the most common Hemoglobinopathy affecting 7% of world's population (i.e. 420 million are carriers).

Thalassemia are inherited autosomal recessive disorders characterized by the absence or reduced synthesis of one or more globin chains. Detailed family& Clinical history, CBC, peripheral smear and ancillary techniques like HPLC are required for diagnosis of disorders of Hb.[1]

The main objective of this study is to evaluate the role of HPLC in characterization of various Hemoglobinopathies/Thalassemic syndrome in correlation with CBC parameters.

HPLC advantages,[2]

  1. HPLC is automated, requires less staff and can run more samples in a short interval of time.
  2. HPLC usually separates Hbs A, A2, F, S, C, D Punjab and G Philadelphia from each other.
  3. HPLC is also applicable for the quantification of HbA1c for the monitoring of DM.
  4. Small quantity of samples (5 ml) is sufficient for analysis; this is especially useful in pediatric patients.
  5. Quantification of normal and variant hemoglobin is available on every sample.


Disadvantages,[3]

  1. HbH and HB Barts are difficult to identify and cannot quantitate.
  2. Minor peaks due to aging and glycosylation.
  3. Hemoglobin A2 cannot be differentiated from Hb E and HbLepore.
  4. Many variants show similar retention times.



   Materials and Methods Top


This is a retrospective One year study (July 2019 to June 2020) carried out in Hematology Dept.

Madinah Maternity and Children Hospital (MMCH) KSA. This is also referral Thalassemia & Hemophilia tertiary care center for Pediatric patients.

BIO RAD Variant II HPLC analyzer is used in this study.

Principle

The Variant II Beta Thalassemia Short Program utilizes principles of ion-exchange HPLC. The samples are automatically mixed and diluted on the Variant II Sampling Station (VSS) and injected to the analytical cartridge. The Variant II Chromatographic Station (VCS) dual pumps deliver a programmed buffer gradient of increasing ionic strength to the cartridge, where HbA2/F are separated based on theirionic interaction with the cartridge material [Figure 1].
Figure 1: BIORAD VARIANT II

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Procedure[4],[5]

  1. 2 ml of venous blood sample in EDTA Tube.
  2. If blood sample is <1.5 ml, dilute (1:200 ie 5μl of blood sample to 1 ml of wash diluent)
  3. Blank – Distilled water.


Controls

  1. Reconstitue with 0.5 ml of distilled water, mix well, allow to stand for 10 min
  2. Dilute 1:200 prior to analysis (5μl of control to 1 ml of distilled water)
  3. Run controls at the beginning & end of run
  4. Stable for 21 days at 2-80C.


Blood primer

Reconstitute with 1 ml distilled water, Mix well, allow to stand for 10 min. Stable for 21 days at 2-80C.

Inclusion criteria

  1. MHA (MCV <80 fl, MCH <27 pg, and RBC count >5 million/l) not responding to conventional iron treatment,
  2. MENTZERIndex : - If MCV/RBC <13 - Thalassemic trait.


Exclusion criteria

  1. IDA cases were excluded on the basis of serum iron profiles & Ferrittin
  2. Other hemolytic anemias were excluded on the basis of CBC, PBS, Retic, DCT, G6PDH assay, Osmotic fragility test etc.


Statistical analysis

Continuous variables were expressed as mean ± SD. Categorical variables were expressed as frequencies and percentages. Two sample t test was applied for significance of correlation between continuous variables. All statistical analysis was performed using SPSS 16.0 statistical software.


   Results Top


[Table 1] shows the distribution of cases with SCA (homozygous state) comprising 29% of total cases & forms the most common disease, next in order being Sickle cell trait (13.6%), Beta thalassemia triat (10.7%), Beta thalassemia major (6.5%) and sickle cell-Beta thalassemia (6.5%).
Table 1: A total of 579 cases of hemoglobinopathies and thalassemic syndromes were studied

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HbF was raised in 50 cases (8.6%) comprising of congenital bone marrow failure syndromes and HPFH & P2 was increased in 14cases (2.4%) signifying Diabetes Mellitus. [Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8],[Figure 9],[Figure 10],[Figure 11] shows peaks and retention times in various hemoglobinopathies. [Figure 2] and [Figure 7] shows Sickle cells and target cells in SCA. [Figure 3] shows Golf ball appearance in Brilliant cresyl blue stain in HBH disease. [Figure 4] shows case of Sickle –Beta Thalassemia with raised HbF and low MCV. [Figure 5] shows P3 Peak with Retention time 21% @ 1.58 min indicating presence of rare HbJ Oxford. [Figure 6] is a case of Diabetes Mellitus showing raised P2 due to ↑ HbA1C.
Figure 2: Sickle cells with target cells (SCA)

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Figure 3: Brilliantcresyl blue - Golf ball appearance (HBH disease)

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Figure 4: Sickle –Beta thalassemia with raised HbF and MCV low

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Figure 5: P3 Peak – 21% with Retention time = 1.58 min indicating presence of HbJ Oxford

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Figure 6: P2 is increased after elution shows ↑ HbA1C (Diabetes Mellitus)

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Figure 7: Sickle cell anemia

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Figure 8: Physiological ↑ HbF in infant

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Figure 9: B-Thalassemia trait

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Figure 10: Hereditary persistent of fetal hemoglobin (HPFH)

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Figure 11: SCA with denatured HbS in A2 region

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Figures 8 and 10 are Physiological ↑ HbF in Infant and Hereditary Persistent of fetal Hemoglobin (HPFH) respectively. [Figure 11] is a case of SCA with denatured HbS in A2 region


   Discussion Top


HPLC is one of the most important tools for accurate diagnosis of hemoglobinopathies and thalassemias

The following supplementary investigations were done

  1. Family studies, CBC, PBS, Retic%, indirect bilirubin, LDH, Serum iron profiles & Serum ferritin levels
  2. For Peaks <0.63 min on HPLC, the presence of HbH was confirmed by using brilliant cresyl blue stain which showed golf ball appearance [Figure 3] and Hb barts were confirmed as fast migrating hemoglobins on Cellulose acetate Hb Electrophoresis (PH-8.6)
  3. Delta beta thalassemia cases showed normal HbA2 with increased HbF. These findings were correlated with MCV and PBS which showed anisopoikilocytosis, hypochromic microcytic red cells, target cells, and basophilic stippling.


In this study Sickle cell syndromes including double heterozygous states accounted for 56.13% of total cases. HomozygousHbSS comprised of 29% of total cases with MCV = 84 ± 6 &HbS 87 ± 6. PBS showed Sickle cells & Sickling test was positive in all the cases. HbS/β0-th &HbS/β+-th comprises 6.5% & 5.1, HbA2 – 4.2 & 4.1 & HbS - 84 ± 9 & 45 ± 12 respectively.

β-thal trait showed HbA2 of 5.1 ± 1.1% & MCV 64 ± 7. Hb Etrait & HbE-β thal showed A2-19 ± 9 & 24 ± 8, respectively. The CBC & HPLC parameters of the present study are in good correlation with the research conducted by Tejinder Singh,[2] Riou J,[3] & AllaJoutovsky.[5]

23 cases showed Peak in <0.63 min area of which 05 cases could be categorized as HBH disease & 18 cases remained undiagnosed in spite of doing supravital stains &Hb electrophoresis. Genetic studies were not done in these cases.

HbF was elevated in 8.6% cases (excluding SC syndromes & β-thal disorders). Of these cases 5.5% were infants & the raised HbF was normal for their age. 12 cases showed Isolated ↑ HbF without other abnormalities in HPLC. Bone marrow aspiration studies showed these as Aplastic Anemias. Our study results are in correlation with Laferty JD et al,[8] Eastman JW et al,[9]& Tyagi et al.[10]

Peak P2 was seen is 16 cases, of which one was diagnosed as Hb HOPE with 48% P2. Other causes of increased P2 >7% were uncontrolled diabetes mellitus which on quantification showed HbA1C = 8 ± 2.1 mmol/L. [Table 2] shows peak names with Retention times in minutes and Hemoglobin variants. Hb A2= 3.5-7% is suggestive of B thal trait. S window with retention time 4.30-4.70 indicates Sickle cell syndrome. [Table 4] shows the pitfalls and Limitations of HPLC in characterisation of Hemoglobinopathies and their drawback in interpretation of results.
Table 2: Manufacturer-assigned windows for bio-rad variant II HPLC system[2],[6]

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Table 3: HPLC findings in various hemoglobinopathies[7]

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Table 4: Pitfalls of HPLC observed in our study

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Role of HB electrophoresis as an aid to HPLC in diagnosis of hemoglobinopathies

The Alkaline Cellulose acetate electrophoresis with pH 8.6 can be used for detection of

fast migrating hemoglobins - H, Barts, N-Baltimore and J-Toronto. The S band shows G-Philadelphia, D-Iran, D-Punjab, Lepore and band A2 shows C, E, O-Arab. [Table 3] shows % HbS, HbA2, HbF in various Hemoglobinopathies.

Acid Agarose gel electrophoresis (pH 6.4) can be used to differentiate HbS and HbD which share the same migration position and hence cannot be separated on cellulose acetate. On Acid electrophoresis band A shows D, G, Lepore, E and A2. All hemoglobins that run in the fast lane in alkaline gel will also be found here.


   Conclusions (Inferences From Our Study) Top


  1. Most common cause of Peaks in <0.63 min is due to Hb barts/HbH disease which are difficult to diagnose by HPLC & needs supravital stains (HbH –Golf ball pattern), Hb electrophoresis & Genetic studies
  2. After Blood transfusion, if HbS <15 then Sickling test can be false negative. Suspected SCA should be screened by sickling test & later confirmed by HPLC
  3. P2 peaks can be seen in uncontrolled diabetics because HbA1C elutes in P2 region & if it is >7% then quantify HbA1C
  4. HPLC in correlation with CBC parameters & family studies can aid in the diagnosis of majority of Hemoglobinopathies and thalassemic syndrome. The merits of HPLC are small quantity of sample, economical, short TAT, accurate categorization of HbS, HbA2, F. But one has to be aware of the limitations and problems associated with this method due to variant hemoglobins within the same retention windows. The present findings show HPLC as an excellent, powerful diagnostic tool for the direct identification of hemoglobin variants with a high degree of precision in the quantification of normal and abnormal hemoglobin fractions.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Bain BJ, Wild BJ. Chapter 14, Investigation of abnormal hemoglobins and thalassemia. Dacie and Lewis Practical Haematology. 11th ed. 2011. p. 301-31.  Back to cited text no. 1
    
2.
Khera R, Singh T. HPLC in characterization of hemoglobin profile in thalassemia syndromes and hemoglobinopathies: A clinicohematological correlation. Indian J Hematol Blood Transfus 2015;31:110-15.  Back to cited text no. 2
    
3.
Wild BJ, Stephens AD. The use of automated HPLC to detect and quantitate haemoglobins. Clin Lab Haematol 1997;19:171-6.  Back to cited text no. 3
    
4.
Riou J, Godart C, Didier H, Mathis M, Bimet C. Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants. Clin Chemistry 1997;43:34-9.  Back to cited text no. 4
    
5.
Joutovsky A, Hadzi-Nesic J. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: A study of 60,000 samples in a clinical diagnostic laboratory. Clin Chem 2004;50:1736-47.  Back to cited text no. 5
    
6.
Working Party of the General Haematology Task Force of the British Committee for Standards in Haemotology. Guideline: The laboratory diagnosis of haemo-globinopathies. Br J Haematol 1998;101:783-92.  Back to cited text no. 6
    
7.
Bain BJ. Hemoglobinopathy Diagnosis. Oxford, England: Blackwell Science Ltd.; 2001. p. 260-71.  Back to cited text no. 7
    
8.
Lafferty JD, McFarlane AG, Chui DHK. Evaluation of a dual hemoglobin A2/A1c quantification kit on the Bio-Rad Variant II Automated Hemoglobin Analyzer. Arch Pathol Lab Med 2002;126:1494-9.  Back to cited text no. 8
    
9.
Eastman JW, Wong R, Liao CL. AutomatedHPLC screening of newborns for sickle cell anemia and otherhemoglobinopathies. Clin Chem 1996;42:704-10.  Back to cited text no. 9
    
10.
Tyagi S, Saxena R, Choudhry VP. HPLC—how necessaryis it for haemoglobinopathy diagnosis in India? Indian J Pathol Microbiology 2003;46:390-3.  Back to cited text no. 10
    

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Correspondence Address:
Mirza A Baig
MD(Pathology), MMCH, KSA, and PhD Lincoln University College, Selangor
KSA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_709_20

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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