Capillary versus hplc 2016

1. Capillary electrophoresis versus HPLC forCapillary electrophoresis versus HPLC for
diagnostic approach of hemoglobinopathiesdiagnostic approach of hemoglobinopathies
By
Prof. Moustafa Rizk
Prof. of Clinical Pathology
Faculty of Medicine, University of Alexandria
1110/11/17
29/09/2016
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2. agenda…agenda…....
 CAPILLARY ELECTROPHORESIS,CAPILLARY ELECTROPHORESIS,
HPLCHPLC
 HEMOGLOBINOPATHIESHEMOGLOBINOPATHIES
Interferences and Limits
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3. CAPILLARY ELECTROPHORESIS
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4. Thermic
bridge
Temperature
Controlled by
Peltier device
Anode +Cathode -
Injection
Silica Capillary in
thermo-conductive
resin (25µm diameter)
CAPILLARYSTM
TECHNOLOGYCAPILLARYSTM
TECHNOLOGY
Migration
Up to 10.000 volts
Deuterium
lamp
Deuterium
lampDetectorDetector
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5. Capillary tube:
• The cross-section of which (typically 50
µm)
• The length of the capillary differs in
different applications, but is normally in
the region of 20 to 50 cm,
• The capillary is filled with running
buffer
• Fused silica is by far the most
frequently used material, due to its
intrinsic properties, which include
transparency over a wide range of the
electromagnetic spectrum and a high
thermal conductance.
• The inner surface of the capillaries can
be untreated or coated .
-+
High voltage
power supply
The ends of a capillary are placed in
separate buffer reservoirs, each containing
an electrode connected to a high-voltage
power supply capable of delivering up to 30
kV.
• The sample is introduced
by placing one end into the
sample and applying an
electric field (electrokinetic
injection) or by applying
external pressure for a few
seconds (Hydrodynamic
injection)
• An electric potential is
applied across the capillary
and the separation is
performed
Detector
Computer
•detection of separated analytes
achieved directly through the
capillary wall near the opposite end
normally near the cathode
•The output of the detector is sent
to a data output and handling
device such as an integrator or
computer.
•The data is then displayed as an
electropherogram
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6. 10/11/1710/11/17 04:1104:1166
Relative migration
times of the
hemoglobin that
past the detector in
zones from Z1
through Z15, Based
on standardizing on
the location of HbA.

7. Normal profileNormal profile
Hb A
Hb A2
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AA
AA22
Alk.Alk.
Anh.Anh.
Car.Car.

8. HbA
HbF
HbS
HbA2
HbC
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9. 10/11/1710/11/17 04:1104:11 99

10. 10/11/1710/11/17 04:1104:11 1010

11. 10/11/1710/11/17 04:1104:11 1111

12. Interferences and LimitsInterferences and Limits
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13. Hb A2Hb A2
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14. 1-Hb S glycated and Hb A21-Hb S glycated and Hb A2
It is well-known and published that with HPLC technology, in
the presence of Hb S, Hb A2 is falsely elevated due to Hb S
adducts such as carbamylated Hb S and/or glycated Hb S.
Labs must take caution in interpreting such Hb A2 results
and not assume that the patient is also beta-thalassemic.
Using Capillary electrophoresis, all glycated fractions co-
migrate with the main corresponding peak and are not
separated. In the presence of Hb S, CE technology will
provide the user with a more accurate Hb A2 quantitation
and is a better indicator of potential beta-thalassemia.
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15. 10/11/1710/11/17 04:1104:11 1515

16. Heterozygous A/S on Capillarys
Hb S
Hb A2
Hb A
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NN
AA
AA22
Anh.Anh.
Car.Car.
Alk.Alk.
SS
FF

17. Hb S glycated and Hb A2Hb S glycated and Hb A2
BibliographyBibliography
““The glycated HbS1c overlaps the HbA2 fraction (The glycated HbS1c overlaps the HbA2 fraction (...)...)
Therefore, an elevated HbA2 fraction, in the presence of HbSTherefore, an elevated HbA2 fraction, in the presence of HbS
and HbA, should not be considered as an indicationfor b-thaland HbA, should not be considered as an indicationfor b-thal
minor but as an artefact”minor but as an artefact”
(described on BioRad Variant II, Menarini HA 8160, Tosoh G7 and Primus Ultra²)(described on BioRad Variant II, Menarini HA 8160, Tosoh G7 and Primus Ultra²)
Evaluating five dedicated automatic devices for haemoglobinopathy diagnosticsEvaluating five dedicated automatic devices for haemoglobinopathy diagnostics
in multi-ethnic populationsin multi-ethnic populations
P. Van Delft, E. Lenters, M. Bakker-Vermeij, M. de Korte, U. Baylan, C. L. Harteveld, P.C. Giordano,P. Van Delft, E. Lenters, M. Bakker-Vermeij, M. de Korte, U. Baylan, C. L. Harteveld, P.C. Giordano,20092009
International Journal of Laboratory HematologyInternational Journal of Laboratory Hematology
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18. Hb S glycated and Hb A2Hb S glycated and Hb A2
BibliographyBibliography
«« High performance liquid chromatography (HPLC) providesHigh performance liquid chromatography (HPLC) provides
precise but inaccurate estimations of haemoglobin A2 in theprecise but inaccurate estimations of haemoglobin A2 in the
presence of haemoglobin S.presence of haemoglobin S.»»
Some observations on the measurement of HbA2 and HbS percentages by HPLC in the presence and absenceSome observations on the measurement of HbA2 and HbS percentages by HPLC in the presence and absence
ofof αα-thalassemia.-thalassemia.C E Head, M Conroy, M Jarvis, L Phelan, BJ Bain,C E Head, M Conroy, M Jarvis, L Phelan, BJ Bain,2004 Journal of Clinical Laboratory2004 Journal of Clinical Laboratory
“Because an increase of only 1% or 2% can be significant for HbA2, we
routinely place a caveat noting this carryover in our interpretive reports
when the HPLC technique is used.”
Comparison of Sebia Capillarys with the Primus HPLC in the evaluation of hemoglobinopathies.D. Keren,
D. Hedstrom, R. Gulbrasnon, C. Ou, R. Bak,2008 American Journal of Clinical Pathology
10/11/1710/11/17 04:1104:11 1818

19. Hb S glycated and Hb A2Hb S glycated and Hb A2
ComparisonComparison
HPLC: BIO-RAD VARIANT II
BETA-THALASSEMIA SHORT PROGRAM
SEBIA CAPILLARYS 2
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20. 2-Hb E and Hb A22-Hb E and Hb A2
Hb E coelutes with Hb A2 on Bio-Rad and Menarini, and
heavily shoulders with Hb A2 with Primus and Tosoh. Either
way, Hb E and Hb A2 cannot be quantified separately with
the use of HPLC technology.
Note: Tosoh just developed a new buffer allowing a clear
separation of HbA2 and HbE. No information about the
limits of this new buffer (resolution, linearity, interferences
in the detection of others Hb variants, throughput…)
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21. Heterozygous A/EHeterozygous A/E
Hb A
Hb A2
Hb E
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22. 10/11/1710/11/17 04:1104:112222

23. 10/11/1710/11/17 04:1104:11 2323

24. Hb E and Hb A2Hb E and Hb A2
ComparisonComparison
HPLC: BIO-RAD VARIANT II
BETA-THALASSEMIA SHORT PROGRAM
SEBIA CAPILLARYS 2
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HbA2 is not completely separated from HbE by HPLC , and the 2
hemoglobins will be measured together. CZE pattern on the same
sample showes that HbA2 does completely separate from HbE.

26. Hb E and Hb A2Hb E and Hb A2
BibliographyBibliography
« In most rapid HPLC gradients, including the Bio-Rad
Laboratories systems, HbE will elute on top of the HbA2
fraction »
Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic
populations.P. Van Delft, E. Lenters, M. Bakker-Vermeij, M. de Korte, U. Baylan, C. L. Harteveld, P.C.
Giordano,2009 International Journal of Laboratory Hematology
« CE has been proven superior to HPLC in the measurement
of HbA2 in the presence of HbE »
The range of Hemoglobin A2 in Hemoglobin E Heterozygotes as Determined by Capillary
Electrophoresis.D.D. Mais, R.D. Gulbranson, D. Keren,2009 American Journal of Clinical Pathology
10/11/1710/11/17 04:1104:11 2626

27. Hb E and Hb A2Hb E and Hb A2
BibliographyBibliography
« The Sebia Capillarys 2 is superior to the Bio-Rad VARIANT II
for the quantification of HbA2 in the presence of HbE and
HbD Punjab »
Quantification of HbA2 in patients with and without β-thalassemia and in the presence of HbS, HbC,
HbE and HbD Punjab Hemoglobin VariantsT. Higgins, A. Khajuria, M. Mack,2009 American Journal of
Clinical Pathology
« the complete separation of HbA2 from HbE by CE
compared with the lack of measurable HbA2 by HPLC
deserves note. Our interpretive report for the HPLC notes
that the HbE value includes HbA2. However, the CE pattern
in this same case demonstrates a clean separation of HbA2
from HbE »
Comparison of Sebia Capillarys with the Primus HPLC in the evaluation of hemoglobinopathies
D. Keren, D. Hedstrom, R. Gulbrasnon, C. Ou, R. Bak,2009 American Journal of Clinical Pathology
10/11/1710/11/17 04:1104:11 2727

28. 3-Hb Lepore and Hb A23-Hb Lepore and Hb A2
As for the HbE, the Hb Lepore co-elute
with HbA2 in the majority of HPLC
systems
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29. Hb Lepore and Hb A2Hb Lepore and Hb A2
ComparisonComparison
HPLC: BIO-RAD VARIANT I
SEBIA CAPILLARYS 2
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30. Hb Lepore and Hb A2Hb Lepore and Hb A2
ComparisonComparison
SEBIA CAPILLARYS 2
Bio-Rad VARIANT II

31. Hb Lepore and Hb A2Hb Lepore and Hb A2
BibliographyBibliography
« HbA2 values around 10–15% with a somewhat asymmetric
A2 peak will indicate Hb Lepore with a reasonable degree of
confidence (…) Hb Lepore, which migrates on top of the HbA2
fraction on the VARIANT IITM apparatus, is well separated on
the G7 apparatus, but within the HbD window »
Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic
populations,P. Van Delft, E. Lenters, M. Bakker-Vermeij, M. de Korte, U. Baylan, C. L. Harteveld, P.C.
Giordano,2009 International Journal of Laboratory Hematology
« HPLC methods, although precise, have some limitations,
including (…) coelution of various hemoglobins, including
HbE, Hb Osu Christianborg, HbG Coushatta, and Hb Lepore
with HbA2»
Quantification of HbA2 in patients with and without β-thalassemia and in the presence of HbS, HbC,
HbE and HbD Punjab hemoglobin variants,T. Higgins, A. Khajuria, M. Mack.2009 American Journal of
Clinical Pathology
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32. 323210/11/1710/11/17 04:1104:11

33. 4-Hb D and Hb A24-Hb D and Hb A2
Many publications reported
underestimation of HbA2 value
in presence of HbD
using HPLC.

34. Hb D and Hb A2Hb D and Hb A2
ComparisonComparison
HPLC: BIO-RAD VARIANT I
SEBIA CAPILLARYS 2

35. Hb D and Hb A2Hb D and Hb A2
BibliographyBibliography
«« The HbA2 fraction is regularly underestimated in theThe HbA2 fraction is regularly underestimated in the
presence of HbD-Punjab, but then again, in these cases, thepresence of HbD-Punjab, but then again, in these cases, the
HbA2 estimation also has no diagnosticHbA2 estimation also has no diagnostic value»value»
Evaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnicEvaluating five dedicated automatic devices for haemoglobinopathy diagnostics in multi-ethnic
populations,P. Van Delft, E. Lenters, M. Bakker-Vermeij, M. de Korte, U. Baylan, C. L. Harteveld, P.C.populations,P. Van Delft, E. Lenters, M. Bakker-Vermeij, M. de Korte, U. Baylan, C. L. Harteveld, P.C.
Giordano,Giordano,2009 International Journal of Laboratory Hematology2009 International Journal of Laboratory Hematology
« HPLC methods, although precise, have some limitations,
including falsely decreased HbA2 levels in patients with the
HbD Punjab trait due to a rising baseline »
Quantification of HbA2 in patients with and without β-thalassemia and in the presence of HbS, HbC, HbE
and HbD Punjab hemoglobin variants,T. Higgins, A. Khajuria, M. Mack,2009 American Journal of Clinical
Pathology

36. 4-Heterozygous A/C4-Heterozygous A/C
Hb A2
Hb A Hb C
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37. 10/11/1710/11/17 04:1104:113737
HbA2 peak shows slight considerable overlap with HbC

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39. 10/11/1710/11/17 04:1104:113939Am J Clin Pathol 2008;130:824-831

40. 404010/11/1710/11/17 04:1104:11

41. 5-Hb A glycated and Hb F5-Hb A glycated and Hb F
Using HPLC, HbF value is
systematically over-estimated
because of overlap of
HbA1c

42. Hb A glycated and Hb FHb A glycated and Hb F
BibliographyBibliography
« Using the Beta Thal programme of the Bio-Rad Variant II HPLC « Using the Beta Thal programme of the Bio-Rad Variant II HPLC
instrument, a false elevation of haemoglobin F as a result of an elevated instrument, a false elevation of haemoglobin F as a result of an elevated
haemoglobin A1c percentage is observed in an appreciable percentage haemoglobin A1c percentage is observed in an appreciable percentage
of diabetic patients (41% in this study) (…) Haemoglobinopathy of diabetic patients (41% in this study) (…) Haemoglobinopathy
laboratories using this programme for thalassemia and variant laboratories using this programme for thalassemia and variant
haemoglobin diagnosis should be alert to this possible discrepancy and haemoglobin diagnosis should be alert to this possible discrepancy and
should use an alternative programme or method for quantifying should use an alternative programme or method for quantifying
haemoglobin F when this problem occurs »haemoglobin F when this problem occurs »
Inaccuracy of HPLC estimation of HbF in the presence of increased HbA1c,Inaccuracy of HPLC estimation of HbF in the presence of increased HbA1c,V. Grey, M. Wilkinson, L.V. Grey, M. Wilkinson, L.
Phelan, C. Hughes, B.J. Bain,Phelan, C. Hughes, B.J. Bain,2006 International Journal of Laboratory Hematology2006 International Journal of Laboratory Hematology

43. 434310/11/1710/11/17 04:1104:11

44. 6-Hb Bart’s & Hb H6-Hb Bart’s & Hb H
With CE, we see the presence of (alpha-thalassemiaWith CE, we see the presence of (alpha-thalassemia
products) Hb H, Hb Bart’s very clearly and separately!products) Hb H, Hb Bart’s very clearly and separately!
Using HPLC, Hb Bart’s is hardly seen and notated by theUsing HPLC, Hb Bart’s is hardly seen and notated by the
user, and, due to the fast elution of Hb H (Hb H anduser, and, due to the fast elution of Hb H (Hb H and
Bart’s might elute “prior to the start of integration withBart’s might elute “prior to the start of integration with
HPLC”), it is either not seen or coelutes with Hb Bart’sHPLC”), it is either not seen or coelutes with Hb Bart’s
and therefore is not notated here by the user. This isand therefore is not notated here by the user. This is
definitely a POSITIVE for CE technology! Also, withdefinitely a POSITIVE for CE technology! Also, with
HPLC, Hb Constant Spring can easily be missed.HPLC, Hb Constant Spring can easily be missed.

45. Hb H & Hb Bart’sHb H & Hb Bart’s
ComparisonComparison
HPLC: BIO-RAD VARIANT I
SEBIA CAPILLARYS 2

46. HbH & Hb Bart’sHbH & Hb Bart’s
BibliographyBibliography
« In conclusion, the advantage of the CE system is its ability
to separate and measure all important Hb fractions. We
expect that CE will measure the levels of Hb Bart’s accurately
in newborns, which will help to diagnose α-thalassemia
syndromes considering levels of Hb Bart’s of 1.8% 0.5% (-
α/αα), 9.2% 1.1% (-α/-α or --/αα), and 24% to 25% in Hb H
diseases»
Rapid diagnosis of thalassemias and other hemoglobinopathies by capillary electrophoresis system,P. WINICHAGOON, S.
SVASTI, T. MUNKONGDEE, W. CHAIYA, P. BOONMONGKOL, N. CHANTRAKUL, and S. FUCHAROEN,2008 Translational
Research

47. Hb H & Hb Bart’sHb H & Hb Bart’s
ComparisonComparison
BIO-RAD VARIANT II SEBIA CAPILLARYS 2

48. This zone corresponding to degraded
Hb is not included in the Hb F value
with Capillarys Neonat
7-Difference to quantify Hb F7-Difference to quantify Hb F
between HPLC and Capillarys Neonatbetween HPLC and Capillarys Neonat
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49. - A very good Hb A- A very good Hb A22 focalization allowing anfocalization allowing an
automatic identification and quantification ofautomatic identification and quantification of
this fraction.this fraction.
- A direct identification of the main- A direct identification of the main
hemoglobin variants.hemoglobin variants.
The Hb Capillarys allows:
CONCLUSION

50. CONCLUSIONCONCLUSION
HPLC Valuable Laboratory TechniqueHPLC Valuable Laboratory Technique
Discussed Common VariantsDiscussed Common Variants
Important To ID A1c VariantsImportant To ID A1c Variants

51. 10/11/1710/11/17 04:1104:115151
Thank you for
listening.
Any Questions?
Prof. Moustafa
Rizk
Thank you for
listening.
Any Questions?
Prof. Moustafa
Rizk