Session Number 2002 Unraveling Hemoglobinopathies with Capillary Electrophoresis David F. Keren, M.D. Professor of Pathology Division Director, Clinical Pathology The University of Michigan [email protected] Financial Disclosure Information In the past 12 months, I have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in my presentation. Hemoglobin Structure Alpha Globin Gene Cluster ( Short arm of Chromosome 16) 5 ’ 3’ 40 kb z LCRA (HS-40) z1 a1 a1 a2 a1 Beta Globin Gene Cluster ( Short arm of Chromosome 11) 3’ 5 ’ LCRB e Gg Ag b d b Embryonic Hemoglobins Hb Gower I (z2e2) Hb Gower II (a2e2) Hb Portland (z2g2) Fetal & Adult Hemoglobins Hb A (a2b2) 95% of adult Hb Hb F (a2g2) 70% neonate <2% adult Hb A2 (a2d2) 2.2-3.4% of adult Hb Nomenclature • Alphabetical – Hb A • Hb A2 (minor fraction seen on Electro in 1955) – – – – – – Hb B ?????????????????? = Hb S Hb C Hb D Hb E Hb F (fetal) →→→→→→→→→→→→→→→→→Hb Q-India • Location: e.g. Hb Ann Arbor Hemoglobin Shorthand Hb Ann Arbor = a80Leu→Arg • a refers to the abnormal chain • 80 is the position with a substitution • Leucine is the normal amino acid • Arginine is the substituted amino acid ~1,000 Variant Hemoglobins • Most Variants are Asymptomatic • Structural Variants – Alpha: Hb G Philadelphia – Beta: Hb S, Hb C, Hb D, Hb O – Delta: Hb A2‘ • Structural & Thalassemia – Constant Spring (alpha variant) – HbE (beta variant) – Lepore (delta-beta fusion protein) Malaria Distribution parallels Major Hemoglobinopathies & Thalassemias Distribution of malaria Harteveld and Higgs Orphanet Journal of Rare Diseases 2010, 5:13 http://www.ojrd.com/content/5/1/13 Investigation of Hemoglobin • Clinical: age, transfusion, race, therapy • Routine: RBC, MCV, MCH, RDW, sickle test • Analytical – Alkaline & Acid electrophoresis – HPLC—(Hb A2 & Hb F) • Cationic exchange: several types – Capillary Electrophoresis (CE) • High pH (10.0) • Confirm Variant: two methods • Referral: Mass spectrometry/Molecular Gel Electrophoresis Alkaline conditions pH 8.6 • Densitometry for fractionation (inadequate for Hb A2 and Hb F) • Cannot differentiate: Hb A2, C, O, or E Hb S, D, G Acid conditions pH 6.5 • Differentiates: D & G from S (but can’t tell D from G) E & O from C Alkaline Gel FASC FAS FS F Köln /A FSC F (C) (F) E/A (F) E/A (F) A G/S (F) A S F A/J (F) A/Chicago (F) A S (A) S C (F) A S ( ) denotes low concentration + Anode Carbonic Anhydrase + Anode CSFA A2 S F A SF Köln F A CSF (C) F EA EA SG S/G A A2 S A FAJ A2 A/Chicago A2 S (F) A C S (A) A2 S A Acid Gel High Performance Liquid Chromatography (HPLC) • • • • • • • • Improved Sensitivity over gels Accurate measurement of Hb A2 and Hb F Complex patterns for interpretation Hb H difficult to measure – Does not separate from A1c on some – Elutes prior to routine measurement on some Bilirubin interferes with Hb Bart’s detection Hb S & Hb C adducts interfere with Hb A2 Cannot separate Hb A2’ from Hb S Cannot separate Hb A2 from Hb E on most Biorad Variant I HPLC Hb A Glycated & Aged Hb A Bilirubin & degradation products Hb A2 Aging HbA Glycated HbA Bio-Rad Variant-II HPLC Hb A Hb A2 Degradation products Peak 1 (glycated A1a,A1b & degradation products) Peak 2 (glycated A1c) BiotechTrinity Ultra HPLC Peak 3 (glycated A1d & degradation products) Peak 5 (Hb A2) Peak 4 (Hb A) Nl <5% Nl 1.7-3.1 Capillary Electrophoresis Positive buffer ions (+) flow to cathode Detector Hb A Hb F Hb S Hb A2 (+) Sample 415nm - Cathode +Anode Sebia Capillarys Hb SC-What to do with no HbA? Use these measurements Overlay with AFSC Controls Use these measurements Mix 1:1 with Normal Sample Do NOT use these measurements Capillary Electrophoresis • Patterns much less complex than HPLC • Accurate quantification of Hb A2, F, and S • No interference of Hb S adducts with Hb A2 • HbA2’ visible in the presence of Hb S • Clear separation of Hb D, G &E from Hb A2 • Detects and measures Hb H and Hb Bart’s • Bilirubin does not interfere with Hb Bart’s 51 y/o man HPLC Pattern RBC 6.14 4.4-5.7 Hgb 13.5 13.5-17 Hct 42.8 40-50 MCV 78.1 79-99 MCH 21.9 27-32 RDW 18.1 11.5-15.0 Hb A Hb ? Position Hb A2 Ultra HPLC Relative Retention RT/S Hb A2 0.85-0.91 Delta G Philadelphia 0.88-0.91 Alpha D Los Angeles 0.91-0.95 Beta 51 y/o man RBC 6.14 4.4-5.7 Hgb 13.5 13.5-17 Hct 42.8 40-50 MCV 78.1 79-99 MCH 21.9 27-32 RDW 18.1 11.5-15.0 Hb G2 HbAA Hb HbA2 A2 Hb 72.6 59.6 ?? >95 >95 1.7-3.1 1.7-3.1 HbFF Hb HbG+A2 G+A2 Hb 00 27.4 27.4 <2.0 <2.0 36-40* 36-40* Hb G2 ? Position for Hb G 0.88-0.92 (Cannot separate HbA2 included) Capillary Electrophoresis HbG Philadelphia Trait Hb A Hb G Philadelphia Hb A2 Hb G2 Hb G (Philadelphia a68Asn→Lys) & a Thalassemia Clinically benign, even with Hb S Associated with a Thalassemia 0 a deletions = 25% Hb G (& G2 relative to A2) 1 a deletion = 33% 2 a deletions = 50% G2 band is always present aA A a A a G a b g d Hb A Hb F Hb A2 Hb A Hb F Hb A2 Hb A Hb F G Hb F Hb A2 Hb G Hb G2 32 y/o woman RBC 4.14 3.9-5.0 Hgb 12.4 12.0-16.0 Hct 36.5 36-48 MCV 88.1 79-99 MCH 30.1 27-32 RDW 12.9 11.5-15.0 Hb A HbA2) Hb ?? Hb ?? Ultra HPLC Relative Retention RT/S Hb A2 0.85-0.91 Delta G Philadelphia 0.88-0.91 Alpha D Los Angeles 0.91-0.95 Beta 32 y/o woman RBC 4.14 3.9-5.0 Hgb 12.4 12.0-16.0 Hb A 58.1 Hct 36.5 36-48 Hb D + A2 41.9 MCV 88.1 79-99 Hb F MCH 30.1 27-32 RDW 12.9 11.5-15.0 0 >95 <2.0 Hb A Hb D (Cannot separate HbA2) Hb D trait b variant Normal MCV Normal RBC No “G2” Hb D Hb D Punjab Hb A Capillary HbD Trait Hb A 51.9 >95 Hb A2 3.2 1.7-3.1 Hb F 0.7 <2.0 Hb D 44.2 Hb A2 Hb F Hemoglobin D (Los Angeles or Punjab) b121 glu→gln Patel et al. Intl J Lab Hematol 2014;36:444-50. Innocuous as Hb D Trait or Homozygote Difficult to distinguish from Hb G by gels Distinction is important: Hb SG behaves like sickle trait Hb SD moderate sickling disorder Hb D with b-thalassemia gives Thalassemia Intermedia or even Thalassemia Major picture Hb SD & a-thalassemia gives microcytosis Comparison of CE to HPLC • Easier pattern to interpret • No glycated products to deal with • But what about Precision in separating variants? • Looked at separation of two closely migrating variants: – 43 consecutive cases of Hb D and HbG traits Ultra HPLC Relative Retention RT/S G Philadelphia 0.88-0.91 Alpha D Los Angeles 0.91-0.95 Beta HPLC (Ultra)-Elution Time Keren et al. Am J Clin Pathol 2012;137:660-4. 30 of 43 samples overlap. Capillarys-Migration Position Keren et al. Am J Clin Pathol 2012;137:660-4. 25 of 43 samples overlap. HPLC (Ultra)-Elution Time/Hb S Keren et al. Am J Clin Pathol 2012;137:660-4. Only 2 of 43 samples overlap. Capillarys-Migration/Hb A2 Keren et al. AJCP 2012 0 of 43 samples overlap. 11 9 7 7 2 1 1 3 Chromosome 11 Beta Thalassemia Trait (Minor) 3’ 5’ LCRB e Gg Ag b d b d Xb 5’ LCRB • • • • e Gg Ag b >200 b+ vs b0 Mutations (deletions uncommon) Lose 30-50% b globin Key is elevated hemoglobin A2 (a2d2) (>3.5%) Low MCV & MCH nl RDW, usually nl hgb, ↑ RBCs 3’ Hb A2 on Variant II HPLC University of Leiden Van Delft et al. Intl J Lab Hematol 2009;31:484-95. HbA2 reduced in d Thalassemia & varriant carriers Specificity and overlap of d/b HbThalassemia A2 values in different cohorts of patients Beta Thal Trait Method HPLC b/a Thalassemia combinations High HbA2 b Thalassemia carriers “Normal” Hb A2 b Thalassemia carriers Hb H Disease a Thalassemia trait a Thalassemia trait Normal a Thalassemia trait Fe Deficiency Normal Range is Method Dependent Precision of Hb A2 CAP Survey 2010 Results on Normal Samples Survey # Gel-1 Gel-2 HPLC CE HG-01 27.4* 26.7 7.5 6.5 HG-02 23.4 21.6 5.6 5.6 HB-03 22.6 21.0 6.8 4.0 * Data is Coefficient of Variation (%) Precision of Hb A2 Paleari et al. Intl J Lab Hematol 2012: 1-7 • Samples (duplicates) run at 2 institutions: • 40 healthy, 29 beta thalassemia & 11 low Hb A2 Method Instrument HPLC BioRad I BioRad II Menarini HA 8160 Tosoh G7 Tosoh G8 Beckman MDQ Beckman PA800 Sebia Capillarys II Capillary Hb A2 <3.5 Hb A2≥3.5 2.7 1.6 0.5 2.8 1.1 4.4 3.3 2.0 4.4 2.0 0.5 1.5 0.8 3.2 1.6 1.2 Beta Thalassemia Trait 56 y/o female RBC 5.0 3.9-5.0 Hgb 10.6 12.0-16.0 Hct 33.9 36-48 MCV 68 79-99 MCH 21.3 27-32 RDW 15.0 11.5-15.0 Hb A 94.4 >95 Hb A2 4.6 1.7-3.1 Hb F 1.0 <2.0 Ref Range A2 1.7-3.1 Same Case Beta Thalassemia Trait Fractions Hb A Hb F Hb A2 % 94.1 1.0 4.9 Ref. % 95-97 <2.0 2.2-3.2 Hb A 94.1 >95 Hb A2 4.9 1.7-3.1 Hb F 1.0 <2.0 Hb A2 (Delta)Variants A2 1.3 A2’ 1.3 Fractions Hb A Hb A2 Hb A2’ % 97.5 1.3 1.2 Ref. % 95-97 <2.0 2.2-3.2 Hb A2’ • Most Common Delta Variant • Present in ~1% of African Americans • Migrates in the same position as Hb S by HPLC (but not by Capillary Electrophoresis) • When present need to add to Hb A2 to assess the complete delta component in: – Beta Thalassemia – Alpha Thalassemia – Iron Deficiency A2 1.6 A2v 0.7 Fractions Fractions Hb A Hb A Hb HbA2 A2 Hb A2v Hb A2’ %% 97.5 97.5 1.8 1.8 0.7 0.7 Ref. Ref.%% 95-97 95-97 2.2-3.2 2.2-3.2 Delta Thalassemia • Clinically Silent trait • Decrease in normally migrating Hb A2 – Structurally normal delta – Suspect with nl CBC & decreased Hb A2 – May give falsely ―normal‖ value in patient with beta thalassemia • Decrease in Hb A2 + Hb A2v – Similar to Hb E a beta variant that is produced in decreased amount A2 1.1 Fractions Hb A Hb F Hb A2v Hb A2 % 97.5 6.9 1.3 1.2 Ref. % 95-97 <2.0 2.2-3.2 Hidden Delta Variant • Clinically Silent trait • Decrease in normally migrating Hb A2 – Structurally abnormal delta – Suspect with nl CBC & decreased Hb A2 – May give falsely ―normal‖ value in patient with beta thalassemia • Repeat with a different technique – Capillary, HPLC, Isoelectric Focusing 18 y/o female sickledex positive RBC 4.2 3.9-5.0 Hgb 12.8 12.0-16.0 Hb A 59.6 >95 Hct 39.4 36-48 Hb A2 3.8 1.7-3.1 MCV 84.0 79-99 Hb F 0 <2.0 MCH 28.4 27-32 Hb S 36.6 36-40* RDW 14.6 11.5-15.0 *Expected for Sickle Trait Same Case: Sickle Trait Hb A2 HPLC CE 3.8 2.9 Fractions Hb A Hb F Hb S Hb A2 % 58.4 0 38.7 2.9 Ref. % 95-97 <2.0 2.2-3.2 HPLC vs CE for Hb A2 Keren et al. AJCP 2008;130:824-31 Effect of Hb S on Hb A2 8.0 7.0 Primus HPLC 6.0 5.0 Hb S Containing Samples No Structural Variant 4.0 3.0 2.0 1.0 0.0 0.0 2.0 4.0 Sebia CE 6.0 8.0 Hb S Trait: HPLC vs CE Keren et al. AJCP 2008;130:824-31 Hemoglobin A2 (%) 6 5 4 3 2 1 0 CE CE-S HPLC HPLC-S Hb S Trait with Elevated Hb A2 • Hb S Trait: Hb S = 36-40%, normal CBC but slight increase in Hb A2 (usually in nl range) • Reasons for Increase Hb A2 1. d globin competes better than bs for a globin - actual increase ~0.5% 2. HPLC artifact: Hb S breakdown products in Hb A2 peak - false increase 1-2% 36 y/o woman Sickledex negative RBC 4.87 3.9-5.0 Hgb 12.1 12.0-16.0 Hct 35.4 36-48 MCV 75.8 79-99 MCH 25.4 27-32 RDW 14.3 11.5-15.0 Rel Rt = S 0.91 Hb? = S 1.01 Ultra HPLC Relative Retention RT/S 0.85-0.91 Delta G Philadelphia 0.88-0.91 Alpha D Los Angeles 0.91-0.95 Beta Hb A2 36 y/o woman RBC 4.87 3.9-5.0 Hgb 12.1 12.0-16.0 Hct 35.4 36-48 MCV 75.8 79-99 MCH 25.4 27-32 RDW 14.3 11.5-15.0 Hb A 60.7 >95 Hb F 1.0 <2.0 Hb G+A2 35.9 Hb G2 2.4 Hb G + A2 = 35.9% Hb G2 = 2.4% Hb G Philadelphia & b & a Thalassemia Hb A2 + G2 HPLC CE ? 5.5 Fractions % Ref. % Hb A Hb F Hb G Hb A2 Hb G2 61.4 0.4 32.7 3.4 2.1 95-97 <2.0 2.2-3.2 Hb G (Philadelphia a68Asn→Lys) & a Thalassemia Clinically benign, even with Hb S Associated with a Thalassemia 0 a deletions = 25% HbG (& G2 relative to A2) 1 a deletion = 33% 2 a deletions = 50% G2 band is always present aA A a A a G a b b d Hb A Hb A Hb A2 Hb A Hb A Hb A2 Hb A Hb A Hb A2 Hb G Hb G Hb G2 Chromosome 16 Hemoglobin H Disease MCR z a2 a1 MCR z a2 • • • • • • a1 Severe microcytosis (MCV 55-64) Hb A2 low (<1.7) Moderate hemolytic anemia, splenomegaly Usually not transfusion dependent Hb Bart’s &/or H is found Can transmit Bart’s Hydrops fetalis Hb H Disease 31 yr woman RBC 5.07 3.9-5.0 Hgb 9.5 12.0-16.0 Hct 31.9 36-48 MCV 63 79-99 MCH 18.8 27-32 RDW 23.8 11.5-15.0 Hb A 89.3 >95 Hb A2 1.0 1.7-3.1 Hb H & A1c 7.9 Hb Bart’s 1.8 Hb Bart’s Hb H & A1c (can’t measure HbH alone) Ref Range A2 1.7-3.1 Bilirubin Masquerading as Barts Howanitz et al. AJCP 2006;125:608-14 Bilirubin Hb H Disease with Hb H and Barts Hb A Hb H Hb Barts Fractions Hb H Hb Bart’s Hb A Hb A2 Hb A2 % Ref. % 16.5 0.7 82.2 0.6 95-97 2.2-3.2 36 y/o woman sickledex positive RBC 4.67 3.9-5.0 Hgb 11.8 12.0-16.0 Hct 35.1 36-48 MCV 75.2 79-99 MCH 25.4 27-32 RDW 15.0 11.5-15.0 Hb A 73.0 >95 Hb A2 4.3 1.7-3.1 Hb S 32.7 35-40* *Expected for Sickle Trait Beta globin products in Thalassemia with & without Hb S Normal b Thal Hb S Trait Hb S/a Thal Hb S/b Thal 24 y/o woman RBC 5.10 3.9-5.0 Hgb 13.8 12.0-16.0 Hct 40.4 36-48 MCV 78.2 79-99 MCH 27.1 27-32 RDW 13.6 11.5-15.0 Hb A2 = 1.4% ? Hb A Rel Rt = A 1.17 Hb E 1.17-1.26 Hb E trait RBC 5.10 3.9-5.0 Hgb 13.8 12.0-16.0 Hct 40.4 MCV Hb A 74.1 36-48 Hb E&A2 22.9 78.2 79-99 Hb F 2.0 MCH 27.1 27-32 RDW 13.6 11.5-15.0 Hb A Hb A2 Hb E >95 <2.0 Capillarys separates Hb E & Hb A2 Hb A Hb E Hb A 71 >95 Hb A2 3.3 2.2-3.2 Hb E 23.8 Hb F 1.9 <2.0 Hb A2 Hb F Fractions Hb A Hb F Hb E Hb A2 % 71.0 1.9 23.8 3.3 Ref. % 95-97 <2.0 2.2-3.2 Hb E = b26glu val Homozygotes and heterozygotes are clinically well with mild microcytosis The mutation activates a cryptic splice site in Exon 1 in the beta globin gene producing a mild b-Thalassemia Hb E/b0Thalassemia patients are anemic (may be as severe as Thalassemia Major) with elevated Hb F 40% or higher BioRad I HPLC Hb E Homozygote 64 y/o woman RBC 5.36 3.9-5.0 Hgb 11.4 12.0-16.0 Hct 34.8 36-48 MCV 70.5 79-99 Hb A MCH 20.9 27-32 Hb E &A2 95.8 RDW 14.6 11.5-15.0 Hb F 4.2 0 >95 <2.0 CZE on Hb E Homozygote Hb E Hb A2 4.0 Hb E 91.2 Hb F 4.8 2.2-3.2 <2.0 Hb A2 Increased Hb A2 is consistent with the b Thalassemia seen in Hb E Hb F Hb F Breakdown Hb E Hb A2 4.8 1.4 89.8 4.0 <2.0 NA 0 2.2-3.2 Table from Steinberg et al. Disorders of Hemoglobin, Ch 43, 2001 UM Hb E/E 11.4 70.5 Hb E/b0 Thalassemia Hb E Hb A2 4.3 Hb E 46.5 Hb F 49.2 2.2-3.2 Hb F <2.0 Hb A2 Mix 1:1 with Control to see Zones Hb E Hb A Hb F Hb A2 Table from Steinberg et al. Disorders of Hemoglobin, Ch 43, 2001 UM Hb E-bo 7.1 73.5 Technique Comparison Parameter Gels HPLC Capillary Automation Interpretation Hb A2 Measure Hb A2 & Hb S Hb A2 & Hb E Bilirubin/Barts Separating Hbs Fair Straightforward Poor at low level No interference Cannot separate No interference Fair Excellent Complex Excellent Adduct issue Some separate Interferes* Excellent Excellent Straightforward Excellent No interference Separates No Interference Excellent *Prewashing of the RBCs removes the interference • http://globin.bx.psu.edu/html/huisman/variants/
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