Unraveling Hemoglobinopathies with Capillary Electrophoresis

Transcription

1 Session Number 2002 Unraveling Hemoglobinopathies with Capillary Electrophoresis David F. Keren, M.D. Professor of Pathology Division Director, Clinical Pathology The University of Michigan

2 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.

3 Hemoglobin Structure

4 Alpha Globin Gene Cluster ( Short arm of Chromosome 16) 5 LCRA (HS-40) 40 kb z z1 a1 a1 a2 a1 3 Beta Globin Gene Cluster ( Short arm of Chromosome 11) 5 LCRB e G g A g b d b 3

5 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) % of adult Hb

6 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

7 Hemoglobin Shorthand Hb Ann Arbor = a80 Leu 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

8 ~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)

9 Malaria Distribution parallels Major Hemoglobinopathies & Thalassemias Distribution of malaria

10 Harteveld and Higgs Orphanet Journal of Rare Diseases 2010, 5:13

11 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

12 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

13 Alkaline Gel Acid Gel C S F A A2 S F A S F Köln F A C S F (C) F E A E A S G S/G A A2 S A F A J A2 A/Chicago A2 S (F) A C S (A) A2 S A + Anode F A S C F A S F S F Köln /A F S C 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 + Anode Carbonic Anhydrase ( ) denotes low concentration

14 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

15 Biorad Variant I HPLC Hb A Glycated & Aged Hb A Bilirubin & degradation products Hb A2

16 Glycated HbA Aging HbA Bio-Rad Variant-II HPLC Hb A Degradation products Hb A2

17 Peak 1 (glycated A1a,A1b & degradation products) Peak 2 (glycated A1c) Biotech- Trinity Ultra HPLC Peak 3 (glycated A1d & degradation products) Peak 5 (Hb A2) Peak 4 (Hb A) Nl <5% Nl

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19 Capillary Electrophoresis Positive buffer ions (+) flow to cathode Detector Hb A Hb F Hb S Hb A2 415nm (+) - Cathode Sample +Anode

20 Sebia Capillarys

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22 Hb SC-What to do with no HbA? Use these measurements

23 Overlay with AFSC Controls Use these measurements

24 Mix 1:1 with Normal Sample Do NOT use these measurements

25 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

26 51 y/o man HPLC Pattern RBC Hgb Hct MCV MCH RDW Hb A Hb? Position Hb A2

27 Ultra HPLC Relative Retention RT/S Hb A Delta G Philadelphia Alpha D Los Angeles Beta

28 51 y/o man RBC Hgb Hct MCV MCH RDW Hb Hb A >95 >95 Hb Hb A2 A2? Hb Hb F 0 <2.0 <2.0 Hb Hb G+A2 G+A * 36-40* Hb G2? Hb G2 Position for Hb G (Cannot separate HbA2 included)

29 Capillary Electrophoresis HbG Philadelphia Trait Hb A Hb G Philadelphia Hb A2 Hb G2

30 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 a A A a A a G a Hb G (Philadelphia a 68Asn Lys ) & a Thalassemia b g d Hb A Hb F Hb A2 Hb A Hb F Hb A2 Hb A Hb F Hb A2 Hb G G Hb F Hb G2

31 32 y/o woman Hb A RBC Hgb Hct MCV MCH RDW HbA2) Hb?? Hb??

32 Ultra HPLC Relative Retention RT/S Hb A Delta G Philadelphia Alpha D Los Angeles Beta

33 32 y/o woman RBC Hgb Hct MCV MCH RDW Hb A Hb A 58.1 >95 Hb D + A Hb F 0 <2.0 Hb D (Cannot separate HbA2) Hb D Hb D trait b variant Normal MCV Normal RBC No G2

34 Hb D Punjab Hb A Capillary HbD Trait Hb A 51.9 >95 Hb A Hb F 0.7 <2.0 Hb D 44.2 Hb F Hb A2

35 Hemoglobin D (Los Angeles or Punjab) b121 glu gln Patel et al. Intl J Lab Hematol 2014;36: 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

36 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

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38 Ultra HPLC Relative Retention RT/S G Philadelphia Alpha D Los Angeles Beta

39 HPLC (Ultra)-Elution Time Keren et al. Am J Clin Pathol 2012;137: of 43 samples overlap.

40 Capillarys-Migration Position Keren et al. Am J Clin Pathol 2012;137: of 43 samples overlap.

41 HPLC (Ultra)-Elution Time/Hb S Keren et al. Am J Clin Pathol 2012;137: Only 2 of 43 samples overlap.

42 Capillarys-Migration/Hb A2 Keren et al. AJCP of 43 samples overlap

43 Chromosome 11 Beta Thalassemia Trait (Minor) 5 3 LCRB 5 3 LCRB >200 b + vs b 0 Mutations (deletions uncommon) Lose 30-50% b globin e G g A g b d b X e G g A g b d b Key is elevated hemoglobin A 2 (a 2 d 2 ) (>3.5%) Low MCV & MCH nl RDW, usually nl hgb, RBCs

44 Hb A2 on Variant II HPLC University of Leiden Van Delft et al. Intl J Lab Hematol 2009;31: HbA2 reduced in d Thalassemia & varriant carriers Specificity and overlap of d/b Hb Thalassemia A2 values in different cohorts of patients b/a Thalassemia combinations High HbA2 b Thalassemia carriers Normal Beta Thal Trait Method HPLC Normal Hb A2 b Thalassemia carriers Hb H Disease a Thalassemia trait a Thalassemia trait a Thalassemia trait Fe Deficiency Normal Range is Method Dependent

45 Precision of Hb A2 CAP Survey 2010 Results on Normal Samples Survey # Gel-1 Gel-2 HPLC CE HG * HG HB * Data is Coefficient of Variation (%)

46 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 Hb A2 <3.5 Hb A2 3.5 HPLC BioRad I BioRad II Menarini HA Tosoh G Tosoh G Capillary Beckman MDQ Beckman PA Sebia Capillarys II

47 Beta Thalassemia Trait 56 y/o female RBC Hgb Hct MCV MCH RDW Hb A 94.4 >95 Hb A Hb F 1.0 <2.0 Ref Range A

48 Same Case Beta Thalassemia Trait Hb A 94.1 >95 Hb A Hb F 1.0 <2.0 Fractions % Ref. % Hb A Hb F Hb A <

49 Hb A2 (Delta)Variants A2 1.3 A2 1.3 Fractions % Ref. % Hb A Hb A2 Hb A <

50 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

51 A2 1.6 A2 v 0.7 Fractions Fractions % Ref. Ref. % Hb A Hb A Hb Hb A2 A2 Hb A2 Hb A2 v

52 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 A2 v Similar to Hb E a beta variant that is produced in decreased amount

53 A2 1.1 Fractions % Ref. % Hb A Hb F Hb A2 v Hb A <

54 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

55 18 y/o female sickledex positive RBC Hgb Hct MCV MCH RDW Hb A 59.6 >95 Hb A Hb F 0 <2.0 Hb S * *Expected for Sickle Trait

56 Same Case: Sickle Trait Hb A2 HPLC CE Fractions % Ref. % Hb A Hb F Hb S Hb A <

57 Primus HPLC HPLC vs CE for Hb A2 Keren et al. AJCP 2008;130: Effect of Hb S on Hb A Hb S Containing Samples No Structural Variant Sebia CE

58 Hemoglobin A2 (%) Hb S Trait: HPLC vs CE Keren et al. AJCP 2008;130: CE CE-S HPLC HPLC-S

59 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 b s for a globin - actual increase ~0.5% 2. HPLC artifact: Hb S breakdown products in Hb A2 peak - false increase 1-2%

60 36 y/o woman Sickledex negative RBC Hgb Hct MCV MCH RDW Hb? = S 1.01 Rel Rt = S 0.91

61 Ultra HPLC Relative Retention RT/S Hb A Delta G Philadelphia Alpha D Los Angeles Beta

62 36 y/o woman RBC Hgb Hct MCV MCH RDW Hb A 60.7 >95 Hb F 1.0 <2.0 Hb G+A Hb G2 2.4 Hb G2 = 2.4% Hb G + A2 = 35.9%

63 Hb G Philadelphia & b & a Thalassemia Hb A2 + G2 HPLC CE? 5.5 Fractions % Ref. % Hb A Hb F Hb G Hb A2 Hb G <

64 a A A a A a G a Hb G (Philadelphia a 68Asn 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 b b Hb A Hb A Hb A Hb A Hb A Hb A Hb G Hb G d Hb A2 Hb A2 Hb A2 Hb G2

65 Chromosome 16 MCR Hemoglobin H Disease 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

66 Hb H Disease 31 yr woman RBC Hgb Hct MCV MCH RDW Hb A 89.3 >95 Hb A Hb H & A1c Hb Bart s Hb Bart s Hb H & A1c (can t measure HbH alone) Ref Range A

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68 Bilirubin Masquerading as Barts Howanitz et al. AJCP 2006;125: Bilirubin

69 Hb H Disease with Hb H and Barts Hb A Hb H Hb Barts Hb A2 Fractions % Ref. % Hb H Hb Bart s Hb A Hb A

70 36 y/o woman sickledex positive RBC Hgb Hct MCV MCH RDW Hb A 73.0 >95 Hb A Hb S * *Expected for Sickle Trait

71 Beta globin products in Thalassemia with & without Hb S Normal b Thal Hb S Trait Hb S/a Thal Hb S/b Thal

72 24 y/o woman RBC Hgb Hct MCV MCH RDW Hb A Hb A2 = 1.4%? Rel Rt = A 1.17

73 Hb E

74 Hb E trait RBC Hgb Hct MCV MCH RDW Hb A 74.1 >95 Hb E&A Hb F 2.0 <2.0 Hb A Hb A2 Hb E

75 Capillarys separates Hb E & Hb A2 Hb A Hb A 71 >95 Hb A Hb E 23.8 Hb F 1.9 <2.0 Hb F Hb E Hb A2 Fractions % Ref. % Hb A Hb F Hb E Hb A <

76 Hb E = b26 glu 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/b 0 Thalassemia patients are anemic (may be as severe as Thalassemia Major) with elevated Hb F 40% or higher

77 BioRad I HPLC Hb E Homozygote 64 y/o woman RBC Hgb Hct MCV MCH RDW Hb A 0 >95 Hb E &A Hb F 4.2 <2.0

78 CZE on Hb E Homozygote Hb E Hb A Hb E 91.2 Hb F 4.8 <2.0 Hb A2 Hb F Hb F 4.8 <2.0 Breakdown 1.4 NA Hb E Hb A Increased Hb A2 is consistent with the b Thalassemia seen in Hb E

79 Table from Steinberg et al. Disorders of Hemoglobin, Ch 43, 2001 UM Hb E/E

80 Hb E/b 0 Thalassemia Hb E Hb A Hb E 46.5 Hb F 49.2 <2.0 Hb F Hb A2

81 Mix 1:1 with Control to see Zones Hb E Hb A Hb F Hb A2

82 Table from Steinberg et al. Disorders of Hemoglobin, Ch 43, 2001 UM Hb E-b o

83 Technique Comparison Parameter Gels HPLC Capillary Automation Fair Excellent Excellent Interpretation Straightforward Complex Straightforward Hb A2 Measure Poor at low level Excellent Excellent Hb A2 & Hb S No interference Adduct issue No interference Hb A2 & Hb E Cannot separate Some separate Separates Bilirubin/Barts No interference Interferes* No Interference Separating Hbs Fair Excellent Excellent *Prewashing of the RBCs removes the interference

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