The Challenging Diagnosis of Treacherous Hemoglobinopathies

Transcription

1 The Challenging Diagnosis of Treacherous Hemoglobinopathies David F. Keren, M.D. Professor of Pathology The University of Michigan

2 Objectives Use Capillary Electrophoresis and HPLC to detect clinically significant variants Decide which cases need molecular confirmation Distinguish carryover and transfused variants from slow eluting treacherous ones

3 Hemoglobin Structure

4 Adult Hemoglobins Alpha Globin Gene Cluster (Chromosome 16) Hb A a2b2 >95% Hb A2 a2d Hb F a2g2 <1% 5 z a2 a1 3 Beta Globin Gene Cluster (Chromosome 11) 5 3 e G g A g d b

5 ~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 A2ꞌ Structural & Thalassemia Constant Spring (alpha variant) HbE (beta variant) Lepore (delta-beta fusion protein)

6 Investigation of Hemoglobin Clinical: age, transfusion, race, therapy Routine: RBC, MCV, MCH, RDW, sickle solubility 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

7 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

8 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) E/A E/A A G/S A S F A/J (F) A/Chicago A S (A) S C (F) A S + Anode Carbonic Anhydrase ( ) denotes low concentration

9 High Performance Liquid Chromatography (HPLC) Improved Sensitivity over gels Accurate measurement of Hb A2 and Hb F Complex patterns for interpretation Hb S adducts interfere with Hb A2 Cannot separate Hb A2 from Hb S Cannot separate Hb A2 from Hb E on most systems

10 Glycated HbA Aged HbA Bio-Rad Variant-II HPLC Hb A Degradation products Hb A2

11 Szuberski et al. Int J Lab Hematol 2013;34: Common Variants in Windows

12 Capillary Electrophoresis Positive buffer ions (+) flow to cathode m diameter Fused silica capillary tube 20 cm long Hb A Hb F b-1 Hb S Hb A2 35 o C ph nm UV Detector Anode + Sample - Cathode

13 Sebia Capillarys

14 Capillarys 2 Precision Riou J et al. Am J Clin Pathol 2018;149:172

15 Variants Zones 2-5 Riou J et al. Am J Clin Pathol 2018;149:172

16 Case year old woman RBC Hgb MCV MCH RDW Hb A 58.5 >95 Hb A Hb F 0 <2.0 Hb

17 Elution time of Unknown = 4.33 Szuberski et al. Int J Lab Hematol 2013;34: Variant Chain Mean Range % in Trait # Cases Stanleyville-II α % 42 Russ α % 40 Richmond β % 26 S β % 38038

18 Capillary Electrophoresis 214 Fractions % Ref. % Hb A Hb F 0.2 <2.0 Hb Hb A

19 Migration of Unknown = 214 Sebia Capillarys X-axis migration positions

20 2 beta genes Beta vs Alpha Variants usually are between 35-45% Hb A2 is unaffected (unless the beta variant elutes on top of it) 4 alpha genes Variants are ~25-30% There is a Variant version of A2 as well as normal A2 (add them together to find total A2

21 What does an Alpha Chain Variant look like? Hb G Philadelphia Hb A Hb G Philadelphia Hb A2 Hb G2

22 Hb G-Philadelphia a68 Asn Lys 1 in 5,000 African Americans Co-inherited with cis 3.7 Kb alpha deletion Benign Variant a genes b d g a1 a 2 b 2 Hb A a 2 d 2 Hb A2 a 2 g 2 Hb F a2 a 2 b 2 Hb A a 2 d 2 Hb A2 a 2 g 2 Hb F a1 G a G 2b 2 Hb G a G 2d 2 Hb G2 a G 2g 2 Hb F G a2 3.7 Kb Del - - -

23 Hb S b6 Glu Val Trait Hb S trait in 8% of African Americans Usually asymptomatic Hematuria ~ 1% of cases Rare sickling (deoxygenation): high altitude, pneumonia, strenuous exertion in warm weather Hb S 35-40% Hb A2 falsely high on HPLC- Hb S glycation Can use sickle solubility as confirmatory

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

25 Case year old African-American female with severe joint and abdominal pain RBC Hgb MCV MCH RDW Hb A 0 >95 A Hb Hb F 10 <2.0

26 Elution time of Unknown = 4.41 Szuberski et al. Int J Lab Hematol 2013;34: Variant Chain Mean Range % in Trait # Cases Stanleyville-II α % 42 Russ α % 40 Richmond β % 26 S β % 38038

27 Case 2. Capillary Electrophoresis Pt mixed with Normal blood Hb 2 Hb A Hb 214 Hb F/var Hb A2 Hb F/var Hb A2 Name % Hb A 0 Hb F or var 8.8 Hb Hb A2 2.9 Name % Hb A 68.4 Hb F or var 3.3 Hb Hb A2 2.7

28 Variants Zones 2-5 Riou J et al. Am J Clin Pathol 2018;149:172

29 Do we need further testing? None needed Sickledex? Confirms Hb S Cannot confirm other variants Mass Spectrometry? Confirms Hb S Detect and identify other variants Molecular? Confirms Hb S Detect and identify other variants Detect Thalassemic deletions

30 Common TreacherousVariants Modified from Williams & Weatherall Cold Spring Harbor Perspectives in Medicine 2012 Variant Amino Acid Pathologic Form Clinical Hb S b6 Glu Val Hb SS, HbS/b-thalassemia* Severe Hb D b121 Gl Gln Hb S/Hb D, Hb D/b-thalassemia* Severe Hb O-Arab b121 Glu Lys Hb S/Hb O-Arab Severe Hb C-Harlem (C-Georgetown) b6 b73 Glu Val Asp Asn Hb S/Hb C-Harlem Severe Hb C b6 Glu Lys Hb S/Hb C Mild- Moderate Hb Lepore (d-b Hybrid) d(1-87) b( ) Hb S/ Hb Lepore Mild- Moderate Hb E b26 Glu Lys Hb E/b-thalassemia* Moderate- Severe *Either b 0 or severe b + thalassemia

31 Hb S accounts for 83.3% Annual Births with Major Hemoglobin Disorders Williams and Weatherall Cold Spring Harbor Perspectives in Medicine 2012 Hb SS disease 217, % Hb SC disease 54, % b-thalssemia Major 22, % Hb E b-thalssemia 19, % Hb S b-thalssemia 11, % Hb H disease 9, % Hb Bart s hydrops (a 0 /a 0 ) 5, %

32 SS Worldwide Location Piel FB, Steinberg MH, Rees DC. NEJM 2017;376:

33 Hb SS b6 Glu Val Prognosis In the US: Life expectancy reduced ~30 years Quality of life often poor Hydroxyurea therapy increases Hb F Hematopoietic stem-cell transplantation In Africa: Mortality younger than 5 yr up to 90%

34 Pathophysiology Deoxygenation-polymerization of Hb S Erythrocyte Damage Hemolytic anemia Vaso-occulsion: Ischemic tissue damage Severe pain Organ failure

35 Case yr old African-American male-acute chest syndrome, Nl ferritin. On Hydroxyurea. RBC Hgb MCV MCH RDW Hb A 0 >95 A Hb S Hb F 15.1 <2.0

36 Case 3 Capillary Electrophoresis Hb 2 Hb A Hb S Hb F Hb A2 Hb F Hb A2 Hb F Hb 2 Hb A

37 Differential for Hb S with Microcytosis with relatively high hemoglobin RBC Hgb MCV MCH RDW Hb A 0 >95 A Hb S Hb F 15.1 <2.0 Hb S/b thalassemia Hb SS/a thalassemia Hb S/db thalassemia Hb S/HPFH Hb SS & Fe deficiency Molecular Tested Needed

38 Molecular Report Hb SS Homozygote gap-pcr: Negative for HPFH, db Thalassemia Promoter regions of g-globin gene: Homozygous C>T polymorph (Xmn I site) Associated with elevated Hb F Alpha gene deletions: Homozygous alpha-globin deletions (- a 3.7 /- a 3.7 ) Associated with microcytosis

39 Hb SS Genetic Modifiers Piel FB, Steinberg MH, Rees DC. NEJM 2017;376: Arab Indian Senegal Haplotypes Cameroon Benin Bantu/CAR * High Levels Hb F Low Levels Number of a-globin genes Two Three Four *CAR: Central African Republic

40 Case y/o female RBC Hgb Hct MCV MCH RDW HbA 97.7 >95 A HbF 0.8 <2.0

41 No variants are assigned by Variant II for Elution time prior to ~40 Seconds Szuberski et al. Int J Lab Hematol 2013;34:

42 Case 4. Capillary Electrophoresis HbA Anode + Hb 38 Hb 92 HbA2 Fractions % Ref Hb H Hb Hb A 82.2 >95 Hb A

43 Migration of Unknowns 38 & 92 Hemoglobin Alternative name Involved Chain migration Hb H b4 7 to 46 Hb Bart's g4 79 to 106 Hb J-Oxford Hb I-Interlaken a1 96

44 Hb H Disease RBC Hgb Hct MCV MCH RDW HbA 97.7* >95 A HbF 0.8 <2.0 Bart s/h/bilirubin HbH * Fast peaks NOT measured

45 Capillary Electrophoresis HbA Hb 38 H Hb 92 Bart s HbA2 Fractions % Ref Hb H Hb Barts Hb A 82.2 >95 Hb A

46 Alpha Thalassemia Affects 5% of the World Population US and Canada increasingly common 95% of cases due to deletion Double deletions- Alpha Thal 1 haplotype Southeast Asian (-- SEA, -- FIL, & -- THAI ) Mediterranean (-- MED, & ) Single Gene- Alpha Thal 2 haplotype (African): 3.7 & 4.2 kb

47 Hemoglobin H Disease a2 X a1 X X Less than 1/3 of alpha globin is produced Unstable high O 2 affinity Hb H (b4) & Bart s (g4) Severe microcytosis (MCV 55-62) & H-bodies HbA2 always low (<2.0) Mild-moderate hemolytic anemia, splenomegaly Usually not requiring transfusion Can transmit Bart s Hydrops fetals (g4)

48 H-bodies

49 Case y/o female RBC Hgb Hct MCV MCH RDW HbA 97.3 >95 A HbF 0 <2.0 Hb C window

50 Elution time of Unknown = 5.07 Szuberski et al. Int J Lab Hematol 2013;34: Variant Chain Mean Range % in Trait # Cases Porto Alegre β % 4 Constant Spring α % 512 C β % 9062

51 Capillary Electrophoresis Winichagoon et al. Translational Research 2008;152: Hb A Hb 38 Hb 92 Hb F Hb A2 Hb 258 Hb Hb Hb A 90.9 >95 Hb A Hb

52 Migration of Unknowns 38, 92 & 258 Hemoglobin Alternative name Involved Chain migration Hb H b4 7 to 46 Hb Bart's g4 79 to 106 Hb J-Oxford Hb I-Interlaken a1 96 Hb Constant Spring a Hb C b 252

53 Nondeletional Hb H Disease RBC Hgb Hct MCV MCH RDW HbA 97.3 >95 A HbF 0 <2.0 H Hb CS 1.7 Hb H/Constant Spring CS

54 Nondeletional Hb H Disease CS X X Relative product 1.0 Relative product 0 Referred to as a T alleles (transformed) TAA CAA Lose stop codon adds 31 C-terminal aa Unstable m-rna produces little CS a globin Hb CS 1% in heterozygote, 2-5% in Hb H disease CS homozygotes also have hemoglobin H disease Can transmit Bart s Hydrops fetals (g4)

55 Dealing with Carryover Kozarski TB et al. Arch Pathol Lab Med 2006;130: Slowly eluting Hb on HPLC-typically Hb C Check prior samples for Hb C If uncertain, repeat the sample No carryover on Capillary Electrophoresis

56 Unstable Hemoglobins Hardison RC et al variant hemoglobins categorized Hb Köln b89val Met - most common De novo Missense mutation leading to decreased solubility Mild to severe hemolytic anemias Detection depends on high index of suspicion Chronic non-spherocytic hemolytic anemia Retics increased out of proportion to Hb

57 Hb Köln by HPLC Li Y et al. Hemoglobin 2016;40:417-9

58 Capillary Electrophoreis Hb Köln Li Y et al. Hemoglobin 2016;40:417-9

59 Detection of Unstable Hemoglobins Positive Heinz body preparation is suggestive May be difficult to detect in young patients and ones with a functional spleen Heat stability &/or the isopropanol precipitation test are the best way to detect Mass spectrometry or Molecular studies are often necessary for specific identification

60 Case year African American male RBC Hgb MCV MCH RDW Hb A >95 Hb F 1.0 <2.0 Hb A

61 Elution of Unknown = 3.53 (8.2%) Szuberski et al. Int J Lab Hematol 2013;34: Variant Chain Mean Range % in Trait # Cases Lepore (δβ fusion) δβ % 523 G Copenhagen β % 30 E β % 5796

62 Capillary Electrophoresis Hb A Hb For var Hb 207 Hb A2 Name % Hb A 90.5 Hb F or var 0.5 Hb Hb A2 2.5

63 Migration of Unknown 207 Hb D-Punjab Hb Osu Christiansborg Hb D-Los Angeles b 208 b 207 Hb Lepore d/b hybrid 207 Hb G-Philadelphia a1 or a2 206

64 Hb Lepore (Boston) d(1-87) b( ) Co-elutes with Hb A2 on HPLC Separates from Hb A2 on Capillary A form of beta thalassemia Microcytosis Mild anemia Lepore/b 0 thalassemia yields severe anemia Hb S/Lepore moderately severe sickling

65 Lepore but why the Erythrocytosis? RBC Hgb MCV MCH RDW Hb A >95 Hb F 1.0 <2.0 Hb A

66 % Oxyhemoglobin Mayo Oxygen Dissociation on Pt 6 Inoue S et al. Hemoglobin 2012;36: P50 = 12 mm Hg P50 = mm Hg po2

67 Hb Johnstown Val Leu Codon 109 (GTG>TTG) Inoue S et al. Hemoglobin 2012;36:

68 Mayo 2011 Pretransfusion study from 2012 report by Inoue et al. Inoue S et al. Hemoglobin 2012;36: Hb Johnstown NOT Hb A Hb Johnstown NOT Hb A Hb Lepore & Hb A2 Hb Lepore Hb A2

69 High Oxygen Affinity Variants Thom CS, et al. Cold Spring Harbor Perspective in Medicine 2013;3: ~100 Variants Autosomal dominant May be alpha or beta variants Left shift in P50 values Many co-migrate with Hb A Compromise of oxygenation when combined with beta thalassemia

70 History Despite phlebotomy to relieve erythrocytosis: severe fatigue shortness of breath chest pain & palpitations Patient placed on monthly RBC exchange transfusions Now doing well

71 Case y/o woman RBC Hgb Hct MCV MCH RDW HbA 69.6 >95 A2&V HbF 1.0 <2.0

72 Elution time of Unknown = 3.69 Szuberski et al. Int J Lab Hematol 2013;34: Variant Chain Mean Range % in Trait # Cases Lepore (δβ fusion) δβ % 523 G Copenhagen β % 30 E β % 5796 Osu- Christiansborg β % 75

73 Same Case Capillary Electrophoresis HbA 228 HbF HbA2 Hb A 71.0 >95 Hb F 1.9 >95 <2.0 Hb <2.0 0 Hb A

74 Migration of Unknown 228 Hemoglobin Alternative name Involved Chain migration Hb Köln (peak 1) Hb Ube-1 b (presence of several peaks) 229 Hb E b 228 Hb M-Saskatoon: minor peak b (presence of several peaks) 227

75 Hb E Trait RBC Hgb Hct MCV MCH RDW HbA 69.6 >95 A2&V HbF 1.0 <2.0

76 Same Case Capillary Electrophoresis HbA Hb E HbF HbA2 Hb A 71.0 >95 Hb F 1.9 >95 <2.0 Hb E 23.8 <2.0 0 Hb A

77 Hemoglobin E b26 Glu Lys The mutation activates a cryptic splice site in Exon 1 in the beta globin gene producing b- Thalassemia Homozygotes and heterozygotes are clinically well with mild microcytosis Homozygotes have less than 10% HbF HbE/b 0 Thalassemia patients are severely anemic (may be as severe as Thalassemia Major) with elevated HbF 40% or higher

78 40 y/o woman Hb E Homozygote RBC Hgb Hct MCV MCH RDW HbA 0 >95 A2&E HbF 8.1 <2.0

79 39 y/o woman with Hb E/b 0 Thalassemia RBC Hgb Hct MCV MCH RDW F 43.8% A2 56.2% HbA 0 >95 A2&E HbF 43.8 <2.0 HbF 43.8% HbE & HbA2

80 Table from Steinberg et al. Disorders of Hemoglobin, Ch 43, 2001

81 Today s goals Capillary Electrophoresis and HPLC to detect clinically significant variants Use of molecular confirmation for some thalassemias and rare clinically significant variants: unstable, high oxygen affinity Distinguish carryover and transfused variants from slow eluting ones