Hb A distribution in cord blood - Enerca ... Hb A distribution in cord blood (normal vs خ²+ or...

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Transcript of Hb A distribution in cord blood - Enerca ... Hb A distribution in cord blood (normal vs خ²+ or...

  • Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers)

    Giovanni Ivaldi

    Laboratorio di Genetica Umana - Settore Microcitemia Ospedali Galliera, Genova - Italy

    2ND European Hemoglobinopathy Forum: Insights on the Diagnosis of Hemoglobin disorders

    November 29th, 2011 Madrid

  • Preliminary remarks:

    Today in Italy the most frequent test is request at birth in:

    • Typing for cord blood collection (International standard, NetCord-FACT)

    • newborn screening programs due to recent migratory flows from Africa, Albania and Asia

  • Moreover for:

    • ascertaining the presence of hemoglobinopathies in newborns, not tested in the prenatal period, with parents who are both carriers of Hb defects

    • confirming the result of the prenatal diagnosis

    The presence of Hb Bart’s in cord blood has been used for early diagnosis and population frequency screening of Alpha Thalassemia in the past. Today is rarely performed.

  • We observe on the cord blood or day-1 fresh blood in EDTA:

    ∗ absence of Hb A2 (

  • The relative percentage of Hb A observed at birth could be due to :

    ∗ gestational age ∗ presence of globin defects ∗ twin condition

    ∗ maternal contamination of the sample (when the blood sample is obtain by umbilical cord)

    ∗ hemolytic anemias

    ∗ the methods used for sample’s collection (analysis of Guthrie card dried blood spots is unsuitable for accurate quantitation)

    ∗ the analytical method applied

  • gestational age: (O.M.S.)

    pre-term: < 37 weeks

    at-term: 37 - 42 weeks

    post-term: > 42 weeks

  • The general screening approach recommend the use of diagnostic technique able to provide suitable results with an optimal grade of cost/benefit ratio (HPLC for example).

    In some cases it is useful to proceed with specific test (electrophoresis, sickling test) before a possible molecular characterization.

  • But it is very important, also at birth,

    a “short anamnesis” concerning:

    ∗ family origin

    ∗ gestational age

    ∗ hemoglobinopathies present in the family

    ∗ possible twin condition

  • Hb A:

    in normal subjects

    - G.Ivaldi, L.Leone et al.

    Biochimica Clinica, 2007; 31(4): 276-9

    - E. Mantikou E, CL Harteveld, PC Giordano

    Clin Biochem 2010; 43

    At birth

    Normal condition

  • At birth

    After 3 weeks

    After 5 weeks

    Normal Subject

  • Not thalassemic condition: twin vs. single subject (pre-term: - 4 weeks)

    Twin N.1 Twin N.2

    Single

  • Hb A:

    in heterozygous β Thalassemia (β° or β+/ βA )

    - G.Ivaldi, L.Leone et al.

    Biochimica Clinica, 2007; 31(4): 276-9

    - Mantikou E, Arkesteijn SG, et al

    Clin Biochem 2009; 42:1284-90.

    At birth

    β-Thalassemia carriers

  • Heterozygous newborn

    β° Thalassemia (cod 39)

    21 weeks, heterozygous fetus

    β° Thalassemia (cod 39)

    β Thal. trait

    Heterozygous newborn

    β+ Thalassemia (IVSI-110)

  • 0 10 20 30 40 50 60 70 80

    0 6 11 16 21 26 31 35

    Beta Thal. Normal

    % Hb A

    N o .

    o f

    C as

    es

    Distribution of Hb A in 445 Newborns on HPLC (VARIANTTM II β-Thal Short Program, Bio-Rad Laboratories Inc. USA)

  • 0 10 20 30 40 50 60 70 80

    0 6 11 16 21 26 31 35

    Beta Thal. Normal

    % Hb A

    A B

    Distribution of Hb A in 445 Newborns on HPLC (VARIANTTM II β-Thal Short Program, Bio-Rad Laboratories Inc. USA)

    A: β°-Thalassemia carriers

    B: β+-Thalassemia carriers

    N o .

    o f

    C as

    es

  • 0

    2

    4

    6

    8

    10

    12

    14

    16

    18

    2

    2, 3

    2, 6

    2, 9

    3, 2

    3, 5

    3, 8

    4, 1

    4, 4

    4, 7 5

    5, 3

    5, 6

    5, 9

    6, 2

    %A2

    % among 825 normal subjects

    % among 240 Beta Thalassemia carriers

    Distribution of Hb A2 in Normal and in Beta Thalassemia Carriers

    % o f C as

    es fo

    r ea

    ch cl

    as s

    o f H

    b A

    2

  • Hb A:

    in heterozygous β Thalassemia (normal β° vs. pre-term β° )

    and Hb Lepore trait (Boston)

  • Hb Lepore trait

    β° thal. trait (cod 39) at term

    β° thal. trait (cod 39) pre-term (-5weeks)

  • Hb A:

    in homozygous β Thalassemia (β°/β°) vs.

    compound β Thalassemia (β°/β+) or (β+/β+)

  • β Thalassemia: (β°/β°) and (β°/β+)

    β°/β° (cod 39)

    β°/β+(cod39 /IVSI-110)

    β+/β+ (IVSI-110 / IVSI-110)

  • 0 10 20 30 40 50 60 70 80

    0 6 11 16 21 26 31 35

    Beta Thal. Normal

    % Hb A

    A B

    Distribution of Hb A in 445 Newborns on HPLC (VARIANTTM II β-Thal Short Program, Bio-Rad Laboratories Inc. USA)

    A: β°-Thalassemia carriers

    B: β+-Thalassemia carriers

    β°/β° or β°/β+

    N o.

    o f C

    as es

  • Hb A:

    in heterozygous Hb S (β° or β+/ βS )

  • Newborns at-term: Hb S trait

    sickle cell trait

    sickle cell trait

    normal

  • Hb A:

    in Hb S / β+ Thal.

  • Hb S + β + Thal. (IVSI-110)

    After 10 months

    At birth

    After 3 months

  • Hb A:

    in Hb S / β+ Thal.

    vs.

    Homozygous Hb S

  • - Different Retention time

    - Similar quantification of the Hb A (apparently)

    Hb S + β + Thal. (IVSI-110)

    Homozygous Hb S Molecular studies are required for a final correct identification

  • Hb A:

    in α Thalassemia Trait

  • Alpha Thalassemia: NCOI/-3.7kb

    Hb Bart’s

    Normal

  • Hb A:

    in Hb H Disease

  • In red is reported the correct percentage of the Hb fractions after the integration of the all picks

    Hb A: 32.7

    Hb F: 44.4

    Hb Bart’s: 12.4

    Hb Facetyl +Hb H : 9.0

    Hb H disease: --Med / -3.7 α

  • Alpha Thalassemia and Hb Bart’s

    - I. Papassotiriou, J. Traeger-Synodinos et al.

    Hemoglobin 1999; 23 (3) 203-11

  • Two rare cases observed on cord blood

    Beta Variant: Hb M Saskatoon

    Alpha Variant: Hb Contaldo

  • Hb M Saskatoon: β 63 His>Tyr

    Newborn

    Adult

  • Hb Contaldo: α 102 Ser>Arg

    Adult

  • CONCLUSIONS • The mesurement of Hb A levels in cord blood by HPLC

    can, with reasonable precision, be used to detect :

    - Normal condition

    - the homozygous state or compound heterozygosity for

    βThal defects - the homozygous state (βS) or compound heterozygosity for βS and βThal

    - the sickle cell trait (confirmed with the sickling test) - many other Hb variants, including the most common

    clinically relevant abnormal hemoglobins like Hb E, Hb C and Hb D Punjab (confirmed with CE)

  • CONCLUSIONS

    • The mesurement of Hb A levels on cord blood by HPLC can be used for a presumptive identification of carrier status in:

    - β-Thal: β+ or β° is not relevant for the newborn - α-Thal: α+ or α° is not relevant for the newborn (while may be important the identification of a child with severe Hb H disease at birth)

    - Hb Lepore trait - δ-β Thal trait

  • THANKS FOR YOUR ATTENTION