Hb A distribution in cord blood

Hb A distribution in cord blood

(normal vs β+ _{or β}o _{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

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

∗ absence of Hb A₂ (<0.5%)

∗ presence of elevated percentages of Hb F

∗ possible presence of Hb variants

∗ RDB indices are not very useful

In this condition:

The correct quantification of Hb A is very important for a presuntive or a conclusive diagnosis at birth

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)

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

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

At birth

After 3 weeks

After 5 weeks

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

Twin N.1 _{Twin N.2}

Hb A:

in

heterozygous

β

Thalassemia

(β° or β+_{/ β}A )

- 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

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

No. of C

ases

Distribution of Hb A in 445 Newborns on HPLC

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}

No. of C

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 A

in Normal and in

Beta Thalassemia Carriers

% of Cases

for

each

class of Hb

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 Thalassemia: (β°/β°) and (β°/β+₎

β°/β° (cod 39)

β°/β+_{(cod39 /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+

Hb A:

in heterozygous

Hb S

Newborns at-term: Hb S trait

sickle cell trait

sickle cell trait

Hb A:

Hb S + β + _{Thal. (IVSI-110)}

After 10 months At birth

Hb A:

in

Hb S /

β

_Thal.

vs.

- 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:

Alpha Thalassemia: NCOI/-3.7kb

Hb Bart’s

Hb A:

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 F_acetyl +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

Hb Contaldo: α 102 Ser>Arg

CONCLUSIONS

•

- Normal condition

- the homozygous state or compound heterozygosity for

βThal _defects

- the homozygous state (βS) or compound heterozygosity for βS _{and β}Thal

- 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

•

- 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