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 recentmigratory flows from Africa, Albania and Asia

Moreover for:

• ascertaining the presence of hemoglobinopathiesin newborns, not tested in the prenatalperiod, with parents who are bothcarriers 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 freshblood 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 veryimportant 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 unsuitablefor 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 withspecific 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)

01020304050607080

0 6 11 16 21 26 31 35

Beta Thal. Normal

% Hb A

No.

of

Cas

es

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

01020304050607080

0 6 11 16 21 26 31 35

Beta Thal. Normal

% Hb A

AB

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

Cas

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 inBeta Thalassemia Carriers

% of Cas

esfo

rea

chcl

ass

of H

bA

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)

01020304050607080

0 6 11 16 21 26 31 35

Beta Thal. Normal

% Hb A

AB

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+

No.

of C

ases

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 SMolecular 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 correctpercentage of the Hb fractionsafter 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 heterozygosityfor βS and βThal

- the sickle cell trait (confirmed with the sickling test)

- many other Hb variants, including the most commonclinically relevant abnormal hemoglobins like Hb E, Hb C andHb D Punjab (confirmed with CE)

CONCLUSIONS

• The mesurement of Hb A levels on cord blood by HPLCcan be used for a presumptive identification of carrierstatus 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 beimportant the identification of a child with severe Hb H disease at birth)

- Hb Lepore trait- δ-β Thal trait

THANKS FOR YOUR ATTENTION