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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