hb a distribution in cord blood - enerca...hb a distribution in cord blood (normal vs β+ or βo...
TRANSCRIPT
![Page 1: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/1.jpg)
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
![Page 2: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/2.jpg)
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
![Page 3: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/3.jpg)
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.
![Page 4: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/4.jpg)
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
![Page 5: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/5.jpg)
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
![Page 6: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/6.jpg)
gestational age: (O.M.S.)
pre-term: < 37 weeks
at-term: 37 - 42 weeks
post-term: > 42 weeks
![Page 7: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/7.jpg)
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.
![Page 8: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/8.jpg)
But it is very important, also at birth,
a “short anamnesis” concerning:
∗ family origin
∗ gestational age
∗ hemoglobinopathies present in the family
∗ possible twin condition
![Page 9: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/9.jpg)
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
![Page 10: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/10.jpg)
At birth
After 3 weeks
After 5 weeks
Normal Subject
![Page 11: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/11.jpg)
Not thalassemic condition: twin vs. single subject (pre-term: - 4 weeks)
Twin N.1 Twin N.2
Single
![Page 12: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/12.jpg)
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
![Page 13: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/13.jpg)
Heterozygous newborn
β° Thalassemia (cod 39)
21 weeks, heterozygous fetus
β° Thalassemia (cod 39)
β Thal. trait
Heterozygous newborn
β+ Thalassemia (IVSI-110)
![Page 14: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/14.jpg)
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)
![Page 15: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/15.jpg)
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
![Page 16: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/16.jpg)
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
![Page 17: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/17.jpg)
Hb A:
in heterozygous β Thalassemia(normal β° vs. pre-term β° )
and Hb Lepore trait (Boston)
![Page 18: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/18.jpg)
Hb Lepore trait
β° thal. trait (cod 39) at term
β° thal. trait (cod 39) pre-term (-5weeks)
![Page 19: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/19.jpg)
Hb A:
in homozygous β Thalassemia(β°/β°) vs.
compound β Thalassemia(β°/β+) or (β+/β+)
![Page 20: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/20.jpg)
β Thalassemia: (β°/β°) and (β°/β+)
β°/β° (cod 39)
β°/β+(cod39 /IVSI-110)
β+/β+ (IVSI-110 / IVSI-110)
![Page 21: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/21.jpg)
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
![Page 22: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/22.jpg)
Hb A:
in heterozygous Hb S(β° or β+/ βS )
![Page 23: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/23.jpg)
Newborns at-term: Hb S trait
sickle cell trait
sickle cell trait
normal
![Page 24: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/24.jpg)
Hb A:
in Hb S / β+ Thal.
![Page 25: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/25.jpg)
Hb S + β + Thal. (IVSI-110)
After 10 months
At birth
After 3 months
![Page 26: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/26.jpg)
Hb A:
in Hb S / β+ Thal.
vs.
Homozygous Hb S
![Page 27: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/27.jpg)
- 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
![Page 28: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/28.jpg)
Hb A:
in α Thalassemia Trait
![Page 29: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/29.jpg)
Alpha Thalassemia: NCOI/-3.7kb
Hb Bart’s
Normal
![Page 30: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/30.jpg)
Hb A:
in Hb H Disease
![Page 31: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/31.jpg)
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
![Page 32: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/32.jpg)
Alpha Thalassemia and Hb Bart’s
- I. Papassotiriou, J. Traeger-Synodinos et al.
Hemoglobin 1999; 23 (3) 203-11
![Page 33: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/33.jpg)
Two rare cases observed on cord blood
Beta Variant: Hb M Saskatoon
Alpha Variant: Hb Contaldo
![Page 34: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/34.jpg)
Hb M Saskatoon: β 63 His>Tyr
Newborn
Adult
![Page 35: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/35.jpg)
Hb Contaldo: α 102 Ser>Arg
Adult
![Page 36: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/36.jpg)
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)
![Page 37: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/37.jpg)
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
![Page 38: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/38.jpg)
THANKS FOR YOUR ATTENTION
![Page 39: Hb A distribution in cord blood - Enerca...Hb A distribution in cord blood (normal vs β+ or βo thalassemia carriers) Giovanni Ivaldi Laboratorio di Genetica Umana - Settore Microcitemia](https://reader030.vdocuments.site/reader030/viewer/2022040808/5e4b5fc51c6106693c54dcf6/html5/thumbnails/39.jpg)