Hb A distribution in cord blood

Transcription

1 2 ND European Hemoglobinopathy Forum: Insights on the Diagnosis of Hemoglobin disorders November 29th, 2011 Madrid 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

2 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

3 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.

4 We observe on the cord blood or day-1 fresh blood in EDTA: absence of Hb A 2 (<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

5 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

6 gestational age: (O.M.S.) pre-term: < 37 weeks at-term: weeks post-term: > 42 weeks

7 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.

8 But it is very important, also at birth, a short anamnesis concerning: family origin gestational age hemoglobinopathies present in the family possible twin condition

9 Hb A: in normal subjects Normal condition At birth - E. Mantikou E, CL Harteveld, PC Giordano Clin Biochem 2010; 43 - G.Ivaldi, L.Leone et al. Biochimica Clinica, 2007; 31(4): 276-9

10 Normal Subject At birth After 3 weeks After 5 weeks

11 Not thalassemic condition: twin vs. single subject (pre-term: - 4 weeks) Twin N.1 Twin N.2 Single

12 Hb A: in heterozygous β Thalassemia (β or β + / β A ) β-thalassemia carriers At birth - Mantikou E, Arkesteijn SG, et al Clin Biochem 2009; 42: G.Ivaldi, L.Leone et al. Biochimica Clinica, 2007; 31(4): 276-9

13 β Thal. trait 21 weeks, heterozygous fetus β Thalassemia (cod 39) Heterozygous newborn β Thalassemia (cod 39) Heterozygous newborn β + Thalassemia (IVSI-110)

14 Distribution of Hb A in 445 Newborns on HPLC (VARIANT TM II β-thal Short Program, Bio-Rad Laboratories Inc. USA) Beta Thal. Normal No. of Cases % Hb A

15 Distribution of Hb A in 445 Newborns on HPLC (VARIANT TM II β-thal Short Program, Bio-Rad Laboratories Inc. USA) A: β -Thalassemia carriers B: β + -Thalassemia carriers Beta Thal. Normal No. of Cases 80 B 70 A % Hb A

16 Distribution of Hb A 2 in Normal and in Beta Thalassemia Carriers % among 825 normal subjects % among 240 Beta Thalassemia carriers %A 2 % of Cases for each class of Hb A2 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

17 Hb A: in heterozygous β Thalassemia (normal β vs. pre-term β ) and Hb Lepore trait (Boston)

18 Hb Lepore trait β thal. trait (cod 39) at term β thal. trait (cod 39) pre-term (-5weeks)

19 Hb A: in homozygous β Thalassemia (β /β ) vs. compound β Thalassemia (β /β + ) or (β + /β + )

20 β Thalassemia: (β /β ) and (β /β + ) β /β (cod 39) β /β + (cod39 /IVSI-110) β + /β + (IVSI-110 / IVSI-110)

21 Distribution of Hb A in 445 Newborns on HPLC (VARIANT TM II β-thal Short Program, Bio-Rad Laboratories Inc. USA) A: β -Thalassemia carriers B: β + -Thalassemia carriers β /β or β /β + Beta Thal. Normal No. of Cases 80 B 70 A % Hb A

22 Hb A: in heterozygous Hb S (β or β + / β S )

23 Newborns at-term: Hb S trait sickle cell trait sickle cell trait normal

24 Hb A: in Hb S / β + Thal.

25 Hb S + β + Thal. (IVSI-110) At birth After 3 months After 10 months

26 Hb A: in Hb S / β + Thal. vs. Homozygous Hb S

27 - Different Retention time - Similar quantification of the Hb A (apparently) Hb S + β + Thal. (IVSI-110) Molecular studies are required for a final correct identification Homozygous Hb S

28 Hb A: in α Thalassemia Trait

29 Alpha Thalassemia: NCOI/-3.7kb Normal Hb Bart s

30 Hb A: in Hb H Disease

31 Hb Bart s: 12.4 Hb A: 32.7 Hb F: 44.4 Hb F acetyl +Hb H : 9.0 In redisreportedthe correct percentage of the Hb fractions after the integration of the all picks Hb H disease: -- Med / α

32 Alpha Thalassemia and Hb Bart s - I. Papassotiriou, J. Traeger-Synodinos et al. Hemoglobin 1999; 23 (3)

33 Two rare cases observed on cord blood Beta Variant: Hb M Saskatoon Alpha Variant: Hb Contaldo

34 Hb M Saskatoon: β 63 His>Tyr Newborn Adult

35 Hb Contaldo: α 102 Ser>Arg Adult

36 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 β Thal defects -the homozygous state (β S ) or compound heterozygosity for β S 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)

37 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) -HbLeporetrait - δ-β Thal trait

38 THANKS FOR YOUR ATTENTION

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