thalassemia

pediatrics

1. Laboratory Diagnosis of Hemoglobinopathies and Thalassemia by HPLC

2. HEMOGLOBIN molecule is a tetramer made up of two pairs polypepetide chains, with each chain Having a iron containig heme group attached. Polypeptide chains are of chemically different. Various globin chain differ in both number and sequence of amino acids and their synthesis by different genes. On chomosone no. 16, 2 sets of gene for alfa chains and zeta chains are located and genes for beta , gama , epsilon and delta globin chains Are located on chromosome 11.

3. Within the red blood cells of an embryo, fetus, child and adult 6 different hemoglobins are detected: embryonic H bs : Gower 1 (zeta 2 and epsilon 2 )and gower 2 ( alfa 2 and epsilon 2)and Portland (zeta 2 and gama 2). Gower H b are predominate in 4 to 8 weeks of gestation and portland H b predominate in 3 rd month of gestation. Fetal H b : HbF ( alfa 2 and gama 2). After 8 th week of gestation it predominates and @ 24 week of gestation it constitutes 90% of total Hb . During third trimester a gradual decline occurs so that @ birth HbF is 70% of total Hb and only trace is present @ 6 to 12 months of age. Adult Hbs : HbA ( alfa 2 and beta 2) and HbA2 ( alfa 2 and delta 2). @24 th of gestation HbA is 5 to 10% and @ birth 30% of total Hb . After 6 to 12 months of life normal adult pattern apppears . HbA2 is <1% @ birth and 2 to 3.4% @ 12 months of age.

4. Hemoglobin -Development Switching

5. Abnormalities in protein part of hemoglobin are reffered to as Hemoglobinopathies , which include: Sickle cell anaemia ( Hb SS), Sickle cell Trait ( Hb AS) , Hb C, Hb D, Hb E, Hb M, HPFH, Thalassemia syndromes and so many(>800 Variants of Hb .). Most of which can be diagnosed by HPLC.

6. High-Pressure Liquid Chromatography

7. Hemoglobins are separated by an analytical cartridge( negetively charged silica) in cation exchange HPLC using a preprogrammed buffer(Na+ and K+ ions) gradient with increasing ionic strength to the cartridge (Figure 3). The hemoglobin fractions separate based on their ionic interaction with the cartridge. The separated fractions pass through a flow cell, where absorbance is measured at 415 nm and again at 690 nm to reduce background noise. Changes in absorbance are monitored over time producing a chromatogram (absorbance vs. time). Each hemoglobin has its own characteristic retention time and is measured from the time of sample injection into the HPLC to the maximum point of each peak

8. Identification of unknown hemoglobin is achieved through comparison with known hemoglobin retention times. If a peak elutes at a retention time not predetermined, it is labeled as an unknown. HPLC achieves good separation and quantitation of HbF and HbA2 in addition to screening for variant hemoglobins along with thalassemia . Chromatography of each sample is completed in 6 min. HPLC is highly reproducible, offers simplicity with automation, superior resolution and rapid results. Some HPLC instrument programs can identify hemoglobinopathies from both newborns and adult specimens while others identify only one or the other. Identification between adult and newborn specimens depends on the algorithm/software/instrument specifications.

9. 9050 Variable UV/Vis Detector 9010 Solvent Delivery System HPLC Solvent Reservoirs Rheodyne Injector HPLC Column 9060 Polychrom (Diode Array) Detector Computer Workstation HPLC MACHINE

10. HPLC PRINCIPLE

11. Advantages •Fast •Small amounts of sample •Accurate quantitation of A2 Disadvantages • Hemoglobin E cannot be separated from A2 • Hemoglobin H and Barts elute too quickly from column

12. INTERPRETATION OF HPLC RESULTS O ne should consider: Hemoglobin Retention Time Variant Hemoglobin Percentage A 2 Percentage Number of Variants CBC Indices Transfusion History Age Clinical Course

13. BIO-RAD VARIANT WINDOWS PEAK NAME RETENTION TIME (MIN) PEAK NAME RETENTION TIME (MIN) F Window 0.98-1.20 A 2 Window 3.30-3.90 P2 Window 1.24-1.40 D Window 3.90-4.30 P3 Window 1.40-1.90 S Window 4.30-4.90 A Window 1.90-3.10 C Window 4.90-5.30

14. Various Hemoglobinopathies and HPLC reports

15. Normal HPLC at birth showing Hb F 75% and Hb A19.3%

16. Normal HPLC report of adult or children beyond 12 months of age.

17. Alfa thalassemia Hemoglobin Bart (all 4 alfa gene deletion) And Hemoglobin H (3 alfa gene deletion) On HPLC

18. Chromatogram of β thalassemia major showing Hb F 92.4% Hb A 2.4% and Hb A2 5.1% β thalassemia

19. Chromatogram of beta thalassemia trait showing elevated HbA2 5.6% (RT 3.68 min) and HbF 0.4%.

20. Chromatogram of E beta thalassemia showing elevated HbA2 51.4%, Hb F 30%.

21. Chromatogram showing elevated Hb F (29.1%) suggestive of HPFH. HPFH

22. Chromatogram showing homozygous sickle cell anaemia Hb SS 88% Sickle cell anaemia Homozygous Hb SS S Disease (Hemoglobin SS ) Severe Symptoms, Sickling in Vivo Hydroxy Urea Treatment →Induces F Crises →Bone Pain, Hemolysis , Stroke, etc

23. Chromatogram of Hb S trait showing Hb S 25.9% (RT 4.42 min). Sickle cell Anaemia Heterozygous Hb SA S Trait (Hemoglobin AS) β 6 Glu→ Val Common In Blacks; Other Populations Asymptomatic , Blood Sickles in Vitro Protective Against Malaria

24. Chromatogram showing Hb C 9% Hemoglobin C   substitution of a  glutamic acid  residue with a  lysine  residue at the 6th position of the  β- globin   chain highest frequencies in  West Africa , where it has been associated with protection against  malaria . Most people do not have  symptoms .

25. Chromatogram of HbD Punjab trait showing HbD 40.5% (RT 4.15 min). Β 121Glu→Gln Found In India (D-Punjab/D-Los Angeles) Most Common D In U.S. Blacks (< 0.02%) Trait Asymtomatic , No Anemia, Normal CBC Disease Asymtomatic , No Anemia/ Hemolysis Hb D

26. Chromatogram of Hb D Punjab homozygous showing Hb D 87.9%. Chromatogram of Hb D Iran showing HbA2 41%.

27. Chromatogram of HbE homozygous showing HbA2 77.5% (RT 3.73 min). Hb E 2nd most prevalent hemoglobin variant –30,000,000 world wide –80% in Southeast Asia •Hb E trait: microcytosis (mean MCV=65fl). No anemia • Hb E disease: MCV =55-65fl with minimal anemia •*On HPLC has similar migration pattern as Hb A2

28. Chromatogram of HbE trait showing HbA2 24.8% (RT 3.72 min).

29. Figure 3: CE-HPLC: Analysis of haemolysates from patients presenting with different associations involving Hb O-Arab. (A) Compound heterozygous Hb O-Arab/ β0 thalassaemia. (B) Compound heterozygous Hb S/HbO-Arab. (C) Compound heterozygous Hb C/Hb O-Arab. (D) Heterozygote Hb O-Arab associated with G-Philadelphia. The hybrid Hb ( α2 G-Phil β2  O-Arab ) elutes in C window.   Figure 3: CE-HPLC: Analysis of haemolysates from patients presenting with different associations involving Hb O-Arab. (A) Compound heterozygous Hb O-Arab/ β0 thalassaemia . (B) Compound heterozygous Hb S/ HbO -Arab. (C) Compound heterozygous Hb C/ Hb O-Arab. (D) Heterozygote Hb O-Arab associated with G-Philadelphia. The hybrid Hb ( α2 G-Phil β2  O-Arab ) elutes in C window.

30. Hemoglobin S/O(Arab ) is a rare compound heterozygous hemoglobinopathy characterized by the presence of two variant beta- globin chains: beta6Glu --> Val ( Hb S) and beta121Glu --> Lys ( Hb O(Arab )).   Hb S/O(Arab) disease is a severe sickling hemoglobinopathy with laboratory and clinical manifestations similar to those of homozygous sickle cell anemia .

31. Hemoglobin G -Philadelphia is a stable, normally-functioning oxygen carrier. G-Philadelphia trait (1 mutated gene) is completely silent. Homozygous G-Philadelphia (2 mutated genes) is rare and, depending on the origin of the mutation and the coinheritance of other alpha thalassemic mutations, produces a spectrum of effects from mild microcytosis to full blown H disease in very rare cases .  Most patients without additional thalassemias adapt well to persistent borderline anemia . Significant hemolysis and/or anemia or microcytosis with hemoglobin G-Philadelphia trait should prompt further investigations for the coinheritance of a thalassemia or sickle hemoglobin

32. Chomatogram showing the Hb Q-India variant haemoglobin  Hemoglobin Q - India (alpha) 64 Asp His is an alpha chain variant which is generally found in heterozygous state and presents normal hematological blood picture. It becomes symptomatic only in a homozygote state and when present in association with other conditions like beta- thalassaemia , alpha- thalassaemia , HbE  

33. Chromatogram of showing HbA2 10.1% presumptive diagnosis of Hb Lepore . Hemoglobin Lepore Hb Lepore is a structurally abnormal hemoglobin in which the abnormal globin chain is a hybrid or fused globin chain (db). Three different Lepore hemoglobins have been identified, differing from each other in the point at which the db fusion occurs; Hb Lepore Hollandia (d22/b50), Hb Lepore Baltimore (d59/b86) Hb Lepore Boston (d87/b116)

34. Chromatogram of Hb Hope showing elevated P2 peak (48.4%). Hb Hope is a clinically asymptomatic β- chain variant [beta136 (H14) Gly→Asp (GGT→GAT)]. It is more prevalent in Mediterranean region of the world than in Asian countries and extremely rare in India. Causes Spuriously Elevated HbA1c Values on HPLC Assay.

35. Chromatogram of Hb J showing elevated P3 peak. Hemoglobin J - Rajappen (alpha)90 Lys  →   Thr is an alpha chain variant found in heterozygous state and presents normal hematological blood picture.