HEMOGLOBIN ELECTROPHORESIS DR ARASH ALGHASI SHAFA HOSPITAL-AHWAZ

Hemoglobin Hemoglobin (Hb), protein constituting 1/3 of the red blood cells Each red cell has 640 million molecules of Hb sites in the cells: Haem in mitochondria Globin in polyribosomes

Synthesis of Haemoglobin Synthesis begins in proerythroblast 65% at erythroblast stage 35% at reticulocyte stage Two parts :Haem,Globin Various types of globin combines with haem to from different hemoglobin Eight functional globin chains, arranged in two clusters the b- cluster (b, g, d and e globin genes) on the short arm of chromosome 11 a- cluster (a and z globin genes) on the short arm of chromosome 16

Structure of Haem Structure 4 polypeptide Subunits Heme group Porphyrin ring Ferrous iron Globin chain 2 Alpha Chains 2 Beta chains

Synthesis of globin Eight functional globin chains, arranged in two clusters the b- cluster (b, g, d and e globin genes) on the short arm of chromosome 11 a- cluster (a and z globin genes) on the short arm of chromosome 16

Synthesis of globin Various types of globin combines with haem to from different hemoglobin Globin synthesis, starts at 3 rd week of gestation Embryonic: Haemoglobin Gower I ( z 2 e 2 ) Haemoglobin Portland ( z 2 g 2 ) Haemoglobin Gower II (a 2 e2) Fetal : HbF (a 2 g 2 ) Adult : HbA, HbA2 ( a 2 d 2 )

Normal Values Newborn Hgb A: 30% Hgb A2: less than 1% Hgb F: 50% to 80% infants 6 month: HBF =8% In infants and children older than 6 _12month; ADULT PICTURE: Hgb A: 95_98% Hgb A2: 2 _3.4% Hgb F: less than 2%

Electrophoresis Electrophoresis is a means of separating hemoglobin's. It depends on the migration of the hemoglobin molecules dissolved in a buffer on, or in, a supporting medium when an electric current is passed through them. Electrophoresis is the migration of charged molecules, particles or ion in a liquid medium under the influence of an electric field separates hemoglobin based on the Charge and size and Shape

General Procedure General operations performed in conventional electrophoresis include: (1) separation (2) staining (3) detection (4) Quantification

The sample Charge Size Shape

Methods of electrophoresis methods for separation of Hemoglobin fractions include : 1-Electrophoresis (Cellulose Acetate At Alkaline ph, Citrate Agar At acid ph) 2-Isoelectric focusing (IEF) 3-Chromatography (HPLC). 4- Capillary Electrophoresis (CE)

1A-Cellulose Acetate At Alkaline ph Cellulose acetate Hb electrophoresis at alkaline ph is the primary screening procedure used to detect variant (abnormal) Hb, of which there are several hundred. The major portion of normal adult Hb is A. In addition, up to 3.5% Hb A 2 is normally present, along with less than 2% Hb F. The more common mutant Hbs are S, C, E, D, G, and lepore. When an abnormal Hb is detected on cellulose acetate electrophoresis at an alkaline ph (8.2-8.6) further testing is frequently indicated: test for Hb S, quantitation of Hb A 2 and F, and citrate agar gel; acid/alkaline globin chain or neutral ph electrophoresis may also be warranted.

1B- Citrate Agar Electrophoresis ( acid ph) Citrate agar separates Hb fractions that migrate together on cellulose acetate agar. all Hb specimens that show an abnormal electrophoretic pattern in alkaline media (cellulose acetate agar) should undergo electrophoresis on an acid citrate agar. Citrate agar electrophoresis is used to confirm variant Hbs and further differentiates Hb S from Hb D and G, and Hb C from Hb E, O Arab, and C Harlem.. The procedure should not be used as a screening procedure because many abnormal Hbs migrate with Hb A. However, this procedure is the method of choice when examining newborns (cord blood specimens) and infants under 3 months of age for some abnormal Hbs such as S and C because the test is able to detect quantities of Hb not easily seen by other techniques.

2-isoelectric focusing electrophoresis(ief) Separates proteins by their isoelectric points (pi) Each protein has own pi = ph at which the protein has equal amount of positive and negative charges (the net charge is zero)

3-HPLC The hemoglobins are eluted by an ionic gradient and detected spectrophotometrically at 415 nm. Importantly, Hb A2 can be accurately quantitated. Typically, whole blood specimens with no pre-preparation can be added to the automated sampler.

4-CAPILLARY ELECTROPHORESIS(CZE): Separation occurs by electrophoresis in a long, but very narrow-bore silica capillary tube. The capillary is filled with sample and buffer and extends between two buffer reservoirs. Separation is effected by application of a very high voltage. The hemoglobin species are detected optically, much the same way as HPLC. CZE of hemoglobins produces a profile similar to the alkaline separation in traditional gels, but at a much higher resolution. Like CE-HPLC, this technique produces a precise Hb A2 quantitation.

Clinical cases: phenotype: major hemoglobin that present in separate bands. report: 1- hemoglobin that has much concentration is writhing first. For example in sickle trait : hb AS 2- IF TWO HEMOGLOBIN HAVE SAME CONCENTRATION THEN ONE WHO HAS MORE CLINICAL IMPORTANT WRIGHTING FIRST. For example: hbsc

Condition with increased HBA2: (α 2 δ 2 ) β-thalassemia trait :3.5 _ 7% Sickle trait PREGNANCY If hba2 BAND >8% maybe HbC or HbE Condition with decreased HBA2: Iron deficiency anemia Alpha thalassemia HPFH Heterozygote

Condition with increased HBF: β-thalassemia trait : 2_5% (IN 50 %CASES) δβ-thalassemia (heterozygote ): 5_15% HPFH (heterozygote :15_30%),HPFH (homozygotes: up to 100%) Lepor Thalassemia:1_5%

Condition with increased HBF:

Courtesy: DR.Youssef Maakaroun

CASE 4 Hb=10 mcv=68 mch=20 SICKLING TEST IS NEGATIVE. DIAGNOSIS?

CASE 4 BAND S/D/G/lepor is increased. 1-Sickling test is negative so HbS is ruled out. 2-lepor α2(δβ)2 : (10% in Heterozygous condition) and lepor (15_20% in Homozygous condition) so Hb LEPOR is ruled out. G G G 3- HbG :(- α /α α 30%) (-α /- α 45%) (-α /-α 90%) 4- HbD : (Heterozygote patients were asymptomatic: 30% ) AND (in Homozygous patients mild hemolytic anemia and chronic non-progressive splenomegaly :90%) THE PATTERN IS CONSISTENT WITH: Heterozygous HB( D) + beta thalassemia trait G

CASE 5 Hb=9 mcv=60 mch=19 Isopropanolol test is negative diagnosis?

CASE 5 Remarkably increased Hb-A2 region should be Hb C or E. But isopropranolol test is negative so Hb E is ruled out. Hb C + beta thalassemia trait

CASE 6 Hb=12 mcv=78 mch=28 Isopropanolol test is negative diagnosis?

CASE 6 Remarkably increased Hb-A2 region should be Hb C or E. But isopropranolol test is negative so Hb E is ruled out. Hb C Homozygote

CASE 8 diagnosis?

CASE 8 FAST BAND IS INCREASED, MICROCYTIC ANEMIA HBH

Courtesy: DR.Youssef Maakaroun

CASE 10 29 DAY S NEONATE : Hb S: 20% HbA:25% HbF:55% SOLUBILITY TEST IS POSITIVE diagnosis?

CASE 10 In this case, the beta chains started functioning and the HbA > HbS; likely to be Sickle trait. therefore, it is THE PATTERN IS CONSISTENT WITH: FAS Diagnosis; Sickle Trait or Sickle/betha plan: genetic counseling, repeat testing at 3 months of age, rule out sickle /betha

CASE 11 15 year girl with bone pain and paleness, without organomegaly Hb S: 78% HbA:2% HbF:20% SOLUBILITY TEST IS POSITIVE Hb=8.5,mcv=62, mch =20 Diagnosis?

CASE 11 SS + Alpha Thalassemia

CASE 12 17 Year s old girl Hb S,D,G: 4% HbA:90% HbF:5% HbA2:1% SOLUBILITY TEST IS NEGATIVE Hb=9,mcv=65, mch =20 Diagnosis?

CASE 12 LEPOR (HETROZYGOTE) lepor α2(δβ)2 : (10% in Heterozygous condition) lepor (15_20% in Homozygous condition).

CASE 13 8 Year s old Boy who is receiving blood transfusion every 3month. HbA: 15% HbF: 15% HbA2: 70% SOLUBILITY TEST IS NEGATIVE Hb=7,mcv=56, mch =20 serum ferritin=3000 Diagnosis?

CASE 13 Hb E/B+

CASE 14 Hb S: 78% HbA: 10% HbF: 8% HbA2 : 4% SOLUBILITY TEST IS POSITIVE Hb=10,mcv=73, mch =24 Diagnosis?

CASE 14 SICKLE/BETHA +

CASE 15 Hb S: 85% HbF: 10% HbA2 : 5% SOLUBILITY TEST IS POSITIVE Hb=10,mcv=73, mch =24

CASE 15 SICKLE/BETHA ZERO