HPLC & Hb electrophoresis

-

What are the methods of hemoglobin analysis? 


2 methods  

Protein based method 

Molecular method (DNA based) 

 

What is protein-based method? 


Gel based electrophoresis 

Capillary zone electrophoresis (CZE) 

Isoelectric focusing (IEF) 

High performance liquid chromatography (HPLC) 

 

What is the principle of protein-based method? 


The basic idea is to separate the negatively charged proteins based on the size or electrical charge and adsorption of certain substance.

These proteins are then detected by photometer and graph is plotted. 

 

Which are the most commonly used these days? 


HPLC, CZE and IEF are commonly used  

Labs prefer HPLC and CZE as they can process large volume of samples in a relatively shorter time. 

 

What are the advantages of HPLC? 


Fully automated 

High precision

Rapid, high throughput (short run times) 

Only a very small sample (e.g., fraction of a blood spot for newborn screening) is required.

Can identify and quantify many variant  hemoglobin 

Can identify and quantify normal hemoglobin (including Hb F, Hb A2 and Hb Barts) 

Information from the pattern of variant peaks guides further testing. 

 

What are the disadvantages of HPLC? 


Capital cost (equipment and reagents) 

Interpretation of results requires technical  skill, experience

Interference/interpretation of results affected by prematurity (in newborn), recent transfusion 

Decreased resolution with sample age due to Hb degradation. 

Cannot distinguish Hb E, from Hb A2 




When results are reported, the Hbs are listed in the order of abundance (from the highest to the lowest amount). As examples: 


HB AS indicates that the amount of Hb A is greater than the amount of Hb S, consistent with sickle cell trait. 

Hb SA indicates the amount of Hb S is greater than the amount of Hb A, consistent with sickle-beta+ thalassemia. 


What are the advantages of gel electrophoresis? 


Low cost 

Separation of major Hbs (Hb A, Hb F, Hb S/D, Hb C/E/O-Arab) and several less common Hb variants 

Acid pH (citrate agar) resolves Hb S and Hb C 

 

What are the disadvantages of gel electrophoresis? 


Poor separation of Hbs other than Hbs A, F, S, C 

Cannot distinguish Hb E from Hb O, or Hb D from Hb G 

Labor intensive 

 

Capillary zone electrophoresis is better than gel-based electrophoresis 


Separates Hb A2 from Hb E (unlike HPLC) 

But poor separation of Hb S from Hb D 

Comments

Post a Comment

Popular posts from this blog

HYPOTHYROIDISM CLINICAL FEATURES

-
Image
     1.       What are the clinical features of congenital hypothyroidism in a newborn? The clinical manifestations are commonly subtle in newborns with congenital hypothyroidism (CH).   Newborns with CH may be asymptomatic because of presence of maternal T 4 (which contributes to approximately one-third of circulating T 4 in a newborn) and residual functioning thyroid tissue.   FEATURES AT BIRTH   The classical symptoms of congenital hypothyroidism include ·          lethargy, ·          hoarse cry, ·          feeding difficulty, ·          constipation not responding to treatment, and increased somnolence.   The characteristic signs of CH include ·          Prolonged neonatal jaundice, ·          Macroglossia, ·          Umbilical hernia, ·          Wide posterior fontanelle, ·          Hypotonia, ·          Dry skin, ·          Head circumference may be slightly increased d/t myxedema of brain ·          Hypothermia. ·          Hirsute for
Read more

Congenital Hypertrophic Pyloric Stenosis

-
Image
What are the parts of pylorus? The incisura angularis divides the stomach into a body to the left and a pyloric portion to the right. The sulcus intermedius further divides the pyloric portion of the stomach: the pyloric vestibule to the left, denoted by an outward convexity of the greater curvature. The pyloric antrum or pyloric canal to the right The pyloric antrum is 2.5 cm and terminates in pyloric orifice into duodenum.   What happens to the normal anatomical structural in CHPS? In infants with IHPS, the pyloric ring is no longer identifiable as a clearly definable separation between the normally distensible pyloric antrum and the duodenal cap. Instead, a channel of variable length (1.5–2.0 cm) corresponding to the pyloric canal separates the normally distensible portion of the antrum from the duodenal cap. ·          Infantile hypertrophic pyloric stenosis  was first fully described by  Harald Hirschsprung  in 1888. ·          IHPS VS CHPS - ????? IHPS is would b
Read more

TURNER SYNDROME

-
Image
What is the other name of Turner Syndrome? Ullrich-Turner syndrome  What is the m/c sex chromosome disorder in female? Turner syndrome and occurs in approximately 1 in 2000 to 1 in 2500 live female births. What are the genotypes of Turner syndrome? 45X monosomy in 45% of cases  45X mosaicism with normal cell line in 15% cases  X chromosome anomalies like - Isochromosome, Ring chromosome, Xp or Xq deletion Y chromosome mosaicism in 10-12% cases. What gene is responsible for short stature in Turner syndrome? SHOX (short stature homeobox-containing gene on the X-chromosome) is associated with short stature in Turner syndrome and in Leri-Weill dyschondrosteosis. What gene is responsible for gonadal insufficiency in Turner syndrome? BMP15 (bone morphogenetic protein 15 (BMP15) on the short arm of the X chromosome (Xp)). What is the frequency of the 45X karyotype at conception? 3% approx, but  99% of these are spontaneously aborted, accounting for 5–10% of all abortuses. Mosaics are found to
Read more