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Heno

This document discusses various hemoglobin assays and their advantages and disadvantages for diagnosing sickle cell disease. It finds that high-performance liquid chromatography allows the most accurate quantification of normal and variant hemoglobins. Isoelectric focusing and cellulose acetate electrophoresis are useful for initial screening but less quantitative. The solubility test alone should never be used for genetic counseling due to limitations but may provide an initial low-cost screen. Definitive diagnosis requires DNA testing or integration of laboratory studies.

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Ahmed Ben Bella
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168 views

Heno

This document discusses various hemoglobin assays and their advantages and disadvantages for diagnosing sickle cell disease. It finds that high-performance liquid chromatography allows the most accurate quantification of normal and variant hemoglobins. Isoelectric focusing and cellulose acetate electrophoresis are useful for initial screening but less quantitative. The solubility test alone should never be used for genetic counseling due to limitations but may provide an initial low-cost screen. Definitive diagnosis requires DNA testing or integration of laboratory studies.

Uploaded by

Ahmed Ben Bella
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Title: Sickle Cell Disease GeneReview Hemoglobin Assays: Advantages and Disadvantages

Author: Bender MA
Updated: August 2017

Hemoglobin Assays: Advantages and Disadvantages

High-performance liquid chromatography (HPLC)

 Readily separates some proteins that cannot be resolved by other means;

 Allows for accurate quantification of normal and variant hemoglobins at low concentrations,
enabling differentiation of Hb S/β+-thalassemia from sickle cell trait (Hb A/S), as well as a
quantitative description of compound heterozygous disorders such as Hb S/HPFH (hereditary
persistence of fetal hemoglobin; see Beta-Thalassemia, Genotype-Phenotype Correlations)
and Hb S/C;

 Does not definitively distinguish Hb S/β°-thalassemia from Hb S/S (identification requires DNA
testing or integration with other laboratory studies).

Isoelectric focusing

 Capable of higher resolution than other hemoglobin electrophoresis

 Routine isoelectric focusing provides an efficient platform for high-throughput screening and
thus is often used for newborn screening, but is less quantitative than HPLC.

 Capillary isoelectric focusing technology allows for separation of very small samples,
quantification, and automation of sampling.

Cellulose acetate electrophoresis and citrate agar electrophoresis

 Useful for quick screening of a small number of samples

 Protein bands are relatively wide and many abnormal hemoglobins overlap.

 Quantitative densitometry of abnormal hemoglobins is inaccurate at low concentrations (e.g.,


HbA2 and HbF in adults).

Kleihauer-Betke test

 Acid-elution test that detects the presence of cells with high fetal hemoglobin content.

 Can be used to characterize coexistent hereditary persistence of fetal hemoglobin (HPFH) with
SCD.
Solubility test (i.e., Sickledex, Sickleprep, or Sicklequik)

 Utilizes the relative insolubility of deoxygenated HbS in solutions of high molarity. Hemoglobin
S in hemolysates precipitate in the test solution while other hemoglobins remain in solution.

 Solubility tests alone should NEVER be used for genetic counseling assessments if one
partner is known to have HbS, as this test will not detect β°-thalassemia, HbC, or other
hemoglobin variants that can lead to compound heterozygous forms of SCD.

The solubility test has no place in the definitive diagnosis of SCD because:

o It does not differentiate SCD from sickle cell trait (Hb A/S);

o False positives have been reported [Hara 1973];

o High levels of HbF may cause false negative results in infants with SCD;

o It may miss some clinically significant forms of sickle hemoglobinopathies (e.g., Hb S/C)
[Fabry et al 2003].

The main uses for the solubility tests are:

o A low-cost and rapid screen for the presence of HbS prior to investing in definitive
testing; and

o Emergent estimation of whether a clinically significant hemoglobinopathy exists (if


combined with a CBC, blood smear, and reticulocyte count).

References

Fabry ME, Archyra SA, Suzuka SM, Nagel RL. Solubility measurement of the sickle polymer. In: Nagel RL, ed.
Hemoglobin Disorders: Molecular Methods and Protocols. Totowa, NJ: Humana Press; 2003:271-87.
Hara S. Reliability and modification of Sickledex test. J Natl Med Assoc. 1973;1973;65:431.

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