Protocol:ITHANET Laboratory Protocols

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The ITHANET Laboratory Protocols want to provide strategies and reliable methods for diagnostic and research work.
To contribute and add new ITHANET Laboratory Protocols, use the Protocol Template.



The hemoglobinopathies are a diverse group of inherited recessive disorders consisting of the structural hemoglobin variants and the thalassemias. Together they form the commonest single-gene disorder in the world population and they are a serious public health problem in many countries. Although there is no definitive cure for the hemoglobinopathies, the methods of clinical management have improved considerably over the last few years and the life expectancy of affected individuals has been significantly increased. However, the treatment required is very expensive and is not a realistic means of controlling the disorders for many countries. Therefore most countries, especially those with a high incidence of β-thalassemia, are applying prevention programs, which involves screening the population for carriers, identifying the couples at risk, and providing a prenatal diagnosis service. Co-ordination of best practice is essential for prenatal diagnosis laboratories to provide a quality service, and organising best-practice protocols in wiki format, open to scrutiny and correction by the hemoglobinopathy community, is an important step towards a maintainable and up-to-date collection of best-practice protocols.

General strategy for diagnostic procedures

Hematological procedures for the investigation of carrier states

General procedures

  1. Best-practice recommendations
  2. Red blood cell indices
  3. Red blood cell indices morphology
  4. Reticulocyte count and Hb H inclusions
  5. Osmotic fragility test
  6. Hb stability – isopropanol and heat tests
  7. Hb F quantitation by alkali denaturation
  8. F-cells by acid elution technique
  9. F-cells by immunofluorescence
  10. Hb S detection - sickling and solubility tests
  11. Hb E detection by DCIP test
  12. Zinc-protoporphyrin (ZnPP)
  13. Serum iron

Haemoglobin analysis

  1. Hb A2 measurement by column chromatography
  2. Hb A2 variant analysis by HPLC
  3. Cellulose acetate electrophoresis
  4. Agar gel electrophoresis
  5. Isoelectric focussing electrophoresis
  6. Starch gel electrophoresis

Molecular diagnosis procedures for the investigation of genotypes

Globin chain synthesis

  1. Weatherall and Clegg method
  2. HPLC method
  3. Vertical isoelectric focusing method

DNA Preparation

  1. DNA from blood: salting-out method
  2. DNA from blood: phenol-chloroform method
  3. DNA from CVS: phenol-chloroform method
  4. DNA from amniocytes: phenol-chloroform method

Diagnosis of known mutations

  1. Diagnostic strategy
  2. Allele specific oligonucleotide (ASO) dot blot
  3. Reverse dot blot (RDB)
  4. Amplification refractory mutation system (ARMS)
  5. Restriction enzyme PCR
  6. Gap-PCR
  7. Multiplex Ligation-dependent Probe Amplification (MLPA)

Diagnosis of unknown mutations

  1. Denaturing gradient gel electrophoresis (DGGE)
  2. DNA sequencing
  3. cDNA sequencing

Best practice procedures for prenatal diagnosis

  1. Best practice guidelines
  2. Checking for maternal DNA contamination by STR analysis
  3. Checking for maternal DNS contamination by variable number tandem repeat (VNTR) analysis

New methods used in the research studies

  1. Real-time PCR
  2. Single-cell PCR
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