General strategy for diagnostic procedures

Determining carrier status
The key to identifying the globin gene mutations in carriers and affected patients is an understanding of the genotype/phenotype relationships of the various globin gene mutations and the effects of interaction when several mutations are co-inherited. Often the quickest way to identify the mutations in an affected patient is to study the hematology of the patient’s parents and other family members and to screen them for single mutations. Carrier screening and mutation identification also form one of the cornerstones of any prevention program for the hemoglobin disorders. The strategy for carrier screening and mutation analysis is that heterozygotes, although free of symptoms, present specific hematological characteristics that are useful for their identification.

The accurate determination of the carrier phenotype is essential for the selection of the appropriate molecular tests to determine the carrier genotype for patient diagnosis or research studies involving new hemoglobinopathies. The basic hematological tests required are the measurement of the mean corpuscular volume (MCV), the mean corpuscular hemoglobin (MCH) value and the quantity of Hb A2 and Hb F. In addition, the hemoglobin pattern needs to be examined, by electrophoresis methods and high-performance liquid chromatography. Other parameters also need to be examined in many cases, such as iron status and the stability of suspected Hb variants. The ITHANET Protocols aim to provide best-practice guidelines presenting the consensus of the expert laboratories involved in the ITHANET project.

Determining the mutation
The second phase of any investigation is to look at the hemoglobinopathy disorder at the molecular level. This can be either at the level of mRNA expression in the erythrocytes or at the level of the DNA sequence change which is responsible for the disorder.

A variety of techniques based on the amplification of DNA by the polymerase chain reaction (PCR) have been developed to identify the globin gene mutations. The techniques commonly used are dot blot analysis, reverse dot blot analysis, the amplification refractory mutation system (ARMS), denaturing gradient gel electrophoresis, gap PCR, Multiplex ligation-dependent probe amplification (MLPA) and restriction endonuclease analysis. Each method has its own advantages and disadvantages and all are recommended for use in best-practice guidelines. Which particular one is chosen by a given laboratory depends not only on the technical expertise available in the diagnostic laboratory but also on the type and variety of the mutations likely to be encountered in the individuals being screened. Hence it is necessary to have procedures for all these techniques, both for known mutations and unknown mutations.