Basic Protocol for Laboratory Carrier Diagnostics and Prevention of the hemoglobinopathies
Human en Clinical Genetics,
Leiden University Medical Center
The carrier status for the most frequent and pathologically most relevant Hemoglobinopathy (HbP) traits (HbS, HbE, HbC, HbD, a- and b-thalassemia) is, due to malaria selection, frequently found in individuals from Mediterranean, African and Asiatic origin and the distribution of the different traits is often country dependent. Therefore, the registration of the ethnic origin is not a discriminating action but an important element in the diagnostic process.
Prevention strategies have been applied in different countries and the success of these strategies is based on three elements (information, carrier diagnostics and referral to a Genetic Center) and on well proved actions. The most effective action is consist of offering the three elements of prevention at the individual level to young persons or young couples in the pre-marital or pre-conception phase. This action is usually initiated by the GP or specialist when referred. After basis information carrier analysis is offered to the individual or to one of the partners, preferably the woman. If carrier analysis is negative the (future) couple is not at risk. If carrier analysis will result positive the partner is controlled. If the partner is not a carrier the (future) couple will not be at risk. If also the partner is found to be carrier referral to the "couple at risk" to a Genetic Center will follow for risk assessment in a specialized lab and for prevention if wished.
How do you recognize a carrier by laboratory analysis?
Carriers of a- of b-thalassemia and of HbE present with variable microcytic hypochromic parameters (Table 1). Carriers of HbS, C en D present with border line or normal indices, usually without anemia. In these cases no significant indication can be obtained from the hematological indices and the family history or the ethnic origin are the only criteria.
For Hb chain synthesis you can make an appointment by telephone with the Hemoglobinopathies Laboratory in Leiden.
You can order a DNA analysis by sending one tube of EDTA blood together with the completed requestform by fast mail.
Detection of HbS by the Sicklecelltest
Hemoglobin S (HbS) is a common abnormal hemoglobin which can be detected by a simple test. HbS forms long polymers in oxygen-poor conditions. This phenomenon causes malformation of the erythrocytes to sickle-like abnormal cells. Is is possible to reproduce this phenomenon in vitro by the sickletest:
Detection of a° thalassemia by the inclusionbodies test
With alfa° thalassemia alleles (--/aa) and rare somatic (-a/) mutations, sporadic erythrocyten can come to exist with the same excess of b globin as with HbH disease (--/-a).
These red cells (b4 inclusion bodies) can be detected on a smear of EDTA blood, after 30 minutes of 1:1 incubation with filtered 1% brilliant cresyl blue solution in PBS at 37° C. Two hours of incubation at room temperature with the same solution is also possible.
Click here for a picture of inclusionbodies.
How to proceed with the interpretation of your results?
The technical approach and material of choice
The methods available for basic HbP analysis are various and some of them are not mentioned on this page. Manual methods such as Hb-electrophoresis and manual estimation of the HbA2 fraction have been automated with the modern HPLC technology.
Thorough evaluations of the 'Variant' HPLC (Bio-Rad) and the Menarini HA 8160 have been done in the Hemoglobinopathies Laboratory at Leiden University and in other specialized centers (Waters et al. 1998). The Bio-Rad apparatus has originally been developed specifically for HbP analysis and is in a more recent version ('Variant' II) also used for routine HbA1c analysis. Conversely, the Menarini HA 8160 is born as an HbA1c analyser but can be used for hemoglobinopathies analyses as well. Both apparatuses are a sensible choice for labs with more than 10 requests for HbP analyses a week. Some examples of HPLC diagnostics done on both apparatuses are shown.
For more information
Hemoglobinopathieën Laboratory tel.: +31(0)71-5269800
Dept of Human and Clinical Genetics
Leiden University Medical Center
P.O.Box 9600, 2300 RC Leiden, Netherlands