Leiden University Medical Center
MQN Best Practice Guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies
This article has been amended since online publication. A corrigendum also appears in this issue.
Joanne Traeger-Synodinos*,1, Cornelis L Harteveld2, John M Old3, Mary Petrou4, Renzo Galanello5, Piero Giordano2, Michael Angastioniotis6, Barbara De la Salle7, Shirley Henderson3 and Alison May8 on behalf of contributors to the EMQN haemoglobinopathies best practice meeting
Haemoglobinopathies constitute the commonest recessive monogenic disorders worldwide, and the treatment of affected individuals presents a substantial global disease burden. Carrier identification and prenatal diagnosis represent valuable procedures that identify couples at risk for having affected children, so that they can be offered options to have healthy offspring. Molecular diagnosis facilitates prenatal diagnosis and definitive diagnosis of carriers and patients (especially ‘atypical’ cases who often have complex genotype interactions). However, the haemoglobin disorders are unique among all genetic diseases in that identification of carriers is preferable by haematological (biochemical) tests rather than DNA analysis. These Best Practice guidelines offer an overview of recommended strategies and methods for carrier identification and prenatal diagnosis of haemoglobinopathies, and emphasize the importance of appropriately applying and interpreting haematological tests in supporting the optimum application and evaluation of globin gene DNA analysis.
European Journal of Human Genetics (2015) 23, 426–437; doi:10.1038/ejhg.2014.131; published online 23 July 2014
The carrier status for the most frequent and pathologically most relevant Hemoglobinopathy (HbP) traits (HbS, HbE, HbC, HbD, α- en β-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 basic information has been given 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 α- of β-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.
- Step 1: Indication based on persisting anemia, abnormal indices, family history or ethnic origin.
Look for microcytic hypochromic parameters, with or without anemia, with normal serum ferritin values, or persisting after treatment with iron supplements, especially in patients from populations at risk for HbP’s. Look into cases without clear hematological indication but with a family history and ethnic background associated with high frequencies for HbS, C of D (Black, Mediterranean or Asiatic) (follow flowchart 1):
- Step 2: The HbP routine analysis.
Apply routine analysis as follow or refer to a specialized laboratory:
Hb-electrophoresis or Hb-chromatography (HPLC) and estimate the level of the HbA2 and HbF fractions. By this method the presence and estimation of the normal and abnormal Hb fraction are semi- or fully automatically obtained. Flowchart 2:
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 request form by fast mail.
Detection of HbS by the Sickle cell test
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. It is possible to reproduce this phenomenon in vitro by the sickle test:
- Bloodsample (full blood)
- Na-metabisulfite, 1% solution in PBS (fresh). At low HbS percentages and/or HbF use a 2% solution. Don’t solve the salt using a vortex but by slowly mixing.
- microscope slides, coverslips
- reaction tube
- Mix 5 drops of Na-Metabisulfite-solution with 1 drop of blood in the reaction tube.
- Put one drop of the solution on the microscope slide.
- Cover the drop with a coverslip (make sure there is no air beneath it).
- Carefully whip away the excess fluid around the coverslip.
- Close the border of the coverslip with the glue. Must be air tight.
- Check the results after 30 minutes under a microscope (100 times enlargement with immersion-oil).
Detection of α° thalassemia by the inclusion bodies test
With α° thalassemia alleles (–/αα) and rare somatic (-α/) mutations, sporadic erythrocytes can come to exist with the same excess of β globin as with HbH disease (–/-α).
These red cells (β4 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.
How to proceed with the interpretation of your results?
- In the presence of an abnormal Hb fraction at position S on electrophoresis or HPLC a confirmation is needed by using the sickle tests. A positive sickle test (see illustration) in the presence of not less than 60% HbA (in a non-transfused patient) indicate HbA/S heterozygosity (sickle cell trait = SCT) (see figure with HPLC example).
- The presence of a confirmed HbS fraction op electrophoresis or HPLC higher than 50% to 90% indicate a Sickle cell disease status (SCD) due to HbS/S homozygosity or HbS/β-thalassemia) (see illustration electrophoresis and HPLC). Please note: HbD/D of HbD/β0-thalassemia combinations are not distinguishable from HbS/S on electrophoresis. The sickle test is equally positive both in the HbS carrier and in the SCD patient.
- Abnormal fractions such as HbE, C and D-Punjab migrate on other positions and cannot be confirmed by simple methods. Due to the high frequency and ethnic association of HbC and HbE (HbC frequent in Blacks, HbE in Asians) presumed identification might be considered acceptable when risk assessment is not required. In our opinion, due to the similar electrophoretic and chromatographic behavior of most abnormal hemoglobins (almost 1,000 to date), molecular characterization is advisable also because a question for risk assessment may come in a late phase at which the presumed identification may be taken for granted. Characterization of all abnormal Hb fractions but HbS is a task for specialized laboratories.
- HbA2 values between 3.5 and 8 % indicate β-thalassemia heterozygosity (The normal HbA2 value has no diagnostic significance in babies younger than 6 months and in rare cases of β-thalassemia heterozygosity associated normal HbA2 values (2,5-3.5%).
- HbA2 values lower than 2.5% in absence of iron depletion may indicate the presence of α-thalassemia (specialized analysis is needed).
- HbF values higher than 1% are unusual after the age of 2. In β-thalassemia heterozygosity the HbF level can be slightly to strongly elevated (1-8% in point mutation carriers 5-30% in deletion carriers)
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 analyzer 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. Recently the Premier High Resolution (trinity Biotech) was introduced in our laboratory for routine hemoglobin fractionation.
- Information and carrier diagnostics can be routinely offered to ethnic minorities at risk in immigration countries via the GP, Obstetrician or Hematologist and the Central Diagnostic Laboratories without fear for stigmatization (Giordano & Harteveld 1998).
- Carrier diagnostics can be achieved in Central Laboratories using manual or automated technologies.
- Referral to specialized laboratories and Genetic Centers is possible at all times and is essential when a couple at risk is found.
- A positive carrier analysis result should be sent to the physician together with a short prevention message indicating the importance of partner, parents or family analysis.
- Patient information can be downloaded from this web page in 11 languages and can be added to the laboratory results to facilitate information and explanation on the genetic risk and the possibilities of primary prevention.
Short information text for the GP which should accompany a positive laboratory diagnosis of HbS, C, E, D, α- en β-thalassemia carrier
When the carrier is a child: Patient is carrier of…… . Due to the possible presence of genetic risk for severe forms of HbP in the future progeny of the parents, it is appropriate to provide information and to offer carrier diagnostics to both parents of this child.
When the carrier is a young adult: Patient is carrier of …… . Due to the possible presence of genetic risk for severe forms of HbP in the progeny it is appropriate to provide genetic information and to offer carrier diagnostics to the eventual partner and to the family of this patient.
When the carrier is an elderly person: Patient is carrier of …… . Due to the possible presence of genetic risk for severe HbP is the progeny of this patient it is appropriate to provide information and to offer carrier diagnostics to his/her children. For all young HbP carriers partner analysis is appropriate.
Brochures and Information
For more information about laboratory diagnostics, prevention and informative brochures for patients in different languages please click here.
Cornelis L. Harteveld (PhD, Assoc. Prof.)
Hemoglobinopathies Laboratory tel.: 071-5269817
Dpt. Of Clinical Genetics/LDGA (LUMC)
P.O.box 9600, 2300 RC Leiden