Protocol:Best-practice recommendations

Genetic counselling
It is best practice for all couples undergoing prenatal diagnosis to be counselled by a qualified health professional well versed in the molecular diversity of the hemoglobinopathies. No woman should undergo prenatal diagnosis unless she has been counselled by a qualified health professional. There should be a consent form signed by patient and counsellor accompanying the fetal sample.

Analysis of parental blood samples
Copies of hematology results should be sent to the molecular diagnostic laboratory. Blood samples should be obtained from both parents to confirm the phenotype of parents by carrying out a full blood count and haemoglobinopathy screen such as electrophoresis, and also to provide a source of control DNA for the molecular analysis. This should be repeated with every prenatal diagnosis that any couple undergoes.

Partner not available for testing
There are cases where a carrier woman requests prenatal diagnosis although her partner is unavailable for testing. In such situations it is important to evaluate the risk of a major haemoglobinopathy in the fetus:


 * For a sickle cell trait mother and untested partner. If a sickle cell trait genotype is diagnosed in the fetus, then test for common &beta;-thalassaemia mutations and any other haemoglobinopathy genes (especially &beta;C or &beta;D) known to exist in the partner’s ethnic group. Alternatively, the method more commonly used,is fetal DNA sequencing, to detect any rare mutations present.
 * For a &beta;-thalassaemia trait mother and untested partner. If the mother’s β-thalassaemia mutation is diagnosed in fetus, the possibility of the fetus being homozygous or compound heterozygous for β-thalassaemia should be excluded, either by testing for the β-thalassaemia mutations and any other β-gene haemoglobinopahies found in the fathers ethnic group. Alternatively, the method more commonly used, is fetal DNA sequencing to detect any rare mutations present.

Fetal Samples
There are three possible procedures for conventional prenatal diagnosis, chorionic villus sampling, amniocentesis and fetal blood sampling. Prenatal diagnosis of haemoglobinopathies should preferably be carried out by a chorionic-villus sample in the first trimester of pregnancy (10–12 weeks), as it allows earlier diagnosis than the use of amniocentesis samples, both techniques in turn being more reliable than analyses based on fetal blood sampling.

Chorionic Villus Sampling
Chorionic villus sampling (CVS) provides a good source of DNA. The risk of maternal contamination is low with careful microscopic dissection to remove contaminating maternal decidua. There is a risk of maternal contamination if the sample is cultured, although this should not be necessary if the sample is of adequate size. The risk of miscarriage is low if the sample taken in an experienced center. The result is available early in pregnancy. It is essential that any maternal tissue present in the sample is removed, as the presence of maternal tissue may result in a diagnostic error. The sample should be placed in a Petri dish and washed with saline or culture medium to remove any maternal blood present. The Petri dish should then be placed on a dissecting microscope and any maternal decidua removed with small clean forceps or two needles.

Amniocentesis
Amniocytes can be used for molecular analysis directly spun down from the amniocentesis sample. This usually yields sufficient DNA for analysis with PCR-based methods. For greater amounts of fetal DNA, samples have to be cultured for 10–14 days. Culture of the cells reduces risk of maternal contamination, but the result is delayed. The risk of miscarriage following amniocentesis is low if the sample is taken in an experienced center. The result is available later in pregnancy, compared to CVS-based analyses, as amniocentesis cannot be taken earlier than about the 16th week.

Take Care Direct analysis should be carried out with caution as the fetal cells are invariably contaminated with maternal cells.

Fetal Blood Sampling
1–2 mL of fetal blood is usually obtained, which can be used for molecular analysis or globin chain biosynthesis studies. The latter can be used when parental mutations are not known, if a couple present late, or if partner is unavailable for testing. The diagnosis is based on the relative synthesis of &beta;-globin (representing HbA) and &gamma;-globin (representing HbF). A &beta;/&gamma; chain synthesis ratio above 0.02–0.03 (slightly variable between laboratories) indicates an unaffected fetus. Take Care When using this technique, results should be interpreted with care, as some mild &beta;-globin mutations can produce higher levels of &beta;-globin, leading to a risk of misdiagnosis.

Recommendations
Prenatal diagnosis using globin chain biosynthesis in fetal blood is no longer recommended by most centres. The approach is associated with a higher rate of miscarriage and is carried out late in pregnancy (after 18–20 weeks) compared to other approaches.

Chorionic villi sampling is the recommended approach for fetal DNA diagnosis. However fetal blood sampling has been used in some centres when alpha thalassaemia hydrops fetalis is suspected by ultrasound examination in the fetus. The advantage is that the sample may be tested by HPLC or similar quick method for the presence of HbF, with a result available within 30 min.

Genotype Analysis

 * The laboratory carrying out the molecular analysis should choose the technique(s) most suitable for their laboratory, expertise and population.
 * Always analyse parental and the appropriate control DNAs simultaneously with the fetal DNA and use a blank control sample.
 * Perform duplicate tests to minimize human errors.
 * To monitor potential laboratory errors such as partial digestion or allele drop-outs use two independent diagnostic methods on each sample for each mutation being investigated.
 * Use a limited number of amplification cycles to minimise co-amplification of any maternal DNA.

Maternal DNA contamination
PCR-based prenatal diagnoses are highly sensitive, which may lead to the detection of maternal DNA contamination in fetal samples. The following procedures are intended to minimise the diagnostic error rate. Routine examination of short tandem repeats (STRs) or Variable Number of Tandem Repeats (VNTRs) in fetal and parental DNA samples by PCR analysis is essential to rule out maternal contamination.

The tandem repeated minisatellite regions of DNA are an ideal method to identify maternal contamination. These highly polymorphic DNA regions show alleleic variation in the number of repeated units. Because of the large number of different alleles these repetitive DNA areas provide informative genetic markers. Since these variations are inherited according to Mendelian genetics, they can be used to test for maternal contamination and indeed will also identify non-paternity.

It is recommended to check for maternal DNA contamination in every case of prenatal diagnosis especially when the fetal genotype is the same as mother's genotype. The choice of polymorphic markers available is wide, including the short tandem repeat (STR) markers such as D21S11, D21S1414, D18S535  or Variable Number Tandem Repeat (VNTR) markers such as ApoB, IgJH and Has-ras.

Using the quantitative fluorescent PCR (QF-PCR) described below, trisomies may also be detected, particularly tri-allelic patterns

Recommendations

 * Use polymorphism analysis to exclude maternal contamination (and may also identify non-paternity).
 * Check for maternal DNA contamination in every case, especially when the fetal genotype is the same as the mother’s genotype. The choice of polymorphic markers available is wide, including Short Tandem Repeat (STR) markers such as D21S11, D21S1414, D18S535 (31). Commercial kits are also now available (eg. ABI AmpF lSTR Identifiler Kit) that tests for up to 16 STR’s. Alternatevely use Variable Number Tandem Repeat (VNTR’s) markers such as ApoB, IgJH and Has-ras (32). Note that STR kits require sizing on an automatic DNA sequencer, whereas VNTRs can be sized by aga-rose gel electrophoresis.
 * When the fetal genotype is the same as the mother’s, and the VNTR/STR markers do not exclude maternal contamination, the fetal diagnosis report should state these findings and indicate greater risk of error in the fetal result. It is strongly recommended that another fresh fetal sample or a cultured sample should be requested for analysis in these cases.

Reports
The fetal DNA report should detail types of DNA analysis performed and clearly state the risk of misdiagnosis based on reported technical errors of the protocols utilized. Laboratory error rates should be documented and explained to patients for all methods.

Prenatal diagnosis follow-up
Ideally, a fetal DNA diagnosis should be confirmed at birth through a request for a cord blood sample that can be sent out with fetal diagnosis report. Hematological, hemoglobin and DNA analyses also requested by some centers. As part of an internal quality assurance procedure, fetal material should be requested when affected pregnancies are terminated to confirm prenatal diagnosis result.

Summary

 * 1) Parental studies: Ensure that fresh parental blood samples are obtained with the fetal sample in order to check the parental phenotypes and to provide fresh control DNA samples. In cases when the father is not available, as often seems to happen with sickle-cell prenatal diagnoses, copies of all laboratory results should be seen.
 * 2) Partner unavailable: In such cases at risk for a sickle cell disorder, extra tests are carried out when a blood sample from the father is not available. The fetal DNA is always analysed for &beta;S, &beta;C and common &beta;-thalassemia mutations in the parents’ ethnic group when an AS genotype is diagnosed, to avoid an incorrect diagnosis as far as possible.
 * 3) Fetal samples: Ensure that the chorionic-villus sample has undergone careful microscopic dissection to remove any contaminating maternal decidua.
 * 4) Genotype analysis: Always analyse parental and the appropriate control DNAs with the fetal DNA and always repeat the fetal DNA analysis to double check the result.
 * 5) Genotype analysis: Whenever possible, use an alternative diagnostic method to confirm the diagnosis.
 * 6) Maternal DNA contamination: Check for maternal DNA contamination in every case. Polymorphism analysis by PCR is used routinely to exclude error due to maternal DNA contamination or non-paternity.
 * 7) Reporting: The fetal DNA diagnosis report should detail the types of DNA analysis used and clearly state the risk of misdiagnosis due to technical errors based on current data.