Course of LABORATORY MEDICINE
Prenatal and neonatal clinical analyses
Prenatal clinical analyses are most often concerned with genetic disorders of the foetus. In principle several fluids of foetal origin can be obtained and submitted to chemical, biochemical and microbiological analysis, e.g. amniotic fluid and blood. This however is technically difficult and of limited diagnostic value since these fluids equilibrate with maternal blood, thus eventual pathological alterations may be concealed.
INDICATIONS
Obtaining samples of foetal tissues or fluids always entails some risk of abortion (e.g. in the case of amniocentesis estimates range between 0.05% and 0.5%; in villocentesis between 0.5 and 1%), thus the indications must be critically evaluated. Risk factors that constitute strong indications are:
- maternal age > 35 years
- history of miscarriages or neonatal deaths
- exposure to theratogenic agents or infectious diseases during pregnancy
- hereditary diseases in the paternal or maternal lineages
- abnormal nuchal translucency or ultrasound finding
- positive tri-test. The triple test is carried out on maternal blood (starting from the 14th week of gestation) and relies on the measurement of the serum concentrations of (i) chorionic gonadotropin, (ii) alpha fetoprotein, and (iii) non conjugated estriol. The tri-test is positive if (i) is elevated while (ii) and (iii) are decreased; positivity is associated to an increased risk of Down syndrome of the foetus (predictive value = 60%)
PREPARATION OF SAMPLES
Foetal cells can be obtained from the amniotic fluid (amniocentesis), from the chorionic villi (villocentesis, chorionic villus sampling) or from foetal blood (cordocentesis).
Amniocentesis is possible from the 16th to the 22nd week of gestation, and is effected using a specifically designed syringe, under echographic control.
Villocentesis (biopsy of the chorionic villi of the placenta) is possible starting from the 10th-12th week of gestation and is not used after the 15th because at that time amniocentesis (which has the same diagnostic indications less risk) becomes possible.
Cordocentesis (percutaneous umbilical cord blood sampling, PUBS) cannot be performed before the 17th week of gestation and entails a 1-2% risk of miscarriage.
STUDY OF THE CHROMOSOMES
The foetal cells obtained by amniocentesis or villocentesis are cultured in artificial media and, when confluent, they are treated with colchicine to block all mitoses in the metaphase. The samples are then stained using quinacrine or Giemsa, and observed under a microscope. Metaphase chromosomes are well formed and easy to visualize; dedicated computer softwares for image analysis are available.
Pathological conditions that may be diagnosed with this method include aneuploidies (alterations in the number of the chromosomes and/or structural anomalies):
1. Alterations in the number of the sex chromosomes 1a. Monosomy: kariotype X0 (Turner syndrome) 1b. Sexual trisomies: kariotypes XXX, XXY (Klinefelter syndrome) and XYY |
2. Alterations in the number of the chromosomes Trisomies: 21 (Down syndrome); 18 (Edwards syndrome); 13 (Patau syndrome) |
3. Translocations (exchanges of genetic material between different chromosomes) 3a. Balanced translocations 3b. Unbalanced translocations |
4. Partial deletions or partial duplications |
5. Somatic mosaicism (some but not all of the cells present any of the aneuploidies listed above) |
STUDY OF SELECTED GENES
In the presence of familiarity for hereditary diseases, selected genes can be amplified using the Polymerase Chain Reaction method and sequenced, to screen for mutations.
The Polymerase Chain Reaction is carried out by adding to the genetic material to be tested the RNA primers corresponding to the beginning of the gene of interest (of bothDNA strands), a heat-resistant DNA polymerase, and an excess of deoxyribonucleotides tri-phosphate. A thermal cycle is then started in which the mixture is heated to promoted the dissociation of the complementary DNA strands, and cooled to allow the DNA polymerase to synthesize the segment (gene) that follows the chosen primer. After a chosen number of cycles the DNA segment (gene) of interest has been greatly amplified and can be submitted to the detection of its nucleotide sequence (sequencing).
Examples of hereditary genetic diseases that can be diagnosed by selective amplification and sequencing include:
| Disease | Frequency at birth | Hereditary mechanism | Gene/protein involved |
| Familial hypercholesterolemia | 1:500 | autosomal dominant | LDL receptor |
| Polycistic kidney disease | 1:1,200 | autosomal dominant | |
| Marfan syndrome | 1:4,000 | autosomal dominant | Fibrillin-1 |
| Sickle cell anemia | 1:600 (USA) | autosomal recessive | Hemoglobin beta subunits |
| Beta thalassemia | 1:500 | autosomal recessive | Hemoglobin beta chain |
| Cystic fibrosis | 1:2,000 | autosomal recessive | Chloride membrane transporter CFTR |
| Hemophilia A | 1:10,000 | X-linked | Coagulation factor VIII |
MITOCHONDRIAL DISEASES
All the mitochondria of the foetus (or of the adult) cells come from the ovum, i.e. they are of maternal origin. Several genetic diseases due to mitochondrial defects are known and are maternally inherited. They can be diagnosed by studying the mitocondrion DNA extracted from any cell (including those obatined by amniocentesis, or villocentesis). The most common mitochondrial diseases are myopathies and neuropathies.
OTHER DISEASES THAT CAN BE DIAGNOSED IN THE FOETUS
Foetal infections may be diagnosed from the presence of bacteria in the (normally sterile) amniotic fluid. They are severe, life-threatening conditions and should be promptly treated with antibiotics administered to the mother or directly in the amnios.
Lung maturity can be estimated by the lecithin/sphingomyelin ratio (normal value >2:1) and the surfactant/albumin ratio (normal value >55) in the amniotic fluid.
Rh incompatibility and risk of foetal erythroblastosis.
Several clinical analysis are routinely carried out on neonatal blood and urine. Technically these are not different from the equivalent analyses one could carry out on the adult; but some diseases must be diagnosed immediately after birth because they require prompt therapy. Examples of these include:
Congenital hypothyroidism
Phenylketonuria and other metabolic defects
The reason why diagnosis is so urgent is that the foetus suffering of any of these conditions is normal at birth, because the metabolism of the mother (if she is healthy) compensates for the defect. After birth the disease becomes evident and usually begins to produce organ damage (most often in the brain).
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