The length of the whole process depends on how smooth and problem-free its individual steps turn out to be. The basic steps include:
1.
Blood sampling
A 10 ml sample of peripheral maternal blood is taken by a female nurse. This requirement minimises potential male DNA contamination, which might affect the process of determining the sex of the foetus (if requested by the pregnant woman). Blood samples can be taken as early as in the 11th week of pregnancy* and need not be taken on an empty stomach.
TRISOMY test screening can be carried out between the 11th and the 22nd week of pregnancy. The earliest or the latest possible options are subject to the pregnant woman’s preference and her doctor’s recommendation, which depends on the patient’s health condition.
Stored in a 10 ml test tube containing EDTA, the blood sample must be labelled with a bar code sticker (included in the special TRISOMY test order form), with the patient’s name and birth certificate number on it. As soon as the test tube is filled, it must be stirred carefully (by turning it upside down 10 times over – no shaking!). Biological material must generally be stored at a temperature of 4°C. The sample must not be allowed to freeze.
If the transport period is expected to be longer, i.e. more than 36 hours after blood sampling, it is necessary to use the Streck Cell Free DNA BCT system.
*Following a resolution of the Ethics Committee of the Ministry of Health of the Slovak Republic, the Laboratory provides test results only upon completion of the 12th week of pregnancy.
The laboratory will not provide any test results unless the patient has completed her 12th week of pregnancy!
2.
Delivery of the sample to the lab
Along with a correctly completed special order form, every sample is stored in a single transport bag and a thermo bag (at 4°C), which must be delivered to the Medirex, a. s. Central Laboratory in Bratislava within a maximum of 36 hours upon blood sampling.
The transport request must be approved by the Medirex transport control team at least 24 hours prior to blood sampling: 0800 00 30 30, dispecing@medirex.sk.
3.
Payment for the test
He payment of a TRISOMY test, TRISOMY test XY, TRISOMY test + depends on the country specification.
4.
Releasing the blood sample for analysis
As soon as the payment has been identified, the sample is analysed using a special method.
5.
In the lab: analysing and evaluating data using special software
1. Blood Sample Processing
As soon as the test tube is delivered to the laboratory, the sample is processed to isolate the plasma that contains genetic foetal material in extracellular form.
2. DNA Isolation
Free circulating DNA is obtained from the plasma using a precise multistep purification procedure. The DNA comes from both the mother and her foetus. The foetal DNA represents approximately 13% of the DNA obtained from the plasma. If the blood sample was not taken in compliance with the guidelines, however, the blood cells contained in the sample may have disintegrated and the proportion of foetal DNA may have dropped. This makes the detection of possible foetal trisomy impossible. Upon isolation, a sample quality check is performed to see whether a breakdown of blood cells has taken place or not. If it is detected, we contact the attending doctor to ask them to take another blood sample.
3. Preparing DNA Samples for Analysis
The isolated DNA is processed in the laboratory - this process is referred to as sequencing library preparation. These sequencing libraries are required for the purpose of analysing samples using new-generation sequencing. As the total amount of DNA obtained from the plasma is very small, we use a polymerase chain reaction to multiply this amount. The preparation of a sequencing library suitable for the analytical procedure takes 48 hours.
4. Sample Quality Check
TRISOMY test screening is a time-consuming and costly screening procedure, its result being heavily dependent on well-prepared samples for analysis. A sensitive fluorescent electrophoretic analysis, however, allows us to see whether the prepared sequencing library is actually suitable for further analysis. Well-prepared sequencing libraries are subsequently used in the next phase, which is called “sequential analysis”.
5. Final Analysis - Sequencing
By reading 30 million fragments in the course of a 20-hour analytical procedure, we perform low-coverage whole-genome analysis (reading approximately every tenth base, not every single one there is). This sequencing method yields approximately 500 MB of data, which is subsequently evaluated using special software based on a unique mathematical algorithm. Our high-performance server is capable of evaluating data obtained from a single sample within several minutes.
6. Test Results
On the basis of the data obtained, using a state-of-the-art algorithm and a sophisticated statistical and mathematical model, our lab specialists can determine the outcome of the analysis.
6.
Delivery of test results to physicians
Sequencing results are sent from the Trisomy test, s.r.o. laboratory back to Medirex. The test results report is delivered directly to the doctor who referred the patient for the test and took a sample of the patient´s blood.
Patients learn about their TRISOMY test, TRISOMY test XY or TRISOMY test + results from their physicians. Information regarding the sex of their foetuses can only be provided once they have completed their first trimester of pregnancy.
| RESULT | INTERPRETATION | RECOMMENDATION |
|---|---|---|
| Negative result | Trisomy of chromosome 21, 18 or 13 not detected. | A screen-negative result does not have to be validated by another test; the patient's attending physician, usually a gynaecologist, will choose further healthcare to be provided. |
| Positive result | Trisomy detected in one of the examined chromosomes. | A screen-positive TRISOMY test, TRISOMY test XY or TRISOMY test + result must be confirmed by a genetic test based on a sample obtained using invasive sampling. It is vital that the patient consults her results at a clinic specialising in medical genetics. |
| Noninformative result | The sample provided could not be processed in accordance with the principles of good laboratory practice or the result of the screening test does not answer the diagnostic question even if the sample is repeatedly analysed. | Another blood sample must be taken (no additional payment is required), typically 14 days later. The concentration of foetal DNA in maternal blood gets progressively higher further on in pregnancy. |
The method of screening test
Negative results only apply to the trisomy types that the test is designed to monitor (chromosomes 21, 18 and 13). The test does not exclude the presence of other genetic disorders, or other disorders that are outside its scope. The test result can be distorted due to multiple pregnancy, vanishing twin syndrome, or foreign DNA in the maternal blood sample – for example, following a blood transfusion, stem cell transplantation, organ transplantation, malignant diseases of the woman or her foetus. On an individual basis, the detection rate of chromosomal abnormalities can also be affected by placental mosaicism, vanishing twin syndrome, point mutation, gene inactivation, or other types of genetic or epigenetic mechanisms. In such cases, it is advisable that blood sampling is preceded by a consultation with the laboratory.
Despite the high sensitivity and specificity of the tests, a screen-positive TRISOMY test, TRISOMY test XY or TRISOMY test + result must be confirmed by a genetic analysis of the patient’s amniotic fluid or a chorionic villus sample. A screen-positive TRISOMY test result is not a relevant reason for abortion.*
Due to biological and technological limitations, the accuracy of TRISOMY test + microdeletion syndrome screening is relatively lower compared to trisomy 21, 18, and 13. Given the generally low occurrence of microdeletions in the population, there have been no studies that would reliably validate the accuracy of tests targeting these syndromes.
* Before recommending the TRISOMY test, make sure you consider the purpose, advantages and potential risks involved, as well as alternatives to the TRISOMY test, and ensure you have all necessary information about the condition of the pregnant patient that may affect your choice of the best procedure in the provision of healthcare.
* In accordance with the recommendation of the Ethics Committee of the Ministry of Health of the Slovak Republic issued on 4 February 2013 (http://www.health.gov.sk/Clanok?eticka-komisia-uznesenie-40-46).
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