QF-PCR

What is the QF-PCR technique?

Approximately 15% of pregnancies in women result in abortion, and there are numerous factors that contribute to this phenomenon. Among the most significant and prevalent factors leading to early miscarriages and pregnancy terminations are chromosomal abnormalities, with 96% of these abnormalities being attributed to an array of chromosome number irregularities. Thus far, the most frequently observed abnormalities involve chromosomes 13, 15, 16, 18, 21, X, and Y. It is worth noting that many women who have experienced a miscarriage due to chromosome number abnormalities are at an increased risk of encountering this issue in subsequent pregnancies. In such cases, cytogenetic studies play a critical role. The identification of the causes of abortion significantly diminishes the long-term psychological burden on mothers and determines the necessity for genetic counseling in future pregnancies.

Every normal human has 23 pairs of chromosomes, one of which is derived from the maternal lineage while the other is inherited from the paternal lineage. Chromosomal irregularities arise as a consequence of changes in the number or structure of chromosomes. Anomalies in the number or structure of chromosomes constitute a significant etiological factor underlying congenital abnormalities. In approximately 99% of instances, when either the father’s or the mother’s germ cells are generated, there exists a possibility for the presence of an additional chromosome within their cell. If such cells contribute to the formation of the embryo, the resultant embryo will be affected. Cytogenetic investigations, encompassing karyotyping and fluorescence in situ hybridization (FISH) techniques, serve as not only expensive and time-consuming methodologies for identifying aneuploidy, but also due to the complexities inherent in the cellular cultivation process, the potential for contamination, and the suboptimal quality of chromosomal preparation, satisfactory outcomes may not be achieved, thereby necessitating the repetition of experiments.

Principles of QF-PCR teqnique

In general, The QF PCR technique encompasses the processes of amplification, identification, and examination of well-known DNA sequences that are specific to chromosomes. This is achieved through the utilization of markers such as STRs (Short Tandem Repeats). In this particular methodology, primers that have been labeled with fluorescence are employed to amplify each marker, utilizing the PCR method. The number of chromosome copies is indicated by the copy number of each marker. Once the amplification of fragments has been successfully accomplished, the resultant PCR products can be analyzed and quantified using an automated genetic analyzer.

The length of STR markers may vary across chromosomes and individuals based on the number of repeats. The determination of the relative copy number for each allele involves the calculation of the ratio of peak areas or peak heights observed for each marker. A typical diploid sample exhibits two peaks for each individual chromosome. In the case of a heterozygous marker, two alleles of the chromosome-specific marker are observed as two peaks in a consistent 1:1 ratio. Conversely, when the marker is homozygous (meaning it possesses alleles of the same length), only one peak is detected. The identification of an extra allele manifests as either a triple peak in a ratio of 1:1:1 or two peaks in a ratio of 2:1/1:2. These findings suggest the presence of an additional copy of the marker, potentially corresponding to an extra chromosome.

Currently, novel research indicates that the QF-PCR molecular technique has the ability to serve as a complementary approach in the identification of chromosomal aneuploidy. At present, this approach is employed in the aneuploidy detection kit. This particular method relies on the Quantitative Fluorescence-Polymerase Chain Reaction procedure, which examines distinctive chromosomal markers through the utilization of primers that are marked with fluorescent substances.

Samples required for QF-PCR

Ultrasound and the utilization of double, triple or quadruple tests during the 10th and 11th weeks of gestation, administered by a gynecologist, will substantially ascertain the condition of the developing fetus. The precision of these assessments is about 85%. In cases where the outcomes of these evaluations are dubious or if the pregnant mother’s age exceeds 35 years, it becomes indispensable to conduct a genetic analysis of the chromosomes. Due to the prohibition of abortion in Iran beyond the 18th week of pregnancy, it is imperative to be apprised of the findings of all examinations prior to the conclusion of the 18th week of gestation. Consequently, subsequent to the 15th week of pregnancy, it is advised to employ the process of rapid diagnosis of chromosomal abnormalities, denominated as QF-PCR.

Response time of QF-PCR test

The QF-PCR method is characterized by its expeditiousness and relatively low cost, thereby yielding dependable outcomes within a span of 48 hours following sampling. This particular technique surpasses traditional cytogenetic methods in terms of both promptness and affordability, boasting an accuracy rate that exceeds 99%. In cases where the developing embryo shows signs of illness, the process of karyotyping is performed in conjunction with this examination. Currently, within the domain of forensic medicine, relying solely on the results of QF-PCR is inadequate to justify the termination of pregnancy; an additional method, such as MLPA findings, is required to support the decision to proceed with pregnancy termination. In situations with limited options, QF-PCR and MLPA assessments are conducted simultaneously with karyotyping.

Applications of QF-PCR method

Rapid diagnosis of chromosomes number abnormalities including Pato syndrome (trisomy 13), Edward syndrome (trisomy 18), Down syndrome (trisomy 21), Turner syndrome (45, XO), Klinefelter syndrome (47, XXY), Jacobs syndrome (47, XYY) and triple X syndrome (47, XXX)
The examination of the health of the fetus is conducted in cases where the results of the first trimester screening are deemed suspicious.
Determination of fetal sex
Diagnosis of ploidy (triploidy) in a fetus or aborted fetus
Rapid investigation of chromosomal abnormalities when in situations where cell culture is not viable or time constraints prohibit it
Chromosomal testing of fetal samples from pregnant women with possible chromosomal disorders. This includes a request for a chromosome test during pregnancy by a doctor, if the mother is over 35 years old during pregnancy and the observation of the test results is carried out during pregnancy, the suspicion of chromosomal disorders in the fetus, indications of chromosomal disorder in the fetus through ultrasound results during pregnancy, having a previous child with a chromosomal disorder (either living or deceased), or the mother or her husband having chromosomal disorders.

QF-PCR method Advantages in aneuploidy diagnosis

1) The identification of aneuploidy using the karyotype technique requires a time period of approximately 14 days. However, the QF-PCR method produces outcomes in a significantly shorter time frame, requiring only 4-5 hours. Moreover, the results of the QF-PCR technique can be determined within a two-day period following the collection of amniotic fluid (AF) or fetal chorionic villi (CVS), thus expediting the decision-making process for healthcare professionals and geneticists regarding the continuation or termination of a pregnancy. The expediency of this test is particularly crucial within the confines of Iran, where legal abortion is permissible up until the conclusion of the 18th week of gestation. This expeditiousness allows for an ample window of time for potential abortion. On the other hand, the lengthy waiting period of two to three weeks for test results presents a challenging experience for expectant mothers. If pregnant women decide to postpone seeking medical attention due to the limited time allowed for legal abortion, the QF-PCR technique can promptly provide a response. In the event that confirmation of fetal infection is achieved through the MLPA technique, the authorization for legal abortion can be swiftly granted, as both methods concur.
2) The QF-PCR technique represents an uncomplicated, rapid, dependable, and cost-effective means of prenatally detecting aneuploidy.
3) The QF-PCR method serves as a more expeditious diagnostic approach, and does not require cell culture. Consequently, this technique can determine the aneuploidy condition of the fetus within a span of 2-3 working days following the procurement of fetal CVS or amniotic fluid. Conversely, the process of cell culture and subsequent karyotype determination demands approximately 10-14 days.
4) The QF-PCR method has high sensitivity and specificity, and as a result, a very small amount of fetal genomic DNA is required to perform the PCR reaction and detect aneuploidy. That is why it is used in the aneuploidy detection kit.
5) After NIPT screening, the QF-PCR test can accurately and reliably diagnose prenatal aneuploidy.
6) This method has a very high sensitivity, specificity and accuracy (>99%) and in cases where the amniotic fluid or CVS sample is small, the probability of obtaining an answer is high.
7) The QF-PCR method is a more cost-effective diagnostic method, and compared to the relatively high price of prenatal chromosome tests, the cost of this method is lower and cost-effective for applicants.
8) The QF-PCR test can also be performed with a non-living sample (for example, an aborted or dead fetus). When cytogenetic methods are used, the sample must be prepared only from live embryos.
9) The performance quality of the QF-PCR method is such that it is not affected by gestational age and fetuses can be sampled at any age for testing.

QF-PCR test method

Sampling by amniocentesis and CVS after the twelfth week of pregnancy: Amniocentesis and CVC are invasive methods for natural sampling of fetal amniotic fluid or choroid. These two methods are always done by a specialist.
Isolation of DNA from amniotic fluid or choroidal vesicles: Most laboratories use a DNA extraction kit. Various DNA isolation kits are currently available in the market.
Amplification of markers by PCR method using a special QF-PCR kit
Sample analysis by capillary electrophoresis in the Genetic Analyzer device
Final analysis of samples using Gene Mapper or Gene Marker software

Limitation of QF-PCR method

These methods cannot detect low mosaicism and disorders of other chromosomes except 13, 18, 21, X and Y.
In general, the QF-PCR technique is not suitable for detecting deletions.
In some rare cases, markers for some chromosomes may be missing from the examined samples, in which case FISH or karyotyping methods are recommended.