Prof. M.C.Bansal MBBS., MS., FICOG., MICOG. Founder Principal & Controller,Jhalawar Medical College & Hospital Jjalawar.MGMC & Hospital , sitapura ., Jaipur
Prenatal diagnosis employs a variety of techniques to determine the health and condition of an unborn fetus. Without knowledge gained by prenatal diagnosis, there could be an untoward outcome for the fetus or the mother or both. Congenital anomalies account for 20 to 25% of perinatal deaths. Specifically, prenatal diagnosis is helpful for: Managing the remaining weeks of the pregnancy Determining the outcome of the pregnancy Planning for possible complications with the birth process Planning for problems that may occur in the newborn infant Deciding whether to continue the pregnancy Finding conditions that may affect future pregnancies
There are a variety of non-invasive and invasive techniques available for prenatal diagnosis. Each of them can be applied only during specific time periods during the pregnancy for greatest utility. The techniques employed for prenatal diagnosis include: Amniocentesis Chorionic villus sampling Fetal blood cells in maternal blood Maternal serum alpha-fetoprotein Maternal serum beta-HCG Maternal serum estriol Inhibin A Pregnancy associated plasma protein A
AmniocentesisTIME TO PERFORM:-14 and 20 weeks gestation . (Enough amniotic fluid is present for this to be accomplished starting about 14 weeks gestation.) However, an ultrasound examination always proceeds amniocentesis in order to determine gestational age, the position of the fetus and placenta, and determine if enough amniotic fluid is present. Within the amniotic fluid are fetal cells (mostly derived from fetal skin). After the amniotic fluid is extracted, the fetal cells are separated from the sample. The cells are grown in a culture medium, then fixed and stained for chromosome analysis, biochemical analysis, and molecular biologic analysis.
In the third trimester of pregnancy, the amniotic fluid can be analyzed for determination of fetal lung maturity. This is important when the fetus is below 35 to 36 weeks gestation, because the lungs may not be mature enough to sustain life. This is because the lungs are not producing enough surfactant. After birth, the infant will develop respiratory distress syndrome from hyaline membrane disease. The amniotic fluid can be analyzed by fluorescence polarization (fpol), for lecithin:sphingomyelin (LS) ration, and/or for phosphatidyl glycerol (PG).
Chorionic Villus Sampling (CVS) CVS can be safely performed between 9.5 and 12.5 weeks gestation. Sampling of cells from the placental chorionic villi. These cells can then be analyzed by a variety of techniques. The most common test employed on cells obtained by CVS is chromosome analysis to determine the karyotype of the fetus. The cells can also be grown in culture for biochemical or molecular biologic analysis Disadvantage :- invasive procedure, and it has a small but significant rate of morbidity for the fetus; this loss rate is about 0.5 to 1% higher than for women undergoing amniocentesis. Rarely, CVS can be associated with limb defects in the fetus. The possibility of maternal Rh sensitization is present. There is also the possibility that maternal blood cells in the developing placenta will be sampled instead of fetal cells and confound chromosome analysis.
Karyotyping Tissues must be obtained as fresh as possible for culture and without contamination. A useful procedure is to wash the tissue samples in sterile saline prior to placing them into cell culture media. Tissues with the best chance for growth are those with the least maceration: placenta, lung, diaphragm.
Sample Collection A karyotype will be done on the white blood cells which are actively dividing (a state known as mitosis). During pregnancy, the sample can either be amniotic fluid collected during an amniocentesis or a piece of the placenta collected during a chorionic villi sampling test (CVS). The amniotic fluid contains fetal skin cells which are used to generate a karyotype. Separating the Cells In order to analyze chromosomes, the sample must contain cells that are actively dividing (or in mitosis). In blood, the white blood cells are actively dividing cells. Most fetal cells are actively dividing. Once the sample reaches the cytogenetics lab, the non-divided cells are separated from the dividing cells using special chemicals.
Growing Cells In order to have enough cells to analyze, the dividing cells are grown in special media or a cell culture. This media contains chemicals and hormones that enable the cells to divide and multiply. This process of “culturing” the cells can take 3 to 4 days for blood cells, and up to a week for fetal cells. Synchronizing Cells Chromosome are long string of human DNA. In order to see chromosomes under a microscope, chromosomes have to be in their most compact form. This compact form occurs at a specific stage of mitosis called metaphase. In order to get all the cells to this specific stage of cell division, the cells are treated with a chemical which stops cell division at the point where the chromosomes are the most compact.
Releasing the Chromosomes from their Cells In order to see these compact chromosomes under a microscope, the chromosomes have to be out of the cells. This is done by treating the cells with a special solution that causes them to burst. This is done while the cells are on a microscopic slide. The leftover debris from the white blood cells is washed away, and the chromosomes are now fixed (or stuck) to the slide. Staining the Chromosomes Chromosomes are naturally colorless. In order to be able to tell one chromosome from another, a special dye called Giemsa dye is applied to the chromosomes on the slide. Giemsa dye stains regions of chromosomes that are rich in the bases adenine (A) and thymine (T). When stained, the chromosomes look like strings with light and dark bands. Each chromosome has a specific pattern of light and dark bands which enables cytogeneticist to tell one chromosome from another. Each dark or light band actually encompasses hundreds of different genes.
The chromosomes may be stained with aceto- orcein, feulgen or a basophilic dye such as toluidine blue or methylene blue if only the general morphology is desired. If more detail is desired, the chromosomes can be treated with various enzymes in combination with stains to yield banding patterns on each chromosome Q-banding Quinacrine stain Fluorescence microscopy G-banding Giemsa stain Additional Conditions a. Heat hydrolysis b. Trypsin treatment c. Giemsa at pH 9.0 R-banding Giemsa or acridine orange Negative bands of Q and G reversed Heat hydrolysis in buffered salt C-banding Giemsa stain Pretreatment with BaOH or NaOH followed by heat and salt.
Analysis Once chromosomes are stained, the slide is put under the microscope and the analysis of the chromosomes begins. A picture is taken of the chromosomes and at the end of the analysis, the total number of chromosomes will be known and there will be a picture of the chromosomes arranged by size. Counting Chromosomes The first step of the analysis is counting the chromosomes. Most humans have 46 chromosomes. People with Down syndrome have 47 chromosomes. It is also possible for people to have missing chromosomes or more than one extra chromosome. By looking at just the number of chromosomes, it is possible to diagnose different conditions including Down syndrome.
Looking at the Structure In addition to looking at the total number of chromosomes and the sex chromosomes, the cytogeneticist will also look at the structure of the specific chromosomes to make sure that there is no missing or additional material, no structural abnormalities like translocations and a variety of other possible chromosome abnormalities. The Final Result In the end, the final karyotype test shows the total number of chromosomes, the sex of the person being studied, and if there are any structural abnormalities with any of the individual chromosomes. A digital picture of the chromosomes is generated with all of the chromosomes arranged by number.
Spectral karyotype (SKYtechnique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling method is used to generate many different colors.
Neural tube defects can be distinguished from other fetal defects (such as abdominal wall defects) by use of the acetylcholinesterase test performed on amniotic fluid obtained by amniocentesis If the acetylcholinesterase is elevated along with MSAFP then a neural tube defect is likely. If the acetylcholinesterase is not detectable, then some other fetal defect is suggested
DNA Probes Fetal cells obtained via amniocentesis or CVS can be analyzed by probes specific for DNA sequences. One method employs restriction fragment length polymorphism (RFLP) analysis. This method is useful for detection of mutations involving genes that are closely linked to the DNA restriction fragments generated by the action of an endonuclease. The DNA of family members is analyzed to determine differences by RFLP analysis. In some cases, if the DNA sequence of a gene is known, a probe to a DNA sequence specific for a genetic marker is available, and the polymerase chain reaction (PCR) technique can be applied for diagnosis. There are many genetic diseases, but only in a minority have particular genes been identified, and tests to detect them have been developed in some of these. Thus, it is not possible to detect all genetic diseases. Moreover, testing is confounded by the presence of different mutations in the same gene, making testing more complex
In RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. Although now largely obsolete due to the rise of inexpensive DNA sequencing technologies, RFLP analysis was the first DNA profiling technique inexpensive enough to see widespread application. In addition to genetic fingerprinting, RFLP was an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing.
Maternal blood sampling for fetalblood cells This is a new technique that makes use of the phenomenon of fetal blood cells gaining access to maternal circulation through the placental villi. Ordinarily, only a very small number of fetal cells enter the maternal circulation in this fashion (not enough to produce a positive Kleihauer- Betke test for fetal-maternal hemorrhage). The fetal cells can be sorted out and analyzed by a variety of techniques to look for particular DNA sequences, but without the risks that these latter two invasive procedures inherently have. Fluorescence in-situ hybridization (FISH) is one technique that can be applied to identify particular chromosomes of the fetal cells recovered from maternal blood and diagnose aneuploid conditions such as the trisomies and monosomy X. The problem with this technique is that it is difficult to get many fetal blood cells. There may not be enough to reliably determine anomalies of the fetal karyotype or assay for other abnormalities.
FISH (performed on fresh tissue or paraffin blocks) In addition to karyotyping, fluorescence in situ hybridization (FISH) can be useful. A wide variety of probes are available. It is useful for detecting aneuploid conditions (trisomies, monosomies). Fresh cells are desirable, but the method can be applied even to fixed tissues stored in paraffin blocks, though working with paraffin blocks is much more time consuming and interpretation can be difficult The ability to use FISH on paraffin blocks means that archival tissues can be examined in cases where karyotyping was not performed, or cells didnt grow in culture.
A metaphase cell positive for thebcr/abl rearrangement (associatedwithchronic myelogenous leukemia) using FISH. The chromosomes can beseen in blue. The chromosome that is labeled with green and red spots is theone where the wrong rearrangement is present
Maternal serum alpha-fetoprotein(MSAFP) The developing fetus has two major blood proteins--albumin and alpha-fetoprotein (AFP). Since adults typically have only albumin in their blood, the MSAFP test can be utilized to determine the levels of AFP from the fetus. Ordinarily, only a small amount of AFP gains access to the amniotic fluid and crosses the placenta to mothers blood. However, when there is a neural tube defect in the fetus, from failure of part of the embryologic neural tube to close, then there is a means for escape of more AFP into the amniotic fluid.
Neural tube defects include anencephaly .Also, if there is an omphalocele or gastroschisis (both are defects in the fetal abdominal wall), the AFP from the fetus will end up in maternal blood in higher amounts. The blood taken is that from mom, but a sample can be obtained for testing from amniotic fluid. The AFP test is not diagnostic. It can only be used to test for the increased likelihood of an abnormality or birth defect. Alpha-Fetoprotein is a substance produced by the fetus in utero. AFP stops being produced once the baby is born. The AFP is excreted in the fetal urine which crosses into the mother’s blood stream. This is why AFP can be detected by a blood sample taken from the pregnant mother.
MSAFP may be performed between the 14th and 22nd weeks of pregnancy, however it seems to be most accurate during the 16th to 18th week. Your levels of AFP vary during pregnancy so accurate pregnancy dating is imperative for more reliable screening results. All pregnant women should be offered the MSAFP screening, but it is especially recommended for: Women who have a family history of birth defects Women who are 35 years or older Women who used possible harmful medications or drugs during pregnancy Women who have diabetes
NORMAL VALUES:- Adults: <15 ng/mL (15 mcg/L) Fetal blood (first trimester): Peak 200-400 mg/dL (2-4 g/L) Pregnancy (2nd trimester): 14 weeks gestation: Median 25.6 ng/mL (25.6 mcg/L) 15 weeks gestation: Median 29.9 ng/mL (29.9 mcg/L) 16 weeks gestation: Median 34.8 ng/mL (34.8 mcg/L) 17 weeks gestation: Median 40.6 ng/mL (40.6 mcg/L) 18 weeks gestation: Median 47.3 ng/mL (47.3 mcg/L) 19 weeks gestation: Median 55.1 ng/mL (55.1 mcg/L) 20 weeks gestation: Median 64.3 ng/mL (64.3 mcg/L) 21 weeks gestation: Median 74.9 ng/mL (74.9 mcg/L) The MSAFP is typically reported as multiples of the mean (MoM). The greater the MoM, the more likely a defect is present The multiple of the median (MoM) value is adjusted for maternal weight, race, diabetes mellitus, and twin pregnancy
However, the MSAFP can be elevated for a variety of reasons which are not related to fetal neural tube or abdominal wall defects, so this test is not 100% specific. The most common cause for an elevated MSAFP is a wrong estimation of the gestational age of the fetus.
Using a combination of MSAFP screening and ultrasonography, almost all cases of anencephaly can be found and most cases of spina bifida. The MSAFP can also be useful in screening for Down syndrome and other trisomies. The MSAFP tends to be lower when Down syndrome or other chromosomal abnormalities is present.
Maternal serum beta-HCG The hormone human chorionic gonadotropin (better known as hCG) is produced during pregnancy. It is made by cells that form the placenta, which nourishes the egg after it has been fertilized and becomes attached to the uterine wall. Levels can first be detected by a blood test about 11 days after conception and about 12 - 14 days after conception by a urine test. In general the hCG levels will double every 72 hours. The level will reach its peak in the first 8 - 11 weeks of pregnancy and then will decline and level off for the remainder of the pregnancy.
An hCG level of less than 5mIU/ml is considered negative for pregnancy, and anything above 25mIU/ml is considered positive for pregnancy. The hCG hormone is measured in milli- international units per milliliter (mIU/ml). A transvaginal ultrasound should be able to show at least a gestational sac once the hCG levels have reached between 1,000 - 2,000mIU/ml. There are two common types of hCG tests. A qualitative hCG test detects if hCG is present in the blood. A quantitative hCG test (or beta hCG) measures the amount of hCG actually present in the blood.
What can a low hCG level mean? A low hCG level can mean any number of things and should be rechecked within 48-72 hours to see how the level is changing. A low hCG level could indicate: Miscalculation of pregnancy dating Possible miscarriage or blighted ovum Ectopic pregnancy
What can a high hCG level mean? A high level of hCG can also mean a number of things and should be rechecked within 48-72 hours to evaluate changes in the level. A high hCG level can indicate: Miscalculation of pregnancy dating Molar pregnancy Multiple pregnancy
Maternal serum estriol The amount of estriol in maternal serum is dependent upon a viable fetus, a properly functioning placenta, and maternal well-being. The substrate for estriol begins as dehydroepiandrosterone (DHEA) made by the fetal adrenal glands. This is further metabolized in the placenta to estriol. The estriol crosses to the maternal circulation and. is excreted by the maternal kidney in urine or by the maternal liver in the bile The measurement of serial estriol levels in the third trimester will give an indication of general well-being of the fetus.
If the estriol level drops, then the fetus is threatened and delivery may be necessary emergently. Estriol tends to be lower when Down syndrome is present and when there is adrenal hypoplasia with anencephaly. When it is used this way, each sample should be drawn at the same time each day.
Inhibin-A Inhibin is secreted by the placenta and the corpus luteum. Inhibin A is made by the placenta during pregnancy. The level of inhibin A in the blood is used in a maternal serum quadruple screening test. Generally done between 15 and 20 weeks Inhibin-A can be measured in maternal serum. An increased level of inhibin-A is associated with an increased risk for trisomy 21. A high inhibin-A may be associated with a risk for preterm delivery.
Pregnancy-associated plasmaprotein A (PAPP-A) Pregnancy-associated plasma protein A, pappalysin 1, also known as PAPPA, is a protein used in screening tests for Down syndrome Low levels of PAPP-A as measured in maternal serum during the first trimester may be associated with fetal chromosomal anomalies including trisomies 13, 18, and 21. In addition, low PAPP-A levels in the first trimester may predict an adverse pregnancy outcome, including a small for gestational age (SGA) baby or stillbirth. A high PAPP-A level may predict a large for gestational age (LGA) baby.
Triple" or "Quadruple" screen Combining the maternal serum assays may aid in increasing the sensitivity and specificity of detection for fetal abnormalities. The classic test is the triple screen for alpha- fetoprotein (MSAFP), beta-HCG, and estriol (uE3). The "quadruple screen" adds inhibin-A.
TRIPLE TEST The triple test, also called triple screen, the Kettering test or the Barts test, is an investigation performed during pregnancy in the second trimester to classify a patient as either high-risk or low-risk for chromosomal abnormalities (and neural tube defects). The Triple test measures serum levels of AFP, estriol, and beta-hCG, with a 70% sensitivity and 5% false-positive rate.
The triple test measures the following three levels in the maternal serum: alpha-fetoprotein (AFP) human chorionic gonadotropin (hCG) unconjugated estriol (UE3) AFP UE3 hCG ASSOCIATED CONDITIONS LOW LOW HIGH DOWN SYNDROME LOW LOW LOW TRISOMY 18(EDWARD SYNDROME) HIGH N/A N/A NEURAL TUBE DEFECT LIKE SPINA BIFIDA, MULTIPLE GESTATIONS,OMP HALOCELE
Quadruple test A test of levels of dimeric inhibin A (DIA) is sometimes added to the other three tests, under the name "quadruple test.” Other names used include "quad test", "quad screen", or "tetra screen." Inhibin A (DIA) will be found high in cases of Trisomy 21 and low in cases of Trisomy 18.
Biochemical Analysis Tissues can be obtained for cell culture or for extraction of compounds that can aid in identification of inborn errors of metabolism. Examples include: long-chain fatty acids (adrenoleukodystrophy) amino acids (aminoacidurias)